Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Long non-coding RNA BACE1-AS plays an oncogenic role in hepatocellular carcinoma cells through miR-214-3p/APLN axis

Long non-coding RNA BACE1-AS plays an oncogenic role in hepatocellular carcinoma cells through... Abstract BACE1 antisense RNA (BACE1-AS) is implicated in promoting cell proliferation in different types of tumors. However, the function and mechanism of BACE1-AS in hepatocellular carcinoma (HCC) are still unclear. In the present study, we found that the relative expression of BACE1-AS in HCC cell lines, HCC tissues, and serum samples of HCC patients was significantly increased, and its high expression was correlated with the poor prognosis of HCC patients. In addition, overexpression of BACE1 promoted HCC cell proliferation, cell cycle progression, migration, and invasion, but inhibited cell apoptosis, while knockdown of BACE1 exerted the opposite role. Furthermore, BACE1-AS sponged microRNA-214-3p (miR-214-3p) and inhibited its expression, thus promoting Apelin (APLN) expression. Overexpression or knockdown of miR-214-3p could partially reverse the abnormal proliferation, cell cycle progression, migration, invasion, and apoptosis caused by overexpression or knockdown of BACE1. These findings suggest that the BACE1-AS/miR-214-3p/APLN axis is a novel signaling pathway that facilitates HCC. BACE1-AS, miR-214-3p, APLN, proliferation, cell cycle, HCC Introduction Primary liver cancer is the sixth most common cancer, and hepatocellular carcinoma (HCC) takes up 90% of all liver cancer cases [1,2]. In the early stage, patients with HCC can be cured by liver transplantation or surgical resection, but few therapies are effective for patients with advanced HCC [3]. It is urgent to clarify the mechanism of this complicated disease to further explore more effective therapeutic strategies. Long non-coding RNAs (lncRNAs) are defined as RNA transcripts with more than 200 nucleotides in length that do not encode proteins [4]. Recent research shows the promising diagnostic and therapeutic value of lncRNAs for HCC. For example, lncRNA RGMB-AS1 is found to be lowly expressed in HCC tissues and cell lines, and RGMB-AS1 overexpression repressed HCC cell proliferation, migration, and invasion [5]. BACE1 antisense RNA (BACE1-AS) is found to play a regulatory role in some tumors. For example, anisomycin represses the proliferation and invasion of human ovarian cancer stem cells and promotes BACE1-AS expression; after BACE1-AS is silenced, the antiproliferative and anti-invasive effects of anisomycin are weakened [6]. However, whether BACE1-AS can modulate the progression of HCC warrants further investigation. In addition to lncRNAs, microRNAs (miRNAs) are vital regulators in the development of tumors. miR-214-3p has been widely investigated in a variety of tumors. In breast cancer, miR-214-3p overexpression inhibits the proliferation of cancer cells via targeting survivin [7]. In HCC, it was reported that miR-214-3p expression is downregulated in HCC samples, and upregulated miR-214-3p expression induces HCC cell cycle arrest and increases apoptosis [8]. Apelin (APLN) is an endogenous ligand of the apelin receptor (APJ) [9]. Multiple physiological and pathological processes are mediated by the APLN/APJ system. It has been revealed that APLN mRNA expression is markedly elevated in HCC specimens, and inhibiting APLN expression using short hairpin RNAs (shRNAs) suppresses the growth of HCC cells in vitro and in vivo, which suggests that APLN has the potential to be the diagnostic biomarker and therapeutic target for HCC [10]. But how APLN expression is regulated in HCC has not been fully explored. In this study, by bioinformatics analysis, we found that BACE1-AS expression was remarkably elevated in HCC tissues. In addition, the potential binding sites between BACE1-AS and miR-214-3p and between miR-214-3p and APLN 3′UTR were identified. Thus, we hypothesized that BACE1-AS‒miR-214-3p‒APLN axis could regulate HCC progression. Materials and Methods Samples collection The HCC samples and matched adjacent non-tumorous liver tissues were resected from 28 patients with primary HCC from April 2016 to June 2018, who were also recruited during the same period. Serum samples were collected from both HCC patients and healthy subjects. All serum and tissue samples were stored at −80°C. The research protocol was approved by the Ethics Committee of Tianjin First Central Hospital, and all patients signed informed consent forms. Cell culture and transfection HCC cell lines (Huh6, Huh7, HepG2, and Hep3B) and normal liver epithelial cell line L02 were obtained from the American Type Culture Collection (Manassas, USA). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Life Technologies, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, USA) at 37°C with 5% CO2. Huh7 and HepG2 cells in the logarithmic phase were selected for experiments. When the rate of cell confluence achieved 70%–80%, Huh7 and HepG2 cells were trypsinized and resuspended in culture medium. The cell suspension was plated to a 6-well plate at a density of 1×106 cells/ml. Subsequently, Huh7 cells were transfected with BACE1-AS shRNA (sh-BACE1-AS: 5′-GCTTTGGCACCTCCTAAGTGT-3′), negative control (sh-NC: 5′-GTATGACAACAGCCTCAAGCTCGAGCTTGAGGCTGTTGTCATAC-3′), miR-214-3p mimics (5′-ACAGCAGGCACAGACAGGCAGU-3′), or the negative controls (5′-UUCUCCGAACGUGUCACGUTT-3′), and HepG2 cells were transfected with BACE1-AS overexpression plasmid, empty vector (vector), miR-214-3p inhibitors (5′-ACUGCCUGUCUGUGCCUGCUGU-3′), or the negative controls (5′-TCGTTCATGAACGAACATT-3′) using Lipofectamine™ 2000 (Invitrogen, Carlsbad, USA) to construct the cell models. The transfection efficiency was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) after 24 h. qRT-PCR Total RNA was isolated from cells or tissues using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. The extracted RNA was reversely transcribed to complementary DNA using a SuperScript Reverse Transcriptase Kit (Vazyme, Nanjing, China). qRT-PCR was then performed with the SYBR Green PCR Master Mix kit (Vazyme) on a Fast Real-time PCR 7300 System (Applied Biosystems, Foster City, USA). U6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were employed as the reference genes, and the 2–ΔΔCT method was used to calculate the relative expression. The primers were designed and constructed by BGI (Shenzhen, China). The primer sequences were as follows: BACE1-AS, forward: 5′-CTTGGGCAAACGAAGGTTGG-3′, reverse: 5′-CCCAGAGCCCAGCATCAAAA-3′; miR-214-3p, forward: 5ʹ-GCACAGCAGGCACAGACA-3ʹ, reverse: 5ʹ-CAGAGCAGGGTCAGCGGTA-3ʹ; GAPDH, forward: 5ʹ-GCACCGTCAAGGCTGAGAAC3ʹ, reverse: 5ʹ-TGGTGAAGACGCCAGTGGA-3ʹ; U6, forward: 5ʹ-CTCGCTTCGGCAGCACA-3ʹ, reverse: 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ. The reaction conditions were as follows: Pre-denaturation of 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, primer-specific annealing temperature at 72°C for 30 s, and extension at 72°C for 10 min. Cell counting kit-8 assay Huh7 and HepG2 cells in the logarithmic phase were transferred into a 96-well plate (1×103 cells/well), and then the cell viability was detected at 24, 48, 72, and 96 h. After the cells in each well were incubated with 10 μl of Cell counting kit (CCK)-8 reagent (Dojindo, Tokyo, Japan) at 37°C for 1 h, the optical density (OD) value at 450 nm was measured with a microplate reader. Ultimately, the viability of the cells was calculated. Cell cycle analysis The effects of BACE1-AS and miR-214-3p on the Huh7 and HepG2 cell cycles were evaluated with a flow cytometer (BD Biosciences, Franklin Lakes, USA). Briefly, cells were rinsed twice with phosphate buffered saline (PBS), trypsinized, fixed with 75% ethanol overnight at −20°C, and then incubated with 100 mg/ml RNase A and 50 mg/ml propidium iodide (PI) solution for 30 min. Subsequently, the percentage of cells in the G0/G1, S, and G2/M was assessed in each group by flow cytometry. Apoptosis detection HepG2 and Huh7 cells were planted into a 6-well plate and then cultured for 48 h. After rinsing three times with PBS, the cells were harvested and then resuspended in 500 μl of binding buffer (10×: 0.1 M HEPES, pH 7.4, 1.4 M NaCl, 25 mM CaCl2). Subsequently, 5 μl of Annexin V-FITC kit solution (Southern Biotechnology, Birmingham, USA) and 5 μl of PI solution were incubated with the above mixture in the dark for 30 min. Then, cell apoptosis was analyzed by flow cytometry on a flow cytometer (BD Biosciences), and the apoptosis rate of cells was calculated. Transwell assays Transwell assays were performed using Transwell chambers (pore size: 8 µM, Corning Costar, Cambridge, USA). The Transwell chambers were placed on 24-well plates. Then, the cells were re-suspended in a serum-free medium, and the density was adjusted to 2×105 cells/ml. After that, the cells were added to the upper chamber (4×104 cells/well), and 500 µl of DMEM containing 10% FBS was added to the wells of the plates. After 24 h, a cotton swab was applied to remove the cells on the upper chamber, and the migrated or invaded cells were fixed with 95% ethanol and then stained with crystal violet for 20 min. Next, four random fields of each membrane were observed under an inverted microscope to count the number of the cells. In the invasion assay, Matrigel (BD Biosciences, Franklin Lakes, USA) was used to coat the membrane, while in the migration assay, Matrigel was not used. RNA immunoprecipitation assay To pinpoint the binding relationship between BACE1-AS and miR-214-3p, RNA immunoprecipitation (RIP) assay was conducted using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, USA). In brief, Huh7 and HepG2 cells at 80% confluency were harvested and lysed in RIP lysis