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Syndecan-2, negatively regulated by miR-20b-5p, contributes to 5-fluorouracil resistance of colorectal cancer cells via the JNK/ERK signaling pathway

Syndecan-2, negatively regulated by miR-20b-5p, contributes to 5-fluorouracil resistance of... Abstract 5-Fluorouracil (5-FU) resistance has been long considered as an obstacle to the efficacy of chemotherapy in colorectal cancer (CRC). In this study, we demonstrated the role of miR-20b-5p-regulated syndecan-2 (SDC2) in 5-FU resistance of CRC cells. 5-FU-resistant SW480 CRC cells were established by treatment of SW480 cells with stepwise increase of 5-FU concentration. The results showed that SDC2 was expressed significantly higher in SW480/5-FU cells than in SW480/WT cells as revealed by quantitative real-time polymerase chain reaction and western blot analysis. MTT assay and BrdU assay showed that SDC2 overexpression led to increased cell survival rate, while SDC2 knockdown reversed the drug resistance of SW480/5-FU cells. Wound healing and transwell invasion assays revealed that knockdown of SDC2 inhibited the migratory and invasive ability of SW480/5-FU cells. Moreover, animal experiments indicated that si-SDC2 plays a suppressive role in tumor growth in vivo. We also confirmed that miR-20b-5p interacted with SDC2, which reversed the effect of SDC2 in SW480/5-FU cells via the c-Jun N-terminal kinase (JNK)/extracellular regulated protein kinases (ERK) signaling pathway. These findings showed that JNK/ERK signaling pathway is involved in miR-20b-5p/SDC2 axis-mediated 5-FU resistance in SW480/5-FU cells, indicating that the miR-20b-5p/SDC2 axis is a potential target for reversing 5-FU resistance in CRC. syndecan-2, miR-20b-5p, 5-fluorouracil resistance, colorectal cancer, JNK/ERK Introduction Colorectal cancer (CRC) is the fourth most common cause of cancer-related death among men and the third most widespread cancer among women [1,2]. According to the World Health Organization, ∼945,000 new diagnoses of CRC occur every year, and >492,000 deaths are related to this disease [3,4]. Chemotherapy is an important treatment method for CRC, although chemoresistance usually leads to invalid chemotherapy [5]. The drug resistance mechanism of tumor cells is complex, including the dysregulation of key signaling pathways, changes in cancer targets, frequent drug efflux, and aberrant levels of RNA, DNA, or proteins [6]. One of the most common drugs in CRC chemotherapy is 5-fluorouracil (5-FU) [7]. Nevertheless, resistance to 5-FU often leads to diminished drug efficiency. Therefore, it is critical to find the key molecules that affect 5-FU sensitivity in CRC. Syndecan-2 (SDC2) is a cell surface heparan sulfate proteoglycan (HSPG) that was first discovered in 2007, which participates in various cell functions [8]. A few studies have proved that SDC2 participants in the occurrence, development, metastasis and prognosis of various malignant tumors, including osteosarcoma, esophageal squamous cell carcinoma and non-small cell lung cancer. The role of SDC2 in cancer development appears to be unique among SDC members with significant evidence supporting that SDC2 plays a role in tumor advancement [9,10]. It appears that SDC2 takes part in signaling pathways that are significant to cancer cell behavior in a way that is particular to the initiation and development of cancer tissue [11,12]. SDC2 has been proven to be highly expressed in mesenchymal cells, and there is evidence that SDC2 overexpression in epithelial-origin tumors is related to forceful behavior [13,14]. It was reported that SDC2 promotes cell proliferation and cell cycle progression in a few colon cancer cells [15]. Our group also reported that SDC2 serves as an oncogene via epithelial–mesenchymal transition and mitogen-activated protein kinase (MAPK) pathway in CRC [16]. Nevertheless, there has not been an in-depth study on the mechanism of SDC2 in 5-FU resistance of CRC and is therefore worthy of intensive investigation. In this study, we aimed to elaborate the role and molecular mechanism of SDC2 in 5-FU resistance of CRC cells. First, we determined that SDC2 was highly expressed in the 5-FU-resistant CRC cell line SW480/5-FU. Next, SDC2 knockdown suppressed cell proliferation and metastasis and regulated apoptosis and drug resistance genes in SW480/5-FU cells. Additionally, we demonstrated that SDC2 and miR-20b-5p directly interact with and regulate each other, thereby reversing their respective functions. Our data help clarify the functional role of miR-20b-5b-regulated SDC2 in the 5-FU resistance in CRC and provide a promising novel target for the treatment of CRC. Materials and Methods Cell lines Human CRC cell line SW480 was obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China). SW480 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St Louis, USA) supplemented with 10% foetal bovine serum (FBS; Hyclone, Logan, USA) at 37°C in 5% CO2. As previously reported [17], 5-FU-resistant SW480 cells were generated from 5-FU-sensitive cells by treatment with stepwise increase in 5-FU concentrations from 0.001 to 0.4 mM for 12 months. Additionally, SW480/5-FU cells were also cultured in DMEM supplemented with 2 μg/ml 5-FU and 10% FBS. SP600125 was purchased from MedChemExpress (Monmouth Junction, USA). Cell transfection First, SW480 wide type (SW480/WT) and SW480/5-FU cells were seeded into 6-well plates at 3×105 cells/well. Small interfering RNAs of SDC2 (si-SDC2-1: 5ʹ-CGCUGAAUAUACAGAACAATT-3ʹ, and si-SDC2-2: 5ʹ-CCAAAGAUACUGUUGACUATT-3ʹ), full-length SDC2 (pcDNA3.1-SDC2), and their corresponding negative controls, including si-NC (5ʹ-UUCUCCGAACGUGUCACGUTT-3ʹ) and pcDNA3.1, were purchased from GenePharma (Shanghai, China). Transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocols. Lentiviral vectors containing miR-20b-5p mimics (5ʹ-CAAAGUGCUCAUAGUGCAGGUAG-3ʹ) or miR-NC (5ʹ-UUGUACUACACAAAAGUACUG-3ʹ) were constructed following the manufacturer’s guidelines (GenePharma) and transfected into CRC cells to induce miR-20b-5p overexpression. MTT assay Cell viability and 5-FU sensitivity of SW480/WT cells and SW480/5-FU cells were assessed by MTT assay. In brief, cells from different groups were suspended into complete medium. Then, 100 μl of cell suspension was cultured in a 96-well plate at a cell density of 1×104 cells/well at 37°C in 5% CO2. Cells were co-incubated with various concentrations of 5-FU (0, 0.25, 0.5, 1, 2, 5, 10, and 25 μg/ml for SW480/WT cells; 0, 2.5, 5, 10, 20, 50, 100, and 250 μg/ml for SW480/5-FU cells) for an additional 48 h at 37°C. Subsequently, 20 μl of MTT (5 mg/ml; Sigma-Aldrich) was added into each well, followed by 4 h of incubation. Then, the medium was removed and 100 μl of DMSO was added. The optical density of each well was recorded at 490 nm with a microplate reader (Bio-Rad, Hercules, USA). Each experiment was conducted in triplicate. Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to determine the SDC2 expression level. Briefly, total RNA was extracted from cells using Trizol reagent (Invitrogen, Carlsbad, USA). The RNA was then reverse-transcribed into complementary DNA using PrimeScript RT Reagent Kit (Takara, Dalian, China). Quantitative reverse transcription PCR was carried out on an ABI 7900HT RealTime PCR System using SYBR Green PCR Master Mix (Roche Diagnostic GmbH, Mannheim, Germany) and the following cycle conditions: 94°C for 10 min, followed by 40 cycles of 94°C for 30 s, 55°C–58°C for 30 s, and 72°C for 45 s, and a final cycle at 72°C for 10 min. The 2−ΔΔCT method was used to calculate the relative mRNA level using β-actin as an internal control. TaqMan MicroRNA assay was performed to measure the miR-20b-5p expression level using TaqMan MicroRNA reagents (Takara) according to the manufacturer’s instructions. U6 was used as the internal control. The primers used in the experiment are listed in Table 1. Each experiment was conducted in triplicate. Table 1. Sequences of primers used in this study Gene Primer sequence SDC2 Forward 5ʹ-AAACGGACAGAAGTCCTAGC-3ʹ Reverse 5ʹ-GATAAGCAGCACTGGATGGT-3ʹ miR-20b-5p Forward 5ʹ-CGGTATCATTTGGCAGTGTCT-3ʹ Reverse 5ʹ-GTGCAGGGTCCGAGGTAT-3ʹ U6 Forward 5ʹ-CTCGCTTCGGCAGCACA-3ʹ Reverse 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ β-actin Forward 5ʹ-GATGAGATTGGCATGGCTTT-3ʹ Reverse 5ʹ-GTCACCTTCACCGTTCCAGT-3ʹ Gene Primer sequence SDC2 Forward 5ʹ-AAACGGACAGAAGTCCTAGC-3ʹ Reverse 5ʹ-GATAAGCAGCACTGGATGGT-3ʹ miR-20b-5p Forward 5ʹ-CGGTATCATTTGGCAGTGTCT-3ʹ Reverse 5ʹ-GTGCAGGGTCCGAGGTAT-3ʹ U6 Forward 5ʹ-CTCGCTTCGGCAGCACA-3ʹ Reverse 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ β-actin Forward 5ʹ-GATGAGATTGGCATGGCTTT-3ʹ Reverse 5ʹ-GTCACCTTCACCGTTCCAGT-3ʹ Open in new tab Table 1. Sequences of primers used in this study Gene Primer sequence SDC2 Forward 5ʹ-AAACGGACAGAAGTCCTAGC-3ʹ Reverse 5ʹ-GATAAGCAGCACTGGATGGT-3ʹ miR-20b-5p Forward 5ʹ-CGGTATCATTTGGCAGTGTCT-3ʹ Reverse 5ʹ-GTGCAGGGTCCGAGGTAT-3ʹ U6 Forward 5ʹ-CTCGCTTCGGCAGCACA-3ʹ Reverse 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ β-actin Forward 5ʹ-GATGAGATTGGCATGGCTTT-3ʹ Reverse 5ʹ-GTCACCTTCACCGTTCCAGT-3ʹ Gene Primer sequence SDC2 Forward 5ʹ-AAACGGACAGAAGTCCTAGC-3ʹ Reverse 5ʹ-GATAAGCAGCACTGGATGGT-3ʹ miR-20b-5p Forward 5ʹ-CGGTATCATTTGGCAGTGTCT-3ʹ Reverse 5ʹ-GTGCAGGGTCCGAGGTAT-3ʹ U6 Forward 5ʹ-CTCGCTTCGGCAGCACA-3ʹ Reverse 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ β-actin Forward 5ʹ-GATGAGATTGGCATGGCTTT-3ʹ Reverse 5ʹ-GTCACCTTCACCGTTCCAGT-3ʹ Open in new tab Western blot analysis Western blot analysis was performed as previously reported [18]. The lysis buffer that contains protease inhibitors (Promega, Madison, USA) was used to extract total protein. Bicinchonninic acid (BCA) method was used to measure the protein concentration. In order to determine the core protein of SDC2, samples underwent deglycosylation using 0.1 unit Heparinase I and III (Sigma-Aldrich), 0.1 unit Chondroitinase ABC (Sigma-Aldrich), 0.1 unit Keratanase (Seikagaku, Joetsu, Japan), and 0.001 unit Keratanase II (Seikagaku) per 1 µg non-deglycosylated proteins at 37°C for 4 h [19]. Next, cell lysates (50 μg proteins) were separated by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% skim milk in TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature. Subsequently, the membranes were incubated with primary antibodies of SDC2, MDR1, MRP5, LRP5, Bax, Bcl-2, Survivin, p-ERK1/2, ERK1/2, p-JNK, JNK, and β-actin (Abcam, Cambridge, USA) overnight at 4°C. Thereafter, the membranes were washed with TBST buffer three times and incubated for another 2 h with horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam). After an extensive wash, the membranes were visualized using an ECL kit (EMD Millipore, USA). ImageJ software was used to analyze the brand intensity. β-Actin was used as an internal control. BrdU cell proliferation assay CRC cells in the logarithmic phase were divided into different groups, including si-NC group, si-SDC2 group, miR-NC group, miR-20b-5p mimics group, pcDNA3.1 group, and miR-20b-5p mimics + pcDNA3.1-SDC2 group, treated with 10 μM of BrdU (Thermo Fisher Scientific) for 48 h. Then, CRC cells were collected and incubated