buffer. Whole-cell extracts were coimmunoprecipitated with RIP buffer containing anti-Argonaute2 (Ago2) antibody conjugated with magnetic beads (Millipore) or normal mouse immunoglobulin G (IgG; Millipore). Subsequently, the samples were treated with proteinase K. Coprecipitated RNA was then isolated with the TRIzol method, and qRT-PCR was employed to detect the enrichment of BACE1-AS and miR-214-3p. Dual-Luciferase reporter assay The sequence segments of BACE1-AS and APLN 3ʹuntranslated region (UTR) containing the binding sites of miR-214-3p were amplified by qRT-PCR and then inserted into pmir-GLO luciferase expression vectors to construct BACE1-AS/APLN wild-type vector (pmirGLO-BACE1-AS-WT/pmirGLO-APLN-WT). BACE1-AS/APLN mutant-type vector (pmirGLO-BACE1-AS-MUT/pmirGLO-APLN-MUT) was constructed after the mutation of the binding sites. The luciferase reporter vectors, miR-214-3p mimics, and negative control were mixed with Lipofectamine™ 2000 (Invitrogen) and then co-transfected into HepG2 cells. After 48 h, the luciferase activity was detected using a Dual-Luciferase Reporter Assay System (Promega, Madison, USA). Western blot analysis APLN expression on protein level was detected by western blot analysis. Briefly, HepG2 and Huh7 cells were lysed using radioimmunoprecipitation assay (RIPA) lysis reagent (Beyotime, Shanghai, China) containing 1% phenylmethylsulfonyl fluoride (PMSF; Beyotime), and the supernatant was collected after centrifugation. Moreover, Bradford method was used for protein quantification. After being mixed with 5× loading buffer, the protein samples were denatured by heating in boiling water for 5 min. Subsequently, the samples were subject to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membrane (Millipore). After being blocked with 5% skim milk for 1 h, the membrane was incubated with rabbit polyclonal anti-APLN antibody (1:1000; Abcam, Cambridge, UK) at 4°C overnight. Afterward, the membrane was rinsed twice with Tris buffered saline with Tween (TBST) and then incubated with horseradish-peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Abcam; 1:2000) for 1 h, followed by rinsing with TBST three times. The protein bands on the membrane were visualized using an electrochemiluminescense western blotting substrate kit (Promega). GAPDH was regarded as the internal control. Statistical analysis Statistical analysis was carried out using SPSS 23.0 software (SPSS Inc., Chicago, USA). The data were expressed as mean±standard deviation, and the comparison of means was conducted using one-way analysis of variance or two-tailed Student’s t-test. P<0.05 signified statistical significance. Results Bioinformatics analysis identified BACE1-AS as a biomarker in HCC First of all, we downloaded lncRNA transcriptome data from The Cancer Genome Atlas (TCGA), including 371 HCC samples and 50 adjacent normal liver tissue samples. With edgeR, differentially expressed lncRNAs with |log2FC|≥ 1 and FDR<0.05 were screened out and BACE1-AS expression was found to be significantly increased in HCC samples in comparison with normal liver tissue samples (Fig. 1A–C). In addition, we performed survival analysis using TGCA data, GEPIA database, and ENCORI database, and the results indicated that the high expression of BACE1-AS was related to poor prognosis in HCC patients (Fig. 1D–F). Figure 1. Open in new tabDownload slide Bioinformatics analysis suggests that BACE1-AS expression is dysregulated in HCC (A) lncRNA transcriptome data from TCGA were used to plot the heatmap and the top 50 lncRNAs with the most significant change in HCC tissues are shown (vs. normal liver tissues). (B) lncRNAs with differential expression are shown in a volcano plot. (C) The expression of BACE1-AS in HCC tissue or normal tissue was analyzed using the GEPIA database. (D‒F) The survival analysis of BACE1-AS for HCC patients was performed using the data from the TCGA database, GEPIA database, and ENCORI database. Figure 1. Open in new tabDownload slide Bioinformatics analysis suggests that BACE1-AS expression is dysregulated in HCC (A) lncRNA transcriptome data from TCGA were used to plot the heatmap and the top 50 lncRNAs with the most significant change in HCC tissues are shown (vs. normal liver tissues). (B) lncRNAs with differential expression are shown in a volcano plot. (C) The expression of BACE1-AS in HCC tissue or normal tissue was analyzed using the GEPIA database. (D‒F) The survival analysis of BACE1-AS for HCC patients was performed using the data from the TCGA database, GEPIA database, and ENCORI database. BACE1-AS expression was increased in HCC tissues and cell lines We then measured the expression of BACE1-AS in HCC tissues and cell lines with qRT-PCR. The data revealed that BACE1-AS expression was significantly upregulated in HCC tissue samples and serum samples of HCC patients (Fig. 2A,B) compared with the normal controls. In addition, BACE1-AS expression in HCC cell lines, including Huh-6, Huh7, HepG2, and Hep3B, was significantly upregulated compared with that in normal liver epithelial cell line L02 (Fig. 2C). Figure 2. Open in new tabDownload slide The expression characteristics of BACE1-AS in HCC (A,B) The expression of BACE1-AS in HCC tissues, adjacent normal tissues, and serum samples from HCC patients was quantified by qRT-PCR. (C) BACE1-AS expression in normal liver epithelial cell and HCC cell lines was quantified by qRT-PCR. ***P<0.001. Figure 2. Open in new tabDownload slide The expression characteristics of BACE1-AS in HCC (A,B) The expression of BACE1-AS in HCC tissues, adjacent normal tissues, and serum samples from HCC patients was quantified by qRT-PCR. (C) BACE1-AS expression in normal liver epithelial cell and HCC cell lines was quantified by qRT-PCR. ***P<0.001. BACE1-AS promoted the proliferation, migration, and invasion and inhibited the apoptosis of HCC cells To explore the biological function of BACE1-AS in HCC cells, BACE1-AS overexpression plasmid and shRNA were transfected into HepG2 cells and Huh7 cells, respectively. qRT-PCR was applied to verify the effects of transfection (Fig. 3A). The results of CCK-8 assay showed that overexpression of BACE1-AS significantly promoted cell proliferation and that knockdown of BACE1-AS worked oppositely (Fig. 3B). Flow cytometry results indicated that BACE1-AS induced more cells to enter the S phase and notably suppressed cell apoptosis, while knockdown of BACE1-AS blocked cell cycle progression and promoted cell apoptosis (Fig. 3C,D). In the Transwell assays, it was demonstrated that BACE1-AS overexpression enhanced the migration and invasion ability of HepG2 cells, while knockdown of BACE1-AS repressed the proliferation and migration of Huh7 cells (Fig. 3E). Figure 3. Open in new tabDownload slide BACE1-AS regulates the biological behaviors of HCC cells (A) The transfection efficiency of BACE1-AS overexpression plasmid and BACE1-AS shRNA was validated by qRT-PCR. (B) CCK-8 assay was performed to measure the proliferation of HepG2 and Huh7 cells. (C) Flow cytometry was used to detect the cell cycle of HepG2 and Huh7 cells. (D) Flow cytometry was used to detect the apoptosis of HepG2 and Huh7 cells. (E) Migration and invasion of HepG2 and Huh7 cells were examined by Transwell assays. Magnification fold: 100×. *P<0.05, **P<0.01, and ***P<0.001. Figure 3. Open in new tabDownload slide BACE1-AS regulates the biological behaviors of HCC cells (A) The transfection efficiency of BACE1-AS overexpression plasmid and BACE1-AS shRNA was validated by qRT-PCR. (B) CCK-8 assay was performed to measure the proliferation of HepG2 and Huh7 cells. (C) Flow cytometry was used to detect the cell cycle of HepG2 and Huh7 cells. (D) Flow cytometry was used to detect the apoptosis of HepG2 and Huh7 cells. (E) Migration and invasion of HepG2 and Huh7 cells were examined by Transwell assays. Magnification fold: 100×. *P<0.05, **P<0.01, and ***P<0.001. miR-214-3p was identified as a direct target of BACE1-AS To uncover the underlying mechanism of BACE1-AS in HCC progression, we investigated the target miRNAs of BACE1-AS. By analyzing the data in the StarBase database and Gene Expression Omnibus dataset GSE108724, we found that miR-214-3p, whose expression was downregulated in HCC tissues compared with normal liver tissues, was predicted to be a potential target of BACE1-AS (Fig. 4A–D). Dual-Luciferase Reporter gene experiment revealed that miR-214-3p mimics remarkably weakened the luciferase activity of BACE1-AS wild-type reporter, but exerted no effect on the luciferase activity of the mutant-type reporter (Fig. 4E). RIP assay further revealed the direct binding relationship between BACE1-AS and miR-214-3p (Fig. 4F). BACE1-AS overexpression reduced the expression of miR-214-3p in HepG2 cells, whereas the antagonism of BACE1-AS increased the miR-214-3p expression in Huh7 cells (Fig. 4G). However, the selective regulation of miR-214-3p expression in HCC cells did not influence BACE1-AS expression (Fig. 4H). Furthermore, in HCC tissues and serum samples of HCC patients, BACE1-AS and miR-214-3p expressions were found to be negatively correlated (Fig. 4I,J). The above results confirmed that miR-214-3p expression was negatively regulated by BACE1-AS in HCC. Figure 4. Open in new tabDownload slide Identification of the binding site between BACE1-AS and miR-214-3p (A) Heatmap was plotted using the data from GSE108724 and miRNAs with significant changes (P<0.05) and log2FC>1.5 or <−1.5 are shown. (B) miRNAs in GSE108724 are shown in the volcano plot. The downregulated miRNA expressions are marked blue, the upregulated miRNA expressions are marked red, and miR-214-3p expression is marked green. (C,D) miR-214-3p was selected as a potential target of BACE1-AS and the binding site was predicted