with 4-6-diamidino-2-phenylindole (DAPI) solution (Sigma-Aldrich). The DAPI-stained cells were imaged and examined under a DMI3000B fluorescent microscope (Leica, Wetzlar, Germany). The BrdU-positive cell ratio was calculated in five random fields. Wound healing assay The monolayer of transfected cells in different groups was scratched using a 200-μl micropipette tip. Then, a BX53M inverted microscope (Olympus, Tokyo, Japan) was used to collect the images at 12 h after scratch. The wound healing process was evaluated by ImageJ using the three random microscopic fields. Cell invasion assay SW480/WT and SW480/5-FU cells were seeded into Matrigel-coated transwell inserts (BD Biosciences, San Jose, USA) with 4×105 cells/well. The lower chamber was filled with medium containing 10% FBS. After 24 h of culture, the cells from the top of the filter were removed, and the invaded cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The BX53M inverted microscope was used to record the number of cells in five randomly selected fields. Bioinformatics analysis and luciferase activity assay Targetscan (http://www.targetscan.org/vert_71/), PicTar (https://pictar.mdc-berlin.de/), and miRanda (http://www.bioinformatics.com.cn/local_miranda_miRNA_target_prediction_120) databases were utilized for bioinformatics analysis to seek for the interacted miRNAs of SDC2. To verify the prediction results, luciferase reporter assay was performed. Wild-type (WT) or mutant (MUT) SDC2-bound miR-20b-5p was inserted into a pmirloGLO dual luciferase plasmid (Promega) to establish SDC2/WT or SDC2/MUT reporter vector. SW480 cells were incubated for 48 h after transfection with SDC2/WT, SDC2/MUT, and/or miR-20b-5p mimics or miR-NC. The luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Animal experiment All animals were raised and maintained in a pathogen-free environment at the Nantong University. The animal experiment protocols were approved by the ethics committee of Affiliated Hospital of Nantong University. To explore the role of SDC2 in 5-FU resistance on BALB/c nude mice (Nantong University) with CRC, SW480/5-FU cells transfected with si-SDC2 or si-NC (1×106 cells) were injected into the mammary fat pads of nude mice to construct CRC animal model. When the tumors were palpable, mice were administered with 5-FU (5 mg/kg) every 4 days through intraperitoneal injection. Next, the volumes of the tumor on nude mice were measured every 4 days. After 20 days of observation, all mice were sacrificed, and the CRC tumor tissues were isolated and analyzed. Statistical analysis GraphpadPrism 7.0 and SPSS 21.0 statistical softwares were utilized to analyze the data. Differences between two groups were compared using Student’s t-test. Additionally, one-way analysis of variance was applied to analyze comparisons between multiple groups. Data are shown as the mean±SD. P<0.05 means statistically significant difference. Results Elevated SDC2 expression level was observed in SW480/5-FU cells To study the mechanism of chemo-resistance, the 5-FU resistant CRC cell line (SW480/5-FU) was developed. Next, MTT assay was used to assess the resistance of cells to 5-FU. WT and 5-FU-resistant SW480 cells were incubated with different 5-FU concentrations ranging from 0.25 to 500 μg/ml. The 50% inhibitory concentration (IC50) of WT SW480 cells was 1.8±0.27 μg/ml, while the IC50 of SW480 5-FU-resistant cells was 54.3±8.2 μg/ml (Fig. 1A,B), which indicated that the 5-FU-resistant SW480 cell line was successfully established. Then, the role of SDC2 in 5-FU resistance of SW480 cells was explored. First, we determined the SDC2 mRNA expression level in WT or 5-FU-resistant SW480 cells. Interestingly, both the mRNA level and protein expression level of SDC2 were remarkably increased in 5-FU-resistant cells compared with in SW480/WT cells (P<0.001; Fig. 1C,D). Therefore, SDC2 was knocked down using small interfering RNAs (siRNAs) for subsequent studies. The successful knockdown of SDC-2 was achieved, especially by using si-SDC2-1 that showed a more significant knockdown efficiency (Fig. 1E,F). Therefore, si-SDC2-1 was selected for the subsequent assays. Figure 1. Open in new tabDownload slide DC2 is abnormally expressed in 5-FU-resistant SW480 cells (A) Inhibition rates and (B) IC50 values of 5-FU in SW480/WT cells and SW480/5-FU cells. (C) SDC2 mRNA expression and (D) SDC2 protein expression levels in SW480/WT and SW480/5-FU cells. (E, F) qRT-PCR and western blot analysis were used to check the transfection efficiency of si-SDC2 in SW480/5-FU cells. **P<0.01, ***P<0.001 vs SW480/WT group or si-NC group. Figure 1. Open in new tabDownload slide DC2 is abnormally expressed in 5-FU-resistant SW480 cells (A) Inhibition rates and (B) IC50 values of 5-FU in SW480/WT cells and SW480/5-FU cells. (C) SDC2 mRNA expression and (D) SDC2 protein expression levels in SW480/WT and SW480/5-FU cells. (E, F) qRT-PCR and western blot analysis were used to check the transfection efficiency of si-SDC2 in SW480/5-FU cells. **P<0.01, ***P<0.001 vs SW480/WT group or si-NC group. SDC2 knockdown restrained SW480/5-FU cell proliferation, migration, and invasion in vitro and in vivo Next, in order to explore the role of SDC2 in SW480/5-FU cells in vitro and in vivo, the MTT, BrdU, transwell invasion, wound healing assay, and animal studies were carried out. According to the MTT results, transfection of si-SDC2 led to a decrease in IC50 value in SW480/5-FU cells down to 13.6±4.3 μg/ml, which was remarkably reduced compared with the si-NC SW480/5-FU group (Fig. 2A). The BrdU assay also revealed that 