with the StarBase database. (E) Dual-Luciferase Reporter gene assay was used to identify the binding site between BACE1-AS and miR-214-3p. (F) The binding relationship between BACE1-AS and miR-214-3p was further identified by RIP assay. (G) qRT-PCR was used to detect the regulatory effects of BACE1-AS on miR-214-3p expression in HCC cells. (H) qRT-PCR was performed to explore the effects of miR-214-3p on BACE1-AS expression in HCC cells. (I,J) The correlations between BACE1-AS and miR-214-3p in HCC tissues and serum samples of HCC patients. ***P<0.001. Figure 4. Open in new tabDownload slide Identification of the binding site between BACE1-AS and miR-214-3p (A) Heatmap was plotted using the data from GSE108724 and miRNAs with significant changes (P<0.05) and log2FC>1.5 or <−1.5 are shown. (B) miRNAs in GSE108724 are shown in the volcano plot. The downregulated miRNA expressions are marked blue, the upregulated miRNA expressions are marked red, and miR-214-3p expression is marked green. (C,D) miR-214-3p was selected as a potential target of BACE1-AS and the binding site was predicted with the StarBase database. (E) Dual-Luciferase Reporter gene assay was used to identify the binding site between BACE1-AS and miR-214-3p. (F) The binding relationship between BACE1-AS and miR-214-3p was further identified by RIP assay. (G) qRT-PCR was used to detect the regulatory effects of BACE1-AS on miR-214-3p expression in HCC cells. (H) qRT-PCR was performed to explore the effects of miR-214-3p on BACE1-AS expression in HCC cells. (I,J) The correlations between BACE1-AS and miR-214-3p in HCC tissues and serum samples of HCC patients. ***P<0.001. miR-214-3p suppressed the proliferation, migration, and invasion and accelerated the apoptosis of HCC cells Next, we transfected miR-214-3p mimics and miR-214-3p inhibitors into Huh7 and HepG2 cells, respectively, to detect the effects of miR-214-3p on cell proliferation, apoptosis, cell cycle, migration, and invasion of HC cells (Fig. 5A). In the CCK-8 assay, miR-214-3p mimics markedly repressed the proliferation of Huh7 cells, while miR-214-3p inhibitors promoted the proliferation of HepG2 cells (Fig. 5B). miR-214-3p was found to induce the cell cycle arrest and apoptosis of HCC cells (Fig. 5C,D). In addition, miR-214-3p weakened the migration and invasion of Huh7 cells, and inhibition of miR-214-3p promoted the migration and invasion of HepG2 cells (Fig. 5E). These data suggested that the functions of miR-214-3p were contrary to BACE1-AS in HCC cells. Figure 5. Open in new tabDownload slide miR-214-3p regulates the biological behaviors of HCC cells (A) qRT-PCR was used to verify the transfection efficiency of miR-214-3p mimics and miR-214-3p inhibitors. (B) CCK-8 assay was performed to measure the proliferation of HepG2 and Huh7 cells. (C) Flow cytometry was used to detect the cell cycle of HepG2 and Huh7 cells. (D) Flow cytometry was used to detect the apoptosis of HepG2 and Huh7 cells. (E) Migration and invasion abilities of HepG2 and Huh7 cells were examined by Transwell assays. Magnification fold: 100×. *P<0.05, **P<0.01, and ***P<0.001. Figure 5. Open in new tabDownload slide miR-214-3p regulates the biological behaviors of HCC cells (A) qRT-PCR was used to verify the transfection efficiency of miR-214-3p mimics and miR-214-3p inhibitors. (B) CCK-8 assay was performed to measure the proliferation of HepG2 and Huh7 cells. (C) Flow cytometry was used to detect the cell cycle of HepG2 and Huh7 cells. (D) Flow cytometry was used to detect the apoptosis of HepG2 and Huh7 cells. (E) Migration and invasion abilities of HepG2 and Huh7 cells were examined by Transwell assays. Magnification fold: 100×. *P<0.05, **P<0.01, and ***P<0.001. Augmentation of miR-214-3p expression repressed the effects of BACE1-AS on HCC cells Next, we co-transfected miR-214-3p mimics and BACE1-AS overexpression plasmid into HepG2 cells to further investigate the functional relationship between BACE1-AS and miR-214-3p. It was found that miR-214-3p restoration reversed the promotion of proliferation, the acceleration of cell cycle progression, the enhancement of migration and invasion, and the inhibition of apoptosis caused by BACE1-AS overexpression (Fig. 6A–E). These results demonstrated that miR-214-3p counteracted the effects of BACE1-AS on HCC cells. Figure 6. Open in new tabDownload slide BACE1-AS regulates the phenotypes of HCC cells via miR-214-3p (A) miR-214-3p mimics and BACE1-AS overexpression plasmid were cotransfected into HepG2 cells, and then the expression of miR-214-3p was detected by qRT-PCR. (B) After the transfection, CCK-8 assay was performed to measure the proliferation of HepG2 cells. (C) Flow cytometry was used to detect the cell cycle of HepG2 cells. (D) Flow cytometry was used to detect the apoptosis of HepG2 cells. (E) Migration and invasion of HepG2 cells were detected via Transwell assays. Magnification fold: 100×. **P<0.01 and ***P<0.001. Figure 6. Open in new tabDownload slide BACE1-AS regulates the phenotypes of HCC cells via miR-214-3p (A) miR-214-3p mimics and BACE1-AS overexpression plasmid were cotransfected into HepG2 cells, and then the expression of miR-214-3p was detected by qRT-PCR. (B) After the transfection, CCK-8 assay was performed to measure the proliferation of HepG2 cells. (C) Flow cytometry was used to detect the cell cycle of HepG2 cells. (D) Flow cytometry was used to detect the apoptosis of HepG2 cells. (E) Migration and invasion of HepG2 cells were detected via Transwell assays. Magnification fold: 100×. **P<0.01 and ***P<0.001. BACE1-AS worked by indirectly regulating APLN expression In GSE101728, APLN expression was found to be notably upregulated in human liver cancer tissue compared with that in the adjacent normal tissue (Fig. 7A). Additionally, by searching microT, miRanda, and miRmap databases, APLN was predicted as a target of miR-214-3p (Fig. 7B,C). Dual-Luciferase Reporter experiment results showed that miR-214-3p mimics markedly weakened the luciferase activity in APLN wild-type reporter, but when the two predicted binding sites were simultaneously mutated, the transfection of miR-214-3p failed to reduce the luciferase reporter vector (Fig. 7D). Additionally, BACE1-AS overexpression was found to significantly elevate APLN expression, while BACE1-AS knockdown suppressed it; miR-214-3p could decrease APLN expression (Fig. 7E,F). So far, we concluded that BACE1-AS promoted the proliferation, migration, and invasion and inhibited the apoptosis of HCC cells, and it also repressed the expression of miR-214-3p and upregulated the expression of APLN. Figure 7. Open in new tabDownload slide BACE1-AS indirectly regulates APLN (A) GSE101728 indicated that APLN expression was significantly upregulated in HCC tissue compared with the adjacent normal tissue. (B) The target genes of miR-214-3p were predicted with microT, miRanda, miRmap, and GSE101728. (C,D) Dual-Luciferase Reporter gene assays were performed to confirm the binding between miR-214-3p and APLN. (E,F) Western blot analysis was used to detect APLN expression in HepG2 and Huh7 cells after BACE1-AS, and miR-214-3p expressions were selectively regulated. **P<0.01 and ***P<0.001. Figure 7. Open in new tabDownload slide BACE1-AS indirectly regulates APLN (A) GSE101728 indicated that APLN expression was significantly upregulated in HCC tissue compared with the adjacent normal tissue. (B) The target genes of miR-214-3p were predicted with microT, miRanda, miRmap, and GSE101728. (C,D) Dual-Luciferase Reporter gene assays were performed to confirm the binding between miR-214-3p and APLN. (E,F) Western blot analysis was used to detect APLN expression in HepG2 and Huh7 cells after BACE1-AS, and miR-214-3p expressions were selectively regulated. **P<0.01 and ***P<0.001. Discussion Accumulating evidence has revealed the regulatory functions of lncRNAs in HCC [11–13]. For example, lncTCF7 recruits SWI/SNF complex to the promoter region of TCF7 to trigger TCF7 transcription and activates the Wnt signal pathway, and via this mechanism, lncTCF7 promotes the renewal of liver cancer stem cells and facilitates the progression of HCC [11]. In recent years, BACE1-AS has reportedly been linked to the pathogenesis of neurological diseases, such as Alzheimer’s disease and Parkinson’s disease [14–16]. Also, the role of BACE1-AS in cancer biology is being gradually recognized. Specifically, BACE1-AS expression in gastric cancer tissues is significantly lower than that in the adjacent normal tissues [17]. Anisomycin exerts its antiproliferative and anti-invasive effects on ovarian cancer stem cells by upregulating BACE1-AS expression [6]. The tumor-suppressive role of BACE1-AS in ovarian cancer is different from the oncogenic role in HCC reported in the present work, which implies that the biological function of BACE1-AS is distinct in different cancers. Notably, BACE1-AS was reported to be highly expressed in HCC tissues, and the receiver operating characteristic curve suggested that BACE1-AS has high sensitivity and specificity for the prognosis prediction of HCC patients [18]. Consistently, in the present study, we found that BACE1-AS expression was significantly upregulated in HCC tissue, serum samples of HCC patients, and HCC cell lines, compared with the controls. Functionally, BACE1-AS promotes the malignant biological behaviors of HCC cells. Our data further demonstrate that BACE1-AS may be a promising diagnostic biomarker and therapy target for HCC. In this work, the binding site between BACE1-AS and miR-214-3p was confirmed. Previous studies reported the role of miR-214-3p in HCC and other malignancies [19–24]. For example, by repressing miR-214-3p expression, lncRNA HOXA11-AS enhances HCC cells’ proliferation and invasion and induces epithelial–mesenchymal transition [19]; miR-214-3p suppresses the proliferation and metastatic potential of Huh7 and HCCLM3 cells [20]. In the present study, we reported that miR-214-3p expression was downregulated in HCC tissues, and miR-214-3p suppressed the proliferation, cell cycle progression, migration, and invasion of HCC cells, but promoted apoptosis, which is consistent with the previous reports [19,20]. Notably, in bladder cancer and gastric cancer, miR-214-3p may facilitate disease progression, showing oncogenic effects [22,23]. Collectively, these studies and the present work imply that miR-214-3p exerts different biological functions in different cancers via distinct mechanisms, probably through repressing different downstream target gene expressions. In the present study, we found that APLN expression was negatively regulated by miR-214-3p and positively regulated by BACE1-AS. APLN is composed of 77 amino acid residues, and it takes part in various physiological processes, including regulating blood pressure, internal environment homeostasis, endocrine stress response, cardiac contractility, angiogenesis, and energy metabolism [25,26]. The dysfunction of APLN or the dysregulation of APLN expression participates in the pathogenesis of various diseases, including obesity, cardiovascular diseases, and cancers [25,26]. In HCC, APLN is highly expressed, and APLN activates phosphatidylinositol 3-kinase/protein kinase B pathway via APLN receptor, leading to the increased expressions of phospho-glycogen synthase kinase 3β and cyclin D1, which promotes tumorigenesis [10]. Additionally, accumulating studies reported that APLN is an indicator of the adverse prognosis of HCC patients [27,28]. However, little is known concerning the mechanism of the dysregulation of APLN expression in HCC. Herein, we identified that APLN was a target gene of miR-214-3p, and its expression could be positively regulated by BACE1-AS, which partly explained APLN overexpression in HCC. In summary, we found that BACE1-AS, miR-214-3p, and APLN expressions are dysregulated in HCC tissue and cell lines. Based on in vitro experiments, BACE1-AS was proved to facilitate the proliferation, migration, and invasion and repress the apoptosis of HCC cells by modulating miR-214-3p and APLN expressions. Our data proved that the BACE1-AS–miR-214-3p–APLN axis has the potential value in HCC diagnosis and therapy. In the future studies, in vivo models are required to further validate our findings. Additionally, more patients from different medical centers are needed to evaluate the value of BACE1-AS as a prognosis prediction marker for HCC patients. Funding This work was supported by the grant from Tianjin Science and Technology Plan Project (No. 19ZXDBSY00010). Conflict of Interest The authors declare that they have no conflict of interest. References 1. Grandhi MS , Kim AK, Ronnekleiv-Kelly SM, Kamel IR, Ghasebeh MA, Pawlik TM. Hepatocellular carcinoma: from diagnosis to treatment . Surg Oncol 2016 , 25 : 74 – 85 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Siegel RL , Miller KD, Jemal A. Cancer statistics, 2020 . CA Cancer J Clin 2020 , 70 : 7 – 30 . Google Scholar Crossref Search ADS PubMed WorldCat 3. Sim HW , Knox J. Hepatocellular carcinoma in the era of immunotherapy . Curr Probl Cancer 2018 , 42 : 40 – 48 . Google Scholar Crossref Search ADS PubMed WorldCat 4. Kopp F , Mendell JT. Functional classification and experimental dissection of long noncoding RNAs . Cell 2018 , 172 : 393 – 407 . Google Scholar Crossref Search ADS PubMed WorldCat 5. Sheng N , Li Y, Qian R, Li Y. The clinical significance and biological function of lncRNA RGMB-AS1 in hepatocellular carcinoma . Biomed Pharmacother 2018 , 98 : 577 – 584 . Google Scholar Crossref Search ADS PubMed WorldCat 6. Chen Q , Liu X, Xu L, Wang Y, Wang S, Li Q, Huang Y, et al. Long non-coding RNA BACE1-AS is a novel target for anisomycin-mediated suppression of ovarian cancer stem cell proliferation and invasion . Oncol Rep 2016 , 35 : 1916 – 1924 . Google Scholar Crossref Search ADS PubMed WorldCat 7. Han LC , Wang H, Niu FL, Yan JY, Cai HF. Effect miR-214-3p on proliferation and apoptosis of breast cancer cells by targeting survivin protein . Eur Rev Med Pharmacol Sci 2019 , 23 : 7469 – 7474 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 8. Li Y , Li Y, Chen Y, Xie Q, Dong N, Gao Y, Deng H, et al. MicroRNA-214-3p inhibits proliferation and cell cycle progression by targeting MELK in hepatocellular carcinoma and correlates cancer prognosis . Cancer Cell Int 2017 , 17 : 102. Google Scholar OpenURL Placeholder Text WorldCat 9. Yang Y , Lv SY, Ye W, Zhang L. Apelin/APJ system and cancer . Clin Chim Acta 2016 , 457 : 112 – 116 . Google Scholar Crossref Search ADS PubMed WorldCat 10. Chen H , Wong CC, Liu D, Go MYY, Wu B, Peng S, Kuang M, et al. APLN promotes hepatocellular carcinoma through activating PI3K/Akt pathway and is a druggable target . Theranostics 2019 , 9 : 5246 – 5260 . Google Scholar Crossref Search ADS PubMed WorldCat 11. Wang Y , He L, Du Y, Zhu P, Huang G, Luo J, Yan X, et al. The long noncoding RNA lncTCF7 promotes self-renewal of human liver cancer stem cells through activation of Wnt signaling . Cell Stem Cell 2015 , 16 : 413 – 425 . Google Scholar Crossref Search ADS PubMed WorldCat 12. Wang H , Huo X, Yang XR, He J, Cheng L, Wang N, Deng X, et al. STAT3-mediated upregulation of lncRNA HOXD-AS1 as a ceRNA facilitates liver cancer metastasis by regulating SOX4 . Mol Cancer 2017 , 16 : 136. Google Scholar OpenURL Placeholder Text WorldCat 13. Huang H , Chen J, Ding CM, Jin X, Jia ZM, Peng J. LncRNA NR2F1-AS1 regulates hepatocellular carcinoma oxaliplatin resistance by targeting ABCC1 via miR-363 . J Cell Mol Med 2018 , 22 : 3238 – 3245 . Google Scholar Crossref Search ADS PubMed WorldCat 14. Fotuhi SN , Khalaj-Kondori M, Hoseinpour Feizi MA, Talebi M. Long non-coding RNA BACE1-AS may serve as an Alzheimer’s disease blood-based biomarker . J Mol Neurosci 2019 , 69 : 351 – 359 . Google Scholar Crossref Search ADS PubMed WorldCat 15. Zhang W , Zhao H, Wu Q, Xu W, Xia M. Knockdown of BACE1-AS by siRNA improves memory and learning behaviors in Alzheimer’s disease animal model . Exp Ther Med 2018 , 16 : 2080 – 2086 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 16. Li Y , Fang J, Zhou Z, Zhou Q, Sun S, Jin Z, Xi Z, et al. Downregulation of lncRNA BACE1-AS improves dopamine-dependent oxidative stress in rats with Parkinson’s disease by upregulating microRNA-34b-5p and downregulating BACE1 . Cell Cycle 2020 , 19 : 1 – 14 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 17. Esfandi F , Ghafouri-Fard S, Oskooei VK, Taheri M. β-Secretase 1 and its naturally occurring anti-sense RNA are down-regulated in gastric cancer . Pathol Oncol Res 2019 , 25 : 1627 – 1633 . Google Scholar Crossref Search ADS PubMed WorldCat 18. Nie Y , Li Y, Xu Y, Jiao Y, Li W. Long non-coding RNA BACE1-AS is an independent unfavorable prognostic factor in liver cancer . Oncol Lett 2020 , 20 : 202. Google Scholar OpenURL Placeholder Text WorldCat 19. Zhan M , He K, Xiao J, Liu F, Wang H, Xia Z, Duan X, et al. LncRNA HOXA11-AS promotes hepatocellular carcinoma progression by repressing miR-214-3p . J Cell Mol Med 2018 , 22 : 3758 – 3767 . Google Scholar Crossref Search ADS WorldCat 20. Lin T , Dai Y, Guo X, Chen W, Zhao J, Cao L, Wu Z. Silencing Of hsa_circ_0008450 represses hepatocellular carcinoma progression through regulation of microRNA-214-3p/EZH2 axis . Cancer Manag Res 2019 , 11 : 9133 – 9143 . Google Scholar Crossref Search ADS PubMed WorldCat 21. Fang YY , Tan MR, Zhou J, Liang L, Liu XY, Zhao K, Bao EC. miR-214-3p inhibits epithelial-to-mesenchymal transition and metastasis of endometrial cancer cells by targeting TWIST1 . Onco Targets Ther 2019 , 12 : 9449 – 9458 . Google Scholar Crossref Search ADS PubMed WorldCat 22. Yu H , Wang S, Zhu H, Rao D. LncRNA MT1JP functions as a tumor suppressor via regulating miR-214-3p expression in bladder cancer . J Cell Physiol 2019 , 20 .doi: 10.1002/jcp.28274. Google Scholar OpenURL Placeholder Text WorldCat Crossref 23. He Z , Wang X, Huang C, Gao Y, Yang C, Zeng P, Chen Z. The FENDRR/miR-214-3P/TET2 axis affects cell malignant activity via RASSF1A methylation in gastric cancer . Am J Transl Res 2018 , 10 : 3211 – 3223 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 24. Ecke TH , Stier K, Weickmann S, Zhao Z, Buckendahl L, Stephan C, Kilic E, et al. miR-199a-3p and miR-214-3p improve the overall survival prediction of muscle-invasive bladder cancer patients after radical cystectomy . Cancer Med 2017 , 6 : 2252 – 2262 . Google Scholar Crossref Search ADS PubMed WorldCat 25. Arababadi MK , Asadikaram P, Asadikaram G. APLN/APJ pathway: the key regulator of macrophage functions . Life Sci 2019 , 232 : 116645. Google Scholar OpenURL Placeholder Text WorldCat 26. Wysocka MB , Pietraszek-Gremplewicz K, Nowak D. The role of Apelin in cardiovascular diseases, obesity and cancer . Front Physiol 2018 , 9 : 557. Google Scholar OpenURL Placeholder Text WorldCat 27. Lee T , Park CK, Ha SY. Prognostic role of Apelin receptor expression in hepatocellular carcinoma treated with curative surgical resection . Anticancer Res 2019 , 39 : 3025 – 3031 . Google Scholar Crossref Search ADS PubMed WorldCat 28. Cabiati M , Gaggini M, De Simone P, Del Ry S. Evaluation of Apelin/APJ system expression in hepatocellular carcinoma as a function of clinical severity . Clin Exp Med 2021 , 21 : 269 – 275 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Acta Biochimica et Biophysica Sinica Oxford University Press

Long non-coding RNA BACE1-AS plays an oncogenic role in hepatocellular carcinoma cells through miR-214-3p/APLN axis

Download PDF
9 pages

Loading next page...