5-FU-resistant cells had a higher proliferation rate compared with normal cells, whereas silencing SDC2 resulted in a lower proliferation rate compared with the si-NC SW480/5-FU group (Fig. 2B). The wound healing assay also revealed a similar trend, as si-SDC2 significantly inhibited the migratory ability of SW480/5-FU cells (Fig. 2C). In the transwell assay, a lower capacity of 12-h invasion of SW480/5-FU cells was observed in the si-SDC2 group compared with in the si-NC SW480/5-FU group (Fig. 2D). Further investigation of si-SDC2 in vivo was conducted using the BALB/c nude mice loaded with SW480/5-FU cells transfected with si-SDC2 or si-NC. The volumes of the tumor were recorded for 20 days. Figure 2E,F indicated that down-regulation of SDC2 in SW480/5-FU cells inhibited tumor growth in vivo, since the tumor volume remained small compared with the control group. Taken together, SDC2 knockdown restrained the proliferation, migration and invasion abilities of SW480/5-FU cells. Figure 2. Open in new tabDownload slide SDC2 knockdown restrained proliferation, migration, and invasion of SW480/5-FU cells in vitro and in vivo (A) IC50 values of SW480/5-FU cells in different transfection groups. (B) BrdU assay. (C) Wound healing assay was used to assess cell migration. (D) Transwell assay was used to evaluate cell migration and invasion. (E) The tumor was smaller in the si-SDC2 group than in the si-NC group after 20 days of treatment. (F) Tumor growth was slower in the si-SDC2 group than in the si-NC group. *P<0.05, **P<0.01, ***P<0.001 vs SW480/5-FU group. Figure 2. Open in new tabDownload slide SDC2 knockdown restrained proliferation, migration, and invasion of SW480/5-FU cells in vitro and in vivo (A) IC50 values of SW480/5-FU cells in different transfection groups. (B) BrdU assay. (C) Wound healing assay was used to assess cell migration. (D) Transwell assay was used to evaluate cell migration and invasion. (E) The tumor was smaller in the si-SDC2 group than in the si-NC group after 20 days of treatment. (F) Tumor growth was slower in the si-SDC2 group than in the si-NC group. *P<0.05, **P<0.01, ***P<0.001 vs SW480/5-FU group. SDC2 knockdown inhibited the expressions of drug-resistance-related proteins and regulated the expressions of apoptosis-related proteins As drug resistance is associated with drug-resistance-related and apoptosis-related proteins, we utilized PCR and western blot analysis to test the influence of SDC2 on these proteins. The drug-resistance-related proteins that we evaluated included MDR1, MRP5, and LRP5, and the apoptosis-related proteins included Bax, Bcl-2, and survivin. As expected, MDR1, MRP5, and LRP5 were significantly up-regulated at both the mRNA and protein levels in SW480/5-FU cells compared with in SW480/WT cells (Fig. 3A,B). Hence, si-SDC2 significantly impaired this up-regulation. Figure 3. Open in new tabDownload slide SDC2 knockdown inhibited the expressions of drug-resistance-related proteins and regulated the expressions of apoptosis-related proteins The expressions of drug-resistance-related proteins (MDR1, MRP5, and LRP5) determined by qRT-PCR (A) and western blot analysis (B). The expressions of apoptosis-related proteins (Bax, Bcl-2, and survivin) determined by qRT-PCR (C) and western blot analysis (D). *P<0.05, **P<0.01, ***P<0.001 vs SW480/5-FU group. Figure 3. Open in new tabDownload slide SDC2 knockdown inhibited the expressions of drug-resistance-related proteins and regulated the expressions of apoptosis-related proteins The expressions of drug-resistance-related proteins (MDR1, MRP5, and LRP5) determined by qRT-PCR (A) and western blot analysis (B). The expressions of apoptosis-related proteins (Bax, Bcl-2, and survivin) determined by qRT-PCR (C) and western blot analysis (D). *P<0.05, **P<0.01, ***P<0.001 vs SW480/5-FU group. With regard to apoptosis-related proteins, survivin and Bcl-2 were significantly up-regulated in SW480/5-FU cells compared with those in SW480/WT cells, while Bax expression was significantly reduced (Fig. 3C,D). Additionally, their expressions were remarkably reversed after transfection with si-SDC2. Taken together, SDC2 regulates apoptosis-related proteins in SW480/5-FU cells. miR-20b-5p is a binding target of SDC2 Targetscan, PicTar, and miRanda databases were utilized for bioinformatics analysis, results of which suggested that miR-20b-5p has a binding domain for SDC2 (Fig. 4A). This was confirmed by luciferase reporter assay in SW480/5-FU cells. The luciferase activity in Fig. 4B was distinctly decreased in the SDC2/WT group, but no alterations in the SDC2/MUT group, which indicated that miR-20b-5p is a binding target of SDC2. Figure 4. Open in new tabDownload slide miR-20b-5p is a binding target of SDC2 (A) Prediction of binding site between SDC2 and miR-20b-5p. (B) Direct interaction between miR-20b-5p and SDC2 was determined by dual-luciferase reporter assay. (C) miR-20b-5p levels in SW480/WT cells and SW480/5-FU cells. qRT-PCR was used to examine miR-20b-5p level (D) and SDC2 level (E) in SW480/5-FU cells transfected with different plasmids. *P<0.05, **P<0.01, ***P<0.001, #P<0.05, ##P<0.01. Figure 4. Open in new tabDownload slide miR-20b-5p is a binding target of SDC2 (A) Prediction of binding site between SDC2 and miR-20b-5p. (B) Direct interaction between miR-20b-5p and SDC2 was determined by dual-luciferase reporter assay. (C) miR-20b-5p levels in SW480/WT cells and SW480/5-FU cells. qRT-PCR was used to examine miR-20b-5p level (D) and SDC2 level (E) in SW480/5-FU cells transfected with different