 
/lp/oxford-university-press/long-non-coding-rna-bace1-as-plays-an-oncogenic-role-in-hepatocellular-dr8dlJXwQ7

References (29)

Publisher
Oxford University Press
Copyright
Copyright © 2021 Institute of Biochemistry and Cell Biology, SIBS, CAS
ISSN
1672-9145
eISSN
1745-7270
DOI
10.1093/abbs/gmab134
Publisher site
See Article on Publisher Site

Abstract

Abstract BACE1 antisense RNA (BACE1-AS) is implicated in promoting cell proliferation in different types of tumors. However, the function and mechanism of BACE1-AS in hepatocellular carcinoma (HCC) are still unclear. In the present study, we found that the relative expression of BACE1-AS in HCC cell lines, HCC tissues, and serum samples of HCC patients was significantly increased, and its high expression was correlated with the poor prognosis of HCC patients. In addition, overexpression of BACE1 promoted HCC cell proliferation, cell cycle progression, migration, and invasion, but inhibited cell apoptosis, while knockdown of BACE1 exerted the opposite role. Furthermore, BACE1-AS sponged microRNA-214-3p (miR-214-3p) and inhibited its expression, thus promoting Apelin (APLN) expression. Overexpression or knockdown of miR-214-3p could partially reverse the abnormal proliferation, cell cycle progression, migration, invasion, and apoptosis caused by overexpression or knockdown of BACE1. These findings suggest that the BACE1-AS/miR-214-3p/APLN axis is a novel signaling pathway that facilitates HCC. BACE1-AS, miR-214-3p, APLN, proliferation, cell cycle, HCC Introduction Primary liver cancer is the sixth most common cancer, and hepatocellular carcinoma (HCC) takes up 90% of all liver cancer cases [1,2]. In the early stage, patients with HCC can be cured by liver transplantation or surgical resection, but few therapies are effective for patients with advanced HCC [3]. It is urgent to clarify the mechanism of this complicated disease to further explore more effective therapeutic strategies. Long non-coding RNAs (lncRNAs) are defined as RNA transcripts with more than 200 nucleotides in length that do not encode proteins [4]. Recent research shows the promising diagnostic and therapeutic value of lncRNAs for HCC. For example, lncRNA RGMB-AS1 is found to be lowly expressed in HCC tissues and cell lines, and RGMB-AS1 overexpression repressed HCC cell proliferation, migration, and invasion [5]. BACE1 antisense RNA (BACE1-AS) is found to play a regulatory role in some tumors. For example, anisomycin represses the proliferation and invasion of human ovarian cancer stem cells and promotes BACE1-AS expression; after BACE1-AS is silenced, the antiproliferative and anti-invasive effects of anisomycin are weakened [6]. However, whether BACE1-AS can modulate the progression of HCC warrants further investigation. In addition to lncRNAs, microRNAs (miRNAs) are vital regulators in the development of tumors. miR-214-3p has been widely investigated in a variety of tumors. In breast cancer, miR-214-3p overexpression inhibits the proliferation of cancer cells via targeting survivin [7]. In HCC, it was reported that miR-214-3p expression is downregulated in HCC samples, and upregulated miR-214-3p expression induces HCC cell cycle arrest and increases apoptosis [8]. Apelin (APLN) is an endogenous ligand of the apelin receptor (APJ) [9]. Multiple physiological and pathological processes are mediated by the APLN/APJ system. It has been revealed that APLN mRNA expression is markedly elevated in HCC specimens, and inhibiting APLN expression using short hairpin RNAs (shRNAs) suppresses the growth of HCC cells in vitro and in vivo, which suggests that APLN has the potential to be the diagnostic biomarker and therapeutic target for HCC [10]. But how APLN expression is regulated in HCC has not been fully explored. In this study, by bioinformatics analysis, we found that BACE1-AS expression was remarkably elevated in HCC tissues. In addition, the potential binding sites between BACE1-AS and miR-214-3p and between miR-214-3p and APLN 3′UTR were identified. Thus, we hypothesized that BACE1-AS‒miR-214-3p‒APLN axis could regulate HCC progression. Materials and Methods Samples collection The HCC samples and matched adjacent non-tumorous liver tissues were resected from 28 patients with primary HCC from April 2016 to June 2018, who were also recruited during the same period. Serum samples were collected from both HCC patients and healthy subjects. All serum and tissue samples were stored at −80°C. The research protocol was approved by the Ethics Committee of Tianjin First Central Hospital, and all patients signed informed consent forms. Cell culture and transfection HCC cell lines (Huh6, Huh7, HepG2, and Hep3B) and normal liver epithelial cell line L02 were obtained from the American Type Culture Collection (Manassas, USA). The cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Life Technologies, Darmstadt, Germany) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, USA) at 37°C with 5% CO2. Huh7 and HepG2 cells in the logarithmic phase were selected for experiments. When the rate of cell confluence achieved 70%–80%, Huh7 and HepG2 cells were trypsinized and resuspended in culture medium. The cell suspension was plated to a 6-well plate at a density of 1×106 cells/ml. Subsequently, Huh7 cells were transfected with BACE1-AS shRNA (sh-BACE1-AS: 5′-GCTTTGGCACCTCCTAAGTGT-3′), negative control (sh-NC: 5′-GTATGACAACAGCCTCAAGCTCGAGCTTGAGGCTGTTGTCATAC-3′), miR-214-3p mimics (5′-ACAGCAGGCACAGACAGGCAGU-3′), or the negative controls (5′-UUCUCCGAACGUGUCACGUTT-3′), and HepG2 cells were transfected with BACE1-AS overexpression plasmid, empty vector (vector), miR-214-3p inhibitors (5′-ACUGCCUGUCUGUGCCUGCUGU-3′), or the negative controls (5′-TCGTTCATGAACGAACATT-3′) using Lipofectamine™ 2000 (Invitrogen, Carlsbad, USA) to construct the cell models. The transfection efficiency was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) after 24 h. qRT-PCR Total RNA was isolated from cells or tissues using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. The extracted RNA was reversely transcribed to complementary DNA using a SuperScript Reverse Transcriptase Kit (Vazyme, Nanjing, China). qRT-PCR was then performed with the SYBR Green PCR Master Mix kit (Vazyme) on a Fast Real-time PCR 7300 System (Applied Biosystems, Foster City, USA). U6 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were employed as the reference genes, and the 2–ΔΔCT method was used to calculate the relative expression. The primers were designed and constructed by BGI (Shenzhen, China). The primer sequences were as follows: BACE1-AS, forward: 5′-CTTGGGCAAACGAAGGTTGG-3′, reverse: 5′-CCCAGAGCCCAGCATCAAAA-3′; miR-214-3p, forward: 5ʹ-GCACAGCAGGCACAGACA-3ʹ, reverse: 5ʹ-CAGAGCAGGGTCAGCGGTA-3ʹ; GAPDH, forward: 5ʹ-GCACCGTCAAGGCTGAGAAC3ʹ, reverse: 5ʹ-TGGTGAAGACGCCAGTGGA-3ʹ; U6, forward: 5ʹ-CTCGCTTCGGCAGCACA-3ʹ, reverse: 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ. The reaction conditions were as follows: Pre-denaturation of 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, primer-specific annealing temperature at 72°C for 30 s, and extension at 72°C for 10 min. Cell counting kit-8 assay Huh7 and HepG2 cells in the logarithmic phase were transferred into a 96-well plate (1×103 cells/well), and then the cell viability was detected at 24, 48, 72, and 96 h. After the cells in each well were incubated with 10 μl of Cell counting kit (CCK)-8 reagent (Dojindo, Tokyo, Japan) at 37°C for 1 h, the optical density (OD) value at 450 nm was measured with a microplate reader. Ultimately, the viability of the cells was calculated. Cell cycle analysis The effects of BACE1-AS and miR-214-3p on the Huh7 and HepG2 cell cycles were evaluated with a flow cytometer (BD Biosciences, Franklin Lakes, USA). Briefly, cells were rinsed twice with phosphate buffered saline (PBS), trypsinized, fixed with 75% ethanol overnight at −20°C, and then incubated with 100 mg/ml RNase A and 50 mg/ml propidium iodide (PI) solution for 30 min. Subsequently, the percentage of cells in the G0/G1, S, and G2/M was assessed in each group by flow cytometry. Apoptosis detection HepG2 and Huh7 cells were planted into a 6-well plate and then cultured for 48 h. After rinsing three times with PBS, the cells were harvested and then resuspended in 500 μl of binding buffer (10×: 0.1 M HEPES, pH 7.4, 1.4 M NaCl, 25 mM CaCl2). Subsequently, 5 μl of Annexin V-FITC kit solution (Southern Biotechnology, Birmingham, USA) and 5 μl of PI solution were incubated with the above mixture in the dark for 30 min. Then, cell apoptosis was analyzed by flow cytometry on a flow cytometer (BD Biosciences), and the apoptosis rate of cells was calculated. Transwell assays Transwell assays were performed using Transwell chambers (pore size: 8 µM, Corning Costar, Cambridge, USA). The Transwell chambers were placed on 24-well plates. Then, the cells were re-suspended in a serum-free medium, and the density was adjusted to 2×105 cells/ml. After that, the cells were added to the upper chamber (4×104 cells/well), and 500 µl of DMEM containing 10% FBS was added to the wells of the plates. After 24 h, a cotton swab was applied to remove the cells on the upper chamber, and the migrated or invaded cells were fixed with 95% ethanol and then stained with crystal violet for 20 min. Next, four random fields of each membrane were observed under an inverted microscope to count the number of the cells. In the invasion assay, Matrigel (BD Biosciences, Franklin Lakes, USA) was used to coat the membrane, while in the migration assay, Matrigel was not used. RNA immunoprecipitation assay To pinpoint the binding relationship between BACE1-AS and miR-214-3p, RNA immunoprecipitation (RIP) assay was conducted using the Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore, Billerica, USA). In