plasmids. *P<0.05, **P<0.01, ***P<0.001, #P<0.05, ##P<0.01. According to qRT-PCR data, miR-20b-5p exhibited a remarkable decrease in 5-FU-resistant SW480 cells compared with in SW480/WT cells (Fig. 4C). Further, qRT-PCR was conducted to detect miR-20b-5p level, which reflects the transfection efficiency of miR-20b-5p mimics or SDC2 in SW480/5-FU cells. The data showed that miR-20b-5p level was remarkably up-regulated in miR-20b-5p mimics-transfected resistant cells (Fig. 4D); when co-transfected with full-length SDC2, the miR-20b-5p level was decreased significantly compared with in the miR-20b-5p mimics group, while full-length SDC2 rescued the SDC2 expression that was markedly reduced by miR-20b-5p mimics (Fig. 4E). We also demonstrated the negative association between miR-20b-5p and SDC2. Taken together, our results demonstrate that miR-20b-5p is a binding target of SDC2, and miR-20b-5p is a negative regulator of SDC2. SDC2 reversed miR-20b-5p function in SW480/5-FU cells It has been shown that miR-20b-5p can negatively regulate SDC2. Next, we wanted to verify the mechanism of the biological functions of miR-20b-5p in 5-FU-resistant cells. Previous evidence supported that miR-20b-5p can increase 5-FU-induced inhibition of SW480/5-FU cells, leading to a reduced IC50 value (Fig. 5A). Whereas co-transfected with SDC2, reversal resistance effect led to a significant decrease compared with the miR-20b-5p mimics group. The BrdU result revealed that the cell viability of SW480/5-FU cells was significantly decreased after transfection with miR-20b-5p mimics (Fig. 5B), whereas the cells co-transfected with SDC2 showed increased proliferation ability compared with the cells in the miR-20b-5p mimics group. Wound healing and cell invasion assays also revealed a similar trend (Fig. 5C,D). Figure 5. Open in new tabDownload slide SDC2 reversed miR-20b-5p function in SW480/5-FU cells (A) IC50 values in SW480/5-FU cells in different transfection groups determined by MTT assay. (B) BrdU assay. (C) Wound healing assay was used to assess cell migration. (D) Transwell assay was used to evaluate cell migration and invasion. **P<0.01, ***P<0.001 vs respective control; #P<0.05, ##P<0.01, ###P<0.001 vs miR-20b-5p mimics group. Figure 5. Open in new tabDownload slide SDC2 reversed miR-20b-5p function in SW480/5-FU cells (A) IC50 values in SW480/5-FU cells in different transfection groups determined by MTT assay. (B) BrdU assay. (C) Wound healing assay was used to assess cell migration. (D) Transwell assay was used to evaluate cell migration and invasion. **P<0.01, ***P<0.001 vs respective control; #P<0.05, ##P<0.01, ###P<0.001 vs miR-20b-5p mimics group. SDC2/miR-20b-5p interaction influenced drug-resistance-related proteins and apoptosis-related proteins via ERK/JNK signaling Next, we detected the expression levels of drug-resistance-related and apoptosis-related genes. Our results indicated that the drug-resistance-related proteins, including MRP5, MDR1, and LRP5, were down-expressed in the miR-20b-5p mimics group at both mRNA and protein levels (Fig. 6A–C). On the other hand, transfection with SDC2 reversed the results in gene expression caused by miR-20b-5p mimics. The expressions of apoptosis-related proteins including Bcl-2 and survivin, were remarkably lower at both mRNA and protein levels in the miR-20b-5p mimics group compared with the miR-NC group. The opposite effect was indicated in the Bax expression. Upon transfection with SDC2, the relative expressions of these genes were significantly reversed. These results further confirmed that miR-20b-5p negatively regulated the proliferation, migration, and invasion caused by SDC2 in SW480/5-FU cells. Figure 6. Open in new tabDownload slide SDC2/miR-20b-5p interaction influenced drug-resistance-related proteins and apoptosis-related proteins via ERK/JNK signaling (A) qRT-PCR was used to examine the levels of drug-resistance-related proteins (MDR1, MRP5, and LRP5). (B) qRT-PCR was used to assess the expression levels of apoptosis-related proteins (Bax, Bcl-2, and survivin). (C) Western blot analysis was used to evaluate the relative expressions of drug-resistance-related and apoptosis-related proteins. (D–F) SP600125 significantly inhibited proliferation, migration, and invasion of SW480/5-FU cells compared with NC group. (G) The relative levels of p-JNK, JNK, p-ERK, and ERK determined by densitometry analysis using ImageJ. *P<0.05, **P<0.01, ***P<0.001 vs respective control; #P<0.05, ##P<0.01, ###P<0.001 vs miR-20b-5p mimics group. Figure 6. Open in new tabDownload slide SDC2/miR-20b-5p interaction influenced drug-resistance-related proteins and apoptosis-related proteins via ERK/JNK signaling (A) qRT-PCR was used to examine the levels of drug-resistance-related proteins (MDR1, MRP5, and LRP5). (B) qRT-PCR was used to assess the expression levels of apoptosis-related proteins (Bax, Bcl-2, and survivin). (C) Western blot analysis was used to evaluate the relative expressions of drug-resistance-related and apoptosis-related proteins. (D–F) SP600125 significantly inhibited proliferation, migration, and invasion of SW480/5-FU cells compared with NC group. (G) The relative levels of p-JNK, JNK, p-ERK, and ERK determined by densitometry analysis using ImageJ. *P<0.05, **P<0.01, ***P<0.001 vs respective control; #P<0.05, ##P<0.01, ###P<0.001 vs miR-20b-5p mimics group. As reported previously, the MAPK signal pathway is closely associated with 5-FU resistance in renal carcinoma cells [17]. Therefore, the effect of the MAPK signal pathway was also explored in SW480/5-FU cells using a JNK inhibitor SP600125. As