brief, Huh7 and HepG2 cells at 80% confluency were harvested and lysed in RIP lysis buffer. Whole-cell extracts were coimmunoprecipitated with RIP buffer containing anti-Argonaute2 (Ago2) antibody conjugated with magnetic beads (Millipore) or normal mouse immunoglobulin G (IgG; Millipore). Subsequently, the samples were treated with proteinase K. Coprecipitated RNA was then isolated with the TRIzol method, and qRT-PCR was employed to detect the enrichment of BACE1-AS and miR-214-3p. Dual-Luciferase reporter assay The sequence segments of BACE1-AS and APLN 3ʹuntranslated region (UTR) containing the binding sites of miR-214-3p were amplified by qRT-PCR and then inserted into pmir-GLO luciferase expression vectors to construct BACE1-AS/APLN wild-type vector (pmirGLO-BACE1-AS-WT/pmirGLO-APLN-WT). BACE1-AS/APLN mutant-type vector (pmirGLO-BACE1-AS-MUT/pmirGLO-APLN-MUT) was constructed after the mutation of the binding sites. The luciferase reporter vectors, miR-214-3p mimics, and negative control were mixed with Lipofectamine™ 2000 (Invitrogen) and then co-transfected into HepG2 cells. After 48 h, the luciferase activity was detected using a Dual-Luciferase Reporter Assay System (Promega, Madison, USA). Western blot analysis APLN expression on protein level was detected by western blot analysis. Briefly, HepG2 and Huh7 cells were lysed using radioimmunoprecipitation assay (RIPA) lysis reagent (Beyotime, Shanghai, China) containing 1% phenylmethylsulfonyl fluoride (PMSF; Beyotime), and the supernatant was collected after centrifugation. Moreover, Bradford method was used for protein quantification. After being mixed with 5× loading buffer, the protein samples were denatured by heating in boiling water for 5 min. Subsequently, the samples were subject to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membrane (Millipore). After being blocked with 5% skim milk for 1 h, the membrane was incubated with rabbit polyclonal anti-APLN antibody (1:1000; Abcam, Cambridge, UK) at 4°C overnight. Afterward, the membrane was rinsed twice with Tris buffered saline with Tween (TBST) and then incubated with horseradish-peroxidase-conjugated goat anti-rabbit IgG secondary antibody (Abcam; 1:2000) for 1 h, followed by rinsing with TBST three times. The protein bands on the membrane were visualized using an electrochemiluminescense western blotting substrate kit (Promega). GAPDH was regarded as the internal control. Statistical analysis Statistical analysis was carried out using SPSS 23.0 software (SPSS Inc., Chicago, USA). The data were expressed as mean±standard deviation, and the comparison of means was conducted using one-way analysis of variance or two-tailed Student’s t-test. P<0.05 signified statistical significance. Results Bioinformatics analysis identified BACE1-AS as a biomarker in HCC First of all, we downloaded lncRNA transcriptome data from The Cancer Genome Atlas (TCGA), including 371 HCC samples and 50 adjacent normal liver tissue samples. With edgeR, differentially expressed lncRNAs with |log2FC|≥ 1 and FDR<0.05 were screened out and BACE1-AS expression was found to be significantly increased in HCC samples in comparison with normal liver tissue samples (Fig. 1A–C). In addition, we performed survival analysis using TGCA data, GEPIA database, and ENCORI database, and the results indicated that the high expression of BACE1-AS was related to poor prognosis in HCC patients (Fig. 1D–F). Figure 1. Open in new tabDownload slide Bioinformatics analysis suggests that BACE1-AS expression is dysregulated in HCC (A) lncRNA transcriptome data from TCGA were used to plot the heatmap and the top 50 lncRNAs with the most significant change in HCC tissues are shown (vs. normal liver tissues). (B) lncRNAs with differential expression are shown in a volcano plot. (C) The expression of BACE1-AS in HCC tissue or normal tissue was analyzed using the GEPIA database. (D‒F) The survival analysis of BACE1-AS for HCC patients was performed using the data from the TCGA database, GEPIA database, and ENCORI database. Figure 1. Open in new tabDownload slide Bioinformatics analysis suggests that BACE1-AS expression is dysregulated in HCC (A) lncRNA transcriptome data from TCGA were used to plot the heatmap and the top 50 lncRNAs with the most significant change in HCC tissues are shown (vs. normal liver tissues). (B) lncRNAs with differential expression are shown in a volcano plot. (C) The expression of BACE1-AS in HCC tissue or normal tissue was analyzed using the GEPIA database. (D‒F) The survival analysis of BACE1-AS for HCC patients was performed using the data from the TCGA database, GEPIA database, and ENCORI database. BACE1-AS expression was increased in HCC tissues and cell lines We then measured the expression of BACE1-AS in HCC tissues and cell lines with qRT-PCR. The data revealed that BACE1-AS expression was significantly upregulated in HCC tissue samples and serum samples of HCC patients (Fig. 2A,B) compared with the normal controls. In addition, BACE1-AS expression in HCC cell lines, including Huh-6, Huh7, HepG2, and Hep3B, was significantly upregulated compared with that in normal liver epithelial cell line L02 (Fig. 2C). Figure 2. Open in new tabDownload slide The expression characteristics of BACE1-AS in HCC (A,B) The expression of BACE1-AS in HCC tissues, adjacent normal tissues, and serum samples from HCC patients was quantified by qRT-PCR. (C) BACE1-AS expression in normal liver epithelial cell and HCC cell lines was quantified by qRT-PCR. ***P<0.001. Figure 2. Open in new tabDownload slide The expression characteristics of BACE1-AS in HCC (A,B) The expression of BACE1-AS in HCC tissues, adjacent normal tissues, and serum samples from HCC patients was quantified by qRT-PCR. (C) BACE1-AS expression in normal liver epithelial cell and HCC cell lines was quantified by qRT-PCR. ***P<0.001. BACE1-AS promoted the proliferation, migration, and invasion and inhibited the apoptosis of HCC cells To explore the biological function of BACE1-AS in HCC cells, BACE1-AS overexpression plasmid and shRNA were transfected into HepG2 cells and Huh7 cells, respectively. qRT-PCR was applied to verify the effects of transfection (Fig. 3A). The results of CCK-8 assay showed that overexpression of BACE1-AS significantly promoted cell proliferation and that knockdown of BACE1-AS worked oppositely (Fig. 3B). Flow cytometry results indicated that BACE1-AS induced more cells to enter the S phase and notably suppressed cell apoptosis, while knockdown of BACE1-AS blocked cell cycle progression and promoted cell apoptosis (Fig. 3C,D). In the Transwell assays, it was demonstrated that BACE1-AS overexpression enhanced the migration and invasion ability of HepG2 cells, while knockdown of BACE1-AS repressed the proliferation and migration of Huh7 cells (Fig. 3E). Figure 3. Open in new tabDownload slide BACE1-AS regulates the biological behaviors of HCC cells (A) The transfection efficiency of BACE1-AS overexpression plasmid and BACE1-AS shRNA was validated by qRT-PCR. (B) CCK-8 assay was performed to measure the proliferation of HepG2 and Huh7 cells. (C) Flow cytometry was used to detect the cell cycle of HepG2 and Huh7 cells. (D) Flow cytometry was used to detect the apoptosis of HepG2 and Huh7 cells. (E) Migration and invasion of HepG2 and Huh7 cells were examined by Transwell assays. Magnification fold: 100×. *P<0.05, **P<0.01, and ***P<0.001. Figure 3. Open in new tabDownload slide BACE1-AS regulates the biological behaviors of HCC cells (A) The transfection efficiency of BACE1-AS overexpression plasmid and BACE1-AS shRNA was validated by qRT-PCR. (B) CCK-8 assay was performed to measure the proliferation of HepG2 and Huh7 cells. (C) Flow cytometry was used to detect the cell cycle of HepG2 and Huh7 cells. (D) Flow cytometry was used to detect the apoptosis of HepG2 and Huh7 cells. (E) Migration and invasion of HepG2 and Huh7 cells were examined by Transwell assays. Magnification fold: 100×. *P<0.05, **P<0.01, and ***P<0.001. miR-214-3p was identified as a direct target of BACE1-AS To uncover the underlying mechanism of BACE1-AS in HCC progression, we investigated the target miRNAs of BACE1-AS. By analyzing the data in the StarBase database and Gene Expression Omnibus dataset GSE108724, we found that miR-214-3p, whose expression was downregulated in HCC tissues compared with normal liver tissues, was predicted to be a potential target of BACE1-AS (Fig. 4A–D). Dual-Luciferase Reporter gene experiment revealed that miR-214-3p mimics remarkably weakened the luciferase activity of BACE1-AS wild-type reporter, but exerted no effect on the luciferase activity of the mutant-type reporter (Fig. 4E). RIP assay further revealed the direct binding relationship between BACE1-AS and miR-214-3p (Fig. 4F). BACE1-AS overexpression reduced the expression of miR-214-3p in HepG2 cells, whereas the antagonism of BACE1-AS increased the miR-214-3p expression in Huh7 cells (Fig. 4G). However, the selective regulation of miR-214-3p expression in HCC cells did not influence BACE1-AS expression (Fig. 4H). Furthermore, in HCC tissues and serum samples of HCC patients, BACE1-AS and miR-214-3p expressions were found to be negatively correlated (Fig. 4I,J). The above results confirmed that miR-214-3p expression was negatively regulated by BACE1-AS in HCC. Figure 4. Open in new tabDownload slide Identification of the binding site between BACE1-AS and miR-214-3p (A) Heatmap was plotted using the data from GSE108724 and miRNAs with significant changes (P<0.05) and log2FC>1.5 or <−1.5 are shown. (B) miRNAs in GSE108724 are shown in the volcano plot. The downregulated miRNA expressions are marked blue, the upregulated miRNA expressions are marked red, and miR-214-3p expression is marked green. (C,D) miR-214-3p was selected