shown in Fig. 6D–F, 10 μM SP600125 significantly inhibited the proliferation, migration, and invasion of SW480/5-FU cells compared with NC group, suggesting that the MAPK signal pathway plays a role in 5-FU resistance in CRC. Whether the miR-20b-5p/SDC2 axis affects the MAPK signal pathway was investigated. Our results showed that the phosphorylation of both JNK and ERK was remarkably attenuated in the miR-20b-5p mimics group when compared with in the miR-NC group, and SDC2 reversed this function (Fig. 6G). Hence, the above evidence supports that the SDC2/miR-20b-5p interaction participates in the JNK/ERK pathway to modulate 5-FU-induced resistance in SW480/5-FU cells. Discussion Although anticancer drugs, such as 5-FU, have been widely used in CRC treatment, chemoresistance is still a major problem that limits the efficacy of treatment and the associated molecular mechanisms remain unclear [18,19]. More and more studies have revealed that aberrant SDC2 levels are often accompanied with complex biological behaviors of human tumors, which attracted much attention in the cancer research field. SDC2, a cell surface HSPG, has been identified to be mainly expressed on mesenchymal cells. SDC2 is associated with the development of numerous diseases [12,13]. SDC2 exerts different functions in different cell types. For instance, SDC2 can be used as a common factor for fibroblast growth factor, a receptor for Wnt protein, or a controller of cell adhesion, proliferation, differentiation, and apoptosis [8]. Overexpression of SDC2 has been shown to participate in the occurrence and prognosis of a variety of epithelial-derived malignancies, such as pancreatic cancer, non-small cell lung cancer, renal carcinoma, and glioblastoma [20]. In this study, we found that SDC2 was highly up-regulated in SW480/5-FU cells relative to the normal SW480/WT cells. Additionally, micro RNAs tend to play a crucial role in cancer and other complex diseases. They have been proved to inhibit gene expression at the post-transcription level, thus regulating cell cycle progression, differentiation, and apoptosis [21]. Using the luciferase reporter assay, we validated miR-20b-5p as a binding target of SDC2. miR-20b-5p was proved that it could weaken hypoxia-induced apoptosis in cardiomyocytes via the hypoxia inducible factor-1α (HIF-1α)/nuclear factor kappa-B (NF-κB) pathway and inhibit mitochondrial dysfunction-mediated apoptosis in hyperoxia-induced acute lung injury [22,23]. In this study, SDC2 and miR-20b-5p were demonstrated to be negatively correlated in SW480/5-FU cells. SDC2 level was highly down-regulated after transfection with miR-20b-5p mimics in SW480/5-FU cells. SDC2 knockdown was able to suppress the invasion ability of SW480/5-FU cells. Additionally, SDC2 weakened the effect of miR-20b-5p mimics on 5-FU-resistance in SW480/5-FU cells, indicating that the effect relies on SDC2 expression. JNK and ERK, two major members of the MAPK pathway, play a key role in cell proliferation, apoptosis, and differentiation [24]. MAPK is an intracellular serine/threonine protein kinase found in most cells [25,26]. The MAPK signaling pathway enables eukaryotic cells to transduce extracellular signals into the cells, causing cellular responses and affecting biological behaviors, such as cell proliferation, differentiation, transformation, and apoptosis, by affecting transcription and regulation of genes. After stimulation with external stimuli, JNK is activated to be transferred into the nucleus, which phosphorylates the amino terminal kinase protein of the downstream substrate and participates in a variety of external stimuli [27]. It is closely related to cell proliferation and apoptosis. ERK is widely distributed across various tissues. Upon activation, the signal can be transduced into the nucleus, and the increase in the activity and duration of the reaction helps determine different forms of response to stimulation, which have important regulatory effects on cell proliferation, growth, and differentiation [26]. miR-20b-5p can reduce p-JNK and p-ERK expression and specifically inhibit the phosphorylation of these proteins, which can effectively inhibit SDC2 expression in CRC cells. In summary, in this study, we revealed the role of SDC2 in 5-FU resistance and its underlying potential mechanisms in CRC. Our results revealed that upregulated expression of SDC2 and miR-20b-5p in SW480/5-FU cells compared with in SW480/WT cells. Luciferase reporter assay verified the direct interaction between SDC2 and miR-20b-5p. SDC2 knockdown suppressed proliferation, migration, and invasion and promoted the apoptosis of SW480/5-FU cells, while increased levels of miR-20b-5p reversed 5-FU resistance. After co-transfection with SDC2, the 5-FU reversing activity caused by miR-20b-5p was impaired, indicating that SDC2 and miR-20b-5p reciprocally negatively regulate each other. In addition, SDC2 could regulate JNK and ERK phosphorylation (Fig. 7). This study fully identified and characterized a new molecular target that has the potential to treat 5-FU resistance in CRC therapy. Figure 7. Open in new tabDownload slide A schematic diagram of miR-20b-5p/SDC2 axis in 5-FU resistance in CRC The interaction between miR-20b-5p and SDC2 influences SW480/5-FU cell proliferation, migration, and invasion via EMT progression, drug resistance proteins, and JNK/ERK activation. Figure 7. Open in new tabDownload slide A schematic diagram of miR-20b-5p/SDC2 axis in 5-FU resistance in CRC The interaction between miR-20b-5p and SDC2 influences SW480/5-FU cell proliferation, migration, and invasion via EMT progression, drug resistance proteins, and JNK/ERK activation. Funding This work was supported by the grant from the Social Development Foundation of Nantong city (No. JC2019074). Conflict of Interest The authors declare that they have no conflict of interest. References 1. Bray F , Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries . 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Biosci Rep 2015 , 35 : e00199. doi: 10.1042/BSR20140141 Google Scholar OpenURL Placeholder Text WorldCat Crossref 25. Sun Y , Liu W-Z, Liu T, Feng X, Yang N, Zhou H-F. Signaling pathway of MAPK/ERK in cell proliferation, differentiation, migration, senescence and apoptosis . J Recept Signal Transduct 2015 , 35 : 600 – 604 . Google Scholar Crossref Search ADS WorldCat 26. Meng X , Zhang S. MAPK cascades in plant disease resistance signaling . Annu Rev Phytopathol 2013 , 51 : 245 – 266 . Google Scholar Crossref Search ADS PubMed WorldCat 27. Kumar A , Singh UK, Kini SG, Garg V, Agrawal S, Tomar PK, Pathak P et al. JNK pathway signaling: a novel and smarter therapeutic targets for various biological diseases . Future Med Chem 2015 , 7 : 2065 – 2086 . Google Scholar Crossref Search ADS PubMed WorldCat Author notes † Ruheng Hua1 and Yan Zhang contributed equally to this work. © 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

Syndecan-2, negatively regulated by miR-20b-5p, contributes to 5-fluorouracil resistance of colorectal cancer cells via the JNK/ERK signaling pathway

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Oxford University Press
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Copyright © 2021 Institute of Biochemistry and Cell Biology, SIBS, CAS
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1672-9145
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1745-7270
DOI
10.1093/abbs/gmab124
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Abstract

Abstract 5-Fluorouracil (5-FU) resistance has been long considered as an obstacle to the efficacy of chemotherapy in colorectal cancer (CRC). In this study, we demonstrated the role of miR-20b-5p-regulated syndecan-2 (SDC2) in 5-FU resistance of CRC cells. 5-FU-resistant SW480 CRC cells were established by treatment of SW480 cells with stepwise increase of 5-FU concentration. The results showed that SDC2 was expressed significantly higher in SW480/5-FU cells than in SW480/WT cells as revealed by quantitative real-time polymerase chain reaction and western blot analysis. MTT assay and BrdU assay showed that SDC2 overexpression led to increased cell survival rate, while SDC2 knockdown reversed the drug resistance of SW480/5-FU cells. Wound healing and transwell invasion assays revealed that knockdown of SDC2 inhibited the migratory and invasive ability of SW480/5-FU cells. Moreover, animal experiments indicated that si-SDC2 plays a suppressive role in tumor growth in vivo. We also confirmed that miR-20b-5p interacted with SDC2, which reversed the effect of SDC2 in SW480/5-FU cells via the c-Jun N-terminal kinase (JNK)/extracellular regulated protein kinases (ERK) signaling pathway. These findings showed that JNK/ERK signaling pathway is involved in miR-20b-5p/SDC2 axis-mediated 5-FU resistance in SW480/5-FU cells, indicating that the miR-20b-5p/SDC2 axis is a potential target for reversing 5-FU resistance in CRC. syndecan-2, miR-20b-5p, 5-fluorouracil resistance, colorectal cancer, JNK/ERK Introduction Colorectal cancer (CRC) is the fourth most common cause of cancer-related death among men and the third most widespread cancer among women [1,2]. According to the World Health Organization, ∼945,000 new diagnoses of CRC occur every year, and >492,000 deaths are related to this disease [3,4]. Chemotherapy is an important treatment method for CRC, although chemoresistance usually leads to invalid chemotherapy [5]. The drug resistance mechanism of tumor cells is complex, including the dysregulation of key signaling pathways, changes in cancer targets, frequent drug efflux, and aberrant levels of RNA, DNA, or proteins [6]. One of the most common drugs in CRC chemotherapy is 5-fluorouracil (5-FU) [7]. Nevertheless, resistance to 5-FU often leads to diminished drug efficiency. Therefore, it is critical to find the key molecules that affect 5-FU sensitivity in CRC. Syndecan-2 (SDC2) is a cell surface heparan sulfate proteoglycan (HSPG) that was first discovered in 2007, which participates in various cell functions [8]. A few studies have proved that SDC2 participants in the occurrence, development, metastasis and prognosis of various malignant tumors, including osteosarcoma, esophageal squamous cell carcinoma and non-small cell lung cancer. The role of SDC2 in cancer development appears to be unique among SDC members with significant evidence supporting that SDC2 plays a role in tumor advancement [9,10]. It appears that SDC2 takes part in signaling pathways that are significant to cancer cell behavior in a way that is particular to the initiation and development of cancer tissue [11,12]. SDC2 has been proven to be highly expressed in mesenchymal cells, and there is evidence that SDC2 overexpression in epithelial-origin tumors is related to forceful behavior [13,14]. It was reported that SDC2 promotes cell proliferation and cell cycle progression in a few colon cancer cells [15]. Our group also reported that SDC2 serves as an oncogene via epithelial–mesenchymal transition and mitogen-activated protein kinase (MAPK) pathway in CRC [16]. Nevertheless, there has not been an in-depth study on the mechanism of SDC2 in 5-FU resistance of