as a potential target of BACE1-AS and the binding site was predicted with the StarBase database. (E) Dual-Luciferase Reporter gene assay was used to identify the binding site between BACE1-AS and miR-214-3p. (F) The binding relationship between BACE1-AS and miR-214-3p was further identified by RIP assay. (G) qRT-PCR was used to detect the regulatory effects of BACE1-AS on miR-214-3p expression in HCC cells. (H) qRT-PCR was performed to explore the effects of miR-214-3p on BACE1-AS expression in HCC cells. (I,J) The correlations between BACE1-AS and miR-214-3p in HCC tissues and serum samples of HCC patients. ***P<0.001. Figure 4. Open in new tabDownload slide Identification of the binding site between BACE1-AS and miR-214-3p (A) Heatmap was plotted using the data from GSE108724 and miRNAs with significant changes (P<0.05) and log2FC>1.5 or <−1.5 are shown. (B) miRNAs in GSE108724 are shown in the volcano plot. The downregulated miRNA expressions are marked blue, the upregulated miRNA expressions are marked red, and miR-214-3p expression is marked green. (C,D) miR-214-3p was selected as a potential target of BACE1-AS and the binding site was predicted with the StarBase database. (E) Dual-Luciferase Reporter gene assay was used to identify the binding site between BACE1-AS and miR-214-3p. (F) The binding relationship between BACE1-AS and miR-214-3p was further identified by RIP assay. (G) qRT-PCR was used to detect the regulatory effects of BACE1-AS on miR-214-3p expression in HCC cells. (H) qRT-PCR was performed to explore the effects of miR-214-3p on BACE1-AS expression in HCC cells. (I,J) The correlations between BACE1-AS and miR-214-3p in HCC tissues and serum samples of HCC patients. ***P<0.001. miR-214-3p suppressed the proliferation, migration, and invasion and accelerated the apoptosis of HCC cells Next, we transfected miR-214-3p mimics and miR-214-3p inhibitors into Huh7 and HepG2 cells, respectively, to detect the effects of miR-214-3p on cell proliferation, apoptosis, cell cycle, migration, and invasion of HC cells (Fig. 5A). In the CCK-8 assay, miR-214-3p mimics markedly repressed the proliferation of Huh7 cells, while miR-214-3p inhibitors promoted the proliferation of HepG2 cells (Fig. 5B). miR-214-3p was found to induce the cell cycle arrest and apoptosis of HCC cells (Fig. 5C,D). In addition, miR-214-3p weakened the migration and invasion of Huh7 cells, and inhibition of miR-214-3p promoted the migration and invasion of HepG2 cells (Fig. 5E). These data suggested that the functions of miR-214-3p were contrary to BACE1-AS in HCC cells. Figure 5. Open in new tabDownload slide miR-214-3p regulates the biological behaviors of HCC cells (A) qRT-PCR was used to verify the transfection efficiency of miR-214-3p mimics and miR-214-3p inhibitors. (B) CCK-8 assay was performed to measure the proliferation of HepG2 and Huh7 cells. (C) Flow cytometry was used to detect the cell cycle of HepG2 and Huh7 cells. (D) Flow cytometry was used to detect the apoptosis of HepG2 and Huh7 cells. (E) Migration and invasion abilities of HepG2 and Huh7 cells were examined by Transwell assays. Magnification fold: 100×. *P<0.05, **P<0.01, and ***P<0.001. Figure 5. Open in new tabDownload slide miR-214-3p regulates the biological behaviors of HCC cells (A) qRT-PCR was used to verify the transfection efficiency of miR-214-3p mimics and miR-214-3p inhibitors. (B) CCK-8 assay was performed to measure the proliferation of HepG2 and Huh7 cells. (C) Flow cytometry was used to detect the cell cycle of HepG2 and Huh7 cells. (D) Flow cytometry was used to detect the apoptosis of HepG2 and Huh7 cells. (E) Migration and invasion abilities of HepG2 and Huh7 cells were examined by Transwell assays. Magnification fold: 100×. *P<0.05, **P<0.01, and ***P<0.001. Augmentation of miR-214-3p expression repressed the effects of BACE1-AS on HCC cells Next, we co-transfected miR-214-3p mimics and BACE1-AS overexpression plasmid into HepG2 cells to further investigate the functional relationship between BACE1-AS and miR-214-3p. It was found that miR-214-3p restoration reversed the promotion of proliferation, the acceleration of cell cycle progression, the enhancement of migration and invasion, and the inhibition of apoptosis caused by BACE1-AS overexpression (Fig. 6A–E). These results demonstrated that miR-214-3p counteracted the effects of BACE1-AS on HCC cells. Figure 6. Open in new tabDownload slide BACE1-AS regulates the phenotypes of HCC cells via miR-214-3p (A) miR-214-3p mimics and BACE1-AS overexpression plasmid were cotransfected into HepG2 cells, and then the expression of miR-214-3p was detected by qRT-PCR. (B) After the transfection, CCK-8 assay was performed to measure the proliferation of HepG2 cells. (C) Flow cytometry was used to detect the cell cycle of HepG2 cells. (D) Flow cytometry was used to detect the apoptosis of HepG2 cells. (E) Migration and invasion of HepG2 cells were detected via Transwell assays. Magnification fold: 100×. **P<0.01 and ***P<0.001. Figure 6. Open in new tabDownload slide BACE1-AS regulates the phenotypes of HCC cells via miR-214-3p (A) miR-214-3p mimics and BACE1-AS overexpression plasmid were cotransfected into HepG2 cells, and then the expression of miR-214-3p was detected by qRT-PCR. (B) After the transfection, CCK-8 assay was performed to measure the proliferation of HepG2 cells. (C) Flow cytometry was used to detect the cell cycle of HepG2 cells. (D) Flow cytometry was used to detect the apoptosis of HepG2 cells. (E) Migration and invasion of HepG2 cells were detected via Transwell assays. Magnification fold: 100×. **P<0.01 and ***P<0.001. BACE1-AS worked by indirectly regulating APLN expression In GSE101728, APLN expression was found to be notably upregulated in human liver cancer tissue compared with that in the adjacent normal tissue (Fig. 7A). Additionally, by searching microT, miRanda, and miRmap databases, APLN was predicted as a target of miR-214-3p (Fig. 7B,C). Dual-Luciferase Reporter experiment results showed that miR-214-3p mimics markedly weakened the luciferase activity in APLN wild-type reporter, but when the two predicted binding sites were simultaneously mutated, the transfection of miR-214-3p failed to reduce the luciferase reporter vector (Fig. 7D). Additionally, BACE1-AS overexpression was found to significantly elevate APLN expression, while BACE1-AS knockdown suppressed it; miR-214-3p could decrease APLN expression (Fig. 7E,F). So far, we concluded that BACE1-AS promoted the proliferation, migration, and invasion and inhibited the apoptosis of HCC cells, and it also repressed the expression of miR-214-3p and upregulated the expression of APLN. Figure 7. Open in new tabDownload slide BACE1-AS indirectly regulates APLN (A) GSE101728 indicated that APLN expression was significantly upregulated in HCC tissue compared with the adjacent normal tissue. (B) The target genes of miR-214-3p were predicted with microT, miRanda, miRmap, and GSE101728. (C,D) Dual-Luciferase Reporter gene assays were performed to confirm the binding between miR-214-3p and APLN. (E,F) Western blot analysis was used to detect APLN expression in HepG2 and Huh7 cells after BACE1-AS, and miR-214-3p expressions were selectively regulated. **P<0.01 and ***P<0.001. Figure 7. Open in new tabDownload slide BACE1-AS indirectly regulates APLN (A) GSE101728 indicated that APLN expression was significantly upregulated in HCC tissue compared with the adjacent normal tissue. (B) The target genes of miR-214-3p were predicted with microT, miRanda, miRmap, and GSE101728. (C,D) Dual-Luciferase Reporter gene assays were performed to confirm the binding between miR-214-3p and APLN. (E,F) Western blot analysis was used to detect APLN expression in HepG2 and Huh7 cells after BACE1-AS, and miR-214-3p expressions were selectively regulated. **P<0.01 and ***P<0.001. Discussion Accumulating evidence has revealed the regulatory functions of lncRNAs in HCC [11–13]. For example, lncTCF7 recruits SWI/SNF complex to the promoter region of TCF7 to trigger TCF7 transcription and activates the Wnt signal pathway, and via this mechanism, lncTCF7 promotes the renewal of liver cancer stem cells and facilitates the progression of HCC [11]. In recent years, BACE1-AS has reportedly been linked to the pathogenesis of neurological diseases, such as Alzheimer’s disease and Parkinson’s disease [14–16]. Also, the role of BACE1-AS in cancer biology is being gradually recognized. Specifically, BACE1-AS expression in gastric cancer tissues is significantly lower than that in the adjacent normal tissues [17]. Anisomycin exerts its antiproliferative and anti-invasive effects on ovarian cancer stem cells by upregulating BACE1-AS expression [6]. The tumor-suppressive role of BACE1-AS in ovarian cancer is different from the oncogenic role in HCC reported in the present work, which implies that the biological function of BACE1-AS is distinct in different cancers. Notably, BACE1-AS was reported to be highly expressed in HCC tissues, and the receiver operating characteristic curve suggested that BACE1-AS has high sensitivity and specificity for the prognosis prediction of HCC patients [18]. Consistently, in the present study, we found that BACE1-AS expression was significantly upregulated in HCC tissue, serum samples of HCC patients, and HCC cell lines, compared with the controls. Functionally, BACE1-AS promotes the malignant biological behaviors of HCC cells. Our data further demonstrate that BACE1-AS may be a promising diagnostic biomarker and therapy target for HCC. In this work, the binding site between BACE1-AS and miR-214-3p was confirmed. Previous studies reported the role of miR-214-3p in HCC and other malignancies [19–24]. For example, by repressing miR-214-3p expression, lncRNA HOXA11-AS enhances HCC