CRC and is therefore worthy of intensive investigation. In this study, we aimed to elaborate the role and molecular mechanism of SDC2 in 5-FU resistance of CRC cells. First, we determined that SDC2 was highly expressed in the 5-FU-resistant CRC cell line SW480/5-FU. Next, SDC2 knockdown suppressed cell proliferation and metastasis and regulated apoptosis and drug resistance genes in SW480/5-FU cells. Additionally, we demonstrated that SDC2 and miR-20b-5p directly interact with and regulate each other, thereby reversing their respective functions. Our data help clarify the functional role of miR-20b-5b-regulated SDC2 in the 5-FU resistance in CRC and provide a promising novel target for the treatment of CRC. Materials and Methods Cell lines Human CRC cell line SW480 was obtained from the Institute of Biochemistry and Cell Biology (Shanghai, China). SW480 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich, St Louis, USA) supplemented with 10% foetal bovine serum (FBS; Hyclone, Logan, USA) at 37°C in 5% CO2. As previously reported [17], 5-FU-resistant SW480 cells were generated from 5-FU-sensitive cells by treatment with stepwise increase in 5-FU concentrations from 0.001 to 0.4 mM for 12 months. Additionally, SW480/5-FU cells were also cultured in DMEM supplemented with 2 μg/ml 5-FU and 10% FBS. SP600125 was purchased from MedChemExpress (Monmouth Junction, USA). Cell transfection First, SW480 wide type (SW480/WT) and SW480/5-FU cells were seeded into 6-well plates at 3×105 cells/well. Small interfering RNAs of SDC2 (si-SDC2-1: 5ʹ-CGCUGAAUAUACAGAACAATT-3ʹ, and si-SDC2-2: 5ʹ-CCAAAGAUACUGUUGACUATT-3ʹ), full-length SDC2 (pcDNA3.1-SDC2), and their corresponding negative controls, including si-NC (5ʹ-UUCUCCGAACGUGUCACGUTT-3ʹ) and pcDNA3.1, were purchased from GenePharma (Shanghai, China). Transfections were performed using Lipofectamine 2000 (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocols. Lentiviral vectors containing miR-20b-5p mimics (5ʹ-CAAAGUGCUCAUAGUGCAGGUAG-3ʹ) or miR-NC (5ʹ-UUGUACUACACAAAAGUACUG-3ʹ) were constructed following the manufacturer’s guidelines (GenePharma) and transfected into CRC cells to induce miR-20b-5p overexpression. MTT assay Cell viability and 5-FU sensitivity of SW480/WT cells and SW480/5-FU cells were assessed by MTT assay. In brief, cells from different groups were suspended into complete medium. Then, 100 μl of cell suspension was cultured in a 96-well plate at a cell density of 1×104 cells/well at 37°C in 5% CO2. Cells were co-incubated with various concentrations of 5-FU (0, 0.25, 0.5, 1, 2, 5, 10, and 25 μg/ml for SW480/WT cells; 0, 2.5, 5, 10, 20, 50, 100, and 250 μg/ml for SW480/5-FU cells) for an additional 48 h at 37°C. Subsequently, 20 μl of MTT (5 mg/ml; Sigma-Aldrich) was added into each well, followed by 4 h of incubation. Then, the medium was removed and 100 μl of DMSO was added. The optical density of each well was recorded at 490 nm with a microplate reader (Bio-Rad, Hercules, USA). Each experiment was conducted in triplicate. Quantitative real-time polymerase chain reaction Quantitative real-time polymerase chain reaction (qRT-PCR) was carried out to determine the SDC2 expression level. Briefly, total RNA was extracted from cells using Trizol reagent (Invitrogen, Carlsbad, USA). The RNA was then reverse-transcribed into complementary DNA using PrimeScript RT Reagent Kit (Takara, Dalian, China). Quantitative reverse transcription PCR was carried out on an ABI 7900HT RealTime PCR System using SYBR Green PCR Master Mix (Roche Diagnostic GmbH, Mannheim, Germany) and the following cycle conditions: 94°C for 10 min, followed by 40 cycles of 94°C for 30 s, 55°C–58°C for 30 s, and 72°C for 45 s, and a final cycle at 72°C for 10 min. The 2−ΔΔCT method was used to calculate the relative mRNA level using β-actin as an internal control. TaqMan MicroRNA assay was performed to measure the miR-20b-5p expression level using TaqMan MicroRNA reagents (Takara) according to the manufacturer’s instructions. U6 was used as the internal control. The primers used in the experiment are listed in Table 1. Each experiment was conducted in triplicate. Table 1. Sequences of primers used in this study Gene Primer sequence SDC2 Forward 5ʹ-AAACGGACAGAAGTCCTAGC-3ʹ Reverse 5ʹ-GATAAGCAGCACTGGATGGT-3ʹ miR-20b-5p Forward 5ʹ-CGGTATCATTTGGCAGTGTCT-3ʹ Reverse 5ʹ-GTGCAGGGTCCGAGGTAT-3ʹ U6 Forward 5ʹ-CTCGCTTCGGCAGCACA-3ʹ Reverse 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ β-actin Forward 5ʹ-GATGAGATTGGCATGGCTTT-3ʹ Reverse 5ʹ-GTCACCTTCACCGTTCCAGT-3ʹ Gene Primer sequence SDC2 Forward 5ʹ-AAACGGACAGAAGTCCTAGC-3ʹ Reverse 5ʹ-GATAAGCAGCACTGGATGGT-3ʹ miR-20b-5p Forward 5ʹ-CGGTATCATTTGGCAGTGTCT-3ʹ Reverse 5ʹ-GTGCAGGGTCCGAGGTAT-3ʹ U6 Forward 5ʹ-CTCGCTTCGGCAGCACA-3ʹ Reverse 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ β-actin Forward 5ʹ-GATGAGATTGGCATGGCTTT-3ʹ Reverse 5ʹ-GTCACCTTCACCGTTCCAGT-3ʹ Open in new tab Table 1. Sequences of primers used in this study Gene Primer sequence SDC2 Forward 5ʹ-AAACGGACAGAAGTCCTAGC-3ʹ Reverse 5ʹ-GATAAGCAGCACTGGATGGT-3ʹ miR-20b-5p Forward 5ʹ-CGGTATCATTTGGCAGTGTCT-3ʹ Reverse 5ʹ-GTGCAGGGTCCGAGGTAT-3ʹ U6 Forward 5ʹ-CTCGCTTCGGCAGCACA-3ʹ Reverse 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ β-actin Forward 5ʹ-GATGAGATTGGCATGGCTTT-3ʹ Reverse 5ʹ-GTCACCTTCACCGTTCCAGT-3ʹ Gene Primer sequence SDC2 Forward 5ʹ-AAACGGACAGAAGTCCTAGC-3ʹ Reverse 5ʹ-GATAAGCAGCACTGGATGGT-3ʹ miR-20b-5p Forward 5ʹ-CGGTATCATTTGGCAGTGTCT-3ʹ Reverse 5ʹ-GTGCAGGGTCCGAGGTAT-3ʹ U6 Forward 5ʹ-CTCGCTTCGGCAGCACA-3ʹ Reverse 5ʹ-AACGCTTCACGAATTTGCGT-3ʹ β-actin Forward 5ʹ-GATGAGATTGGCATGGCTTT-3ʹ