cells’ proliferation and invasion and induces epithelial–mesenchymal transition [19]; miR-214-3p suppresses the proliferation and metastatic potential of Huh7 and HCCLM3 cells [20]. In the present study, we reported that miR-214-3p expression was downregulated in HCC tissues, and miR-214-3p suppressed the proliferation, cell cycle progression, migration, and invasion of HCC cells, but promoted apoptosis, which is consistent with the previous reports [19,20]. Notably, in bladder cancer and gastric cancer, miR-214-3p may facilitate disease progression, showing oncogenic effects [22,23]. Collectively, these studies and the present work imply that miR-214-3p exerts different biological functions in different cancers via distinct mechanisms, probably through repressing different downstream target gene expressions. In the present study, we found that APLN expression was negatively regulated by miR-214-3p and positively regulated by BACE1-AS. APLN is composed of 77 amino acid residues, and it takes part in various physiological processes, including regulating blood pressure, internal environment homeostasis, endocrine stress response, cardiac contractility, angiogenesis, and energy metabolism [25,26]. The dysfunction of APLN or the dysregulation of APLN expression participates in the pathogenesis of various diseases, including obesity, cardiovascular diseases, and cancers [25,26]. In HCC, APLN is highly expressed, and APLN activates phosphatidylinositol 3-kinase/protein kinase B pathway via APLN receptor, leading to the increased expressions of phospho-glycogen synthase kinase 3β and cyclin D1, which promotes tumorigenesis [10]. Additionally, accumulating studies reported that APLN is an indicator of the adverse prognosis of HCC patients [27,28]. However, little is known concerning the mechanism of the dysregulation of APLN expression in HCC. Herein, we identified that APLN was a target gene of miR-214-3p, and its expression could be positively regulated by BACE1-AS, which partly explained APLN overexpression in HCC. In summary, we found that BACE1-AS, miR-214-3p, and APLN expressions are dysregulated in HCC tissue and cell lines. Based on in vitro experiments, BACE1-AS was proved to facilitate the proliferation, migration, and invasion and repress the apoptosis of HCC cells by modulating miR-214-3p and APLN expressions. Our data proved that the BACE1-AS–miR-214-3p–APLN axis has the potential value in HCC diagnosis and therapy. In the future studies, in vivo models are required to further validate our findings. Additionally, more patients from different medical centers are needed to evaluate the value of BACE1-AS as a prognosis prediction marker for HCC patients. Funding This work was supported by the grant from Tianjin Science and Technology Plan Project (No. 19ZXDBSY00010). Conflict of Interest The authors declare that they have no conflict of interest. References 1. Grandhi MS , Kim AK, Ronnekleiv-Kelly SM, Kamel IR, Ghasebeh MA, Pawlik TM. Hepatocellular carcinoma: from diagnosis to treatment . Surg Oncol 2016 , 25 : 74 – 85 . Google Scholar Crossref Search ADS PubMed WorldCat 2. Siegel RL , Miller KD, Jemal A. Cancer statistics, 2020 . CA Cancer J Clin 2020 , 70 : 7 – 30 . Google Scholar Crossref Search ADS PubMed WorldCat 3. Sim HW , Knox J. Hepatocellular carcinoma in the era of immunotherapy . Curr Probl Cancer 2018 , 42 : 40 – 48 . Google Scholar Crossref Search ADS PubMed WorldCat 4. Kopp F , Mendell JT. Functional classification and experimental dissection of long noncoding RNAs . Cell 2018 , 172 : 393 – 407 . Google Scholar Crossref Search ADS PubMed WorldCat 5. Sheng N , Li Y, Qian R, Li Y. The clinical significance and biological function of lncRNA RGMB-AS1 in hepatocellular carcinoma . Biomed Pharmacother 2018 , 98 : 577 – 584 . Google Scholar Crossref Search ADS PubMed WorldCat 6. Chen Q , Liu X, Xu L, Wang Y, Wang S, Li Q, Huang Y, et al. Long non-coding RNA BACE1-AS is a novel target for anisomycin-mediated suppression of ovarian cancer stem cell proliferation and invasion . Oncol Rep 2016 , 35 : 1916 – 1924 . Google Scholar Crossref Search ADS PubMed WorldCat 7. Han LC , Wang H, Niu FL, Yan JY, Cai HF. Effect miR-214-3p on proliferation and apoptosis of breast cancer cells by targeting survivin protein . Eur Rev Med Pharmacol Sci 2019 , 23 : 7469 – 7474 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 8. Li Y , Li Y, Chen Y, Xie Q, Dong N, Gao Y, Deng H, et al. MicroRNA-214-3p inhibits proliferation and cell cycle progression by targeting MELK in hepatocellular carcinoma and correlates cancer prognosis . Cancer Cell Int 2017 , 17 : 102. Google Scholar OpenURL Placeholder Text WorldCat 9. Yang Y , Lv SY, Ye W, Zhang L. Apelin/APJ system and cancer . Clin Chim Acta 2016 , 457 : 112 – 116 . Google Scholar Crossref Search ADS PubMed WorldCat 10. Chen H , Wong CC, Liu D, Go MYY, Wu B, Peng S, Kuang M, et al. APLN promotes hepatocellular carcinoma through activating PI3K/Akt pathway and is a druggable target . Theranostics 2019 , 9 : 5246 – 5260 . Google Scholar Crossref Search ADS PubMed WorldCat 11. Wang Y , He L, Du Y, Zhu P, Huang G, Luo J, Yan X, et al. The long noncoding RNA lncTCF7 promotes self-renewal of human liver cancer stem cells through activation of Wnt signaling . Cell Stem Cell 2015 , 16 : 413 – 425 . Google Scholar Crossref Search ADS PubMed WorldCat 12. Wang H , Huo X, Yang XR, He J, Cheng L, Wang N, Deng X, et al. STAT3-mediated upregulation of lncRNA HOXD-AS1 as a ceRNA facilitates liver cancer metastasis by regulating SOX4 . Mol Cancer 2017 , 16 : 136. Google Scholar OpenURL Placeholder Text WorldCat 13. Huang H , Chen J, Ding CM, Jin X, Jia ZM, Peng J. LncRNA NR2F1-AS1 regulates hepatocellular carcinoma oxaliplatin resistance by targeting ABCC1 via miR-363 . J Cell Mol Med 2018 , 22 : 3238 – 3245 . Google Scholar Crossref Search ADS PubMed WorldCat 14. Fotuhi SN , Khalaj-Kondori M, Hoseinpour Feizi MA, Talebi M. Long non-coding RNA BACE1-AS may serve as an Alzheimer’s disease blood-based biomarker . J Mol Neurosci 2019 , 69 : 351 – 359 . Google Scholar Crossref Search ADS PubMed WorldCat 15. Zhang W , Zhao H, Wu Q, Xu W, Xia M. Knockdown of BACE1-AS by siRNA improves memory and learning behaviors in Alzheimer’s disease animal model . Exp Ther Med 2018 , 16 : 2080 – 2086 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 16. Li Y , Fang J, Zhou Z, Zhou Q, Sun S, Jin Z, Xi Z, et al. Downregulation of lncRNA BACE1-AS improves dopamine-dependent oxidative stress in rats with Parkinson’s disease by upregulating microRNA-34b-5p and downregulating BACE1 . Cell Cycle 2020 , 19 : 1 – 14 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 17. Esfandi F , Ghafouri-Fard S, Oskooei VK, Taheri M. β-Secretase 1 and its naturally occurring anti-sense RNA are down-regulated in gastric cancer . Pathol Oncol Res 2019 , 25 : 1627 – 1633 . Google Scholar Crossref Search ADS PubMed WorldCat 18. Nie Y , Li Y, Xu Y, Jiao Y, Li W. Long non-coding RNA BACE1-AS is an independent unfavorable prognostic factor in liver cancer . Oncol Lett 2020 , 20 : 202. Google Scholar OpenURL Placeholder Text WorldCat 19. Zhan M , He K, Xiao J, Liu F, Wang H, Xia Z, Duan X, et al. LncRNA HOXA11-AS promotes hepatocellular carcinoma progression by repressing miR-214-3p . J Cell Mol Med 2018 , 22 : 3758 – 3767 . Google Scholar Crossref Search ADS WorldCat 20. Lin T , Dai Y, Guo X, Chen W, Zhao J, Cao L, Wu Z. Silencing Of hsa_circ_0008450 represses hepatocellular carcinoma progression through regulation of microRNA-214-3p/EZH2 axis . Cancer Manag Res 2019 , 11 : 9133 – 9143 . Google Scholar Crossref Search ADS PubMed WorldCat 21. Fang YY , Tan MR, Zhou J, Liang L, Liu XY, Zhao K, Bao EC. miR-214-3p inhibits epithelial-to-mesenchymal transition and metastasis of endometrial cancer cells by targeting TWIST1 . Onco Targets Ther 2019 , 12 : 9449 – 9458 . Google Scholar Crossref Search ADS PubMed WorldCat 22. Yu H , Wang S, Zhu H, Rao D. LncRNA MT1JP functions as a tumor suppressor via regulating miR-214-3p expression in bladder cancer . J Cell Physiol 2019 , 20 .doi: 10.1002/jcp.28274. Google Scholar OpenURL Placeholder Text WorldCat Crossref 23. He Z , Wang X, Huang C, Gao Y, Yang C, Zeng P, Chen Z. The FENDRR/miR-214-3P/TET2 axis affects cell malignant activity via RASSF1A methylation in gastric cancer . Am J Transl Res 2018 , 10 : 3211 – 3223 . Google Scholar PubMed OpenURL Placeholder Text WorldCat 24. Ecke TH , Stier K, Weickmann S, Zhao Z, Buckendahl L, Stephan C, Kilic E, et al. miR-199a-3p and miR-214-3p improve the overall survival prediction of muscle-invasive bladder cancer patients after radical cystectomy . Cancer Med 2017 , 6 : 2252 – 2262 . Google Scholar Crossref Search ADS PubMed WorldCat 25. Arababadi MK , Asadikaram P, Asadikaram G. APLN/APJ pathway: the key regulator of macrophage functions . Life Sci 2019 , 232 : 116645. Google Scholar OpenURL Placeholder Text WorldCat 26. Wysocka MB , Pietraszek-Gremplewicz K, Nowak D. The role of Apelin in cardiovascular diseases, obesity and cancer . Front Physiol 2018 , 9 : 557. Google Scholar OpenURL Placeholder Text WorldCat 27. Lee T , Park CK, Ha SY. Prognostic role of Apelin receptor expression in hepatocellular carcinoma treated with curative surgical resection . Anticancer Res 2019 , 39 : 3025 – 3031 . Google Scholar Crossref Search ADS PubMed WorldCat 28. Cabiati M , Gaggini M, De Simone P, Del Ry S. Evaluation of Apelin/APJ system expression in hepatocellular carcinoma as a function of clinical severity . Clin Exp Med 2021 , 21 : 269 – 275 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

Acta Biochimica et Biophysica Sinica – Oxford University Press

Published: Oct 12, 2021

Keywords: bace1 gene; apelin; cell cycle; apoptosis; carcinoma, hepatocellular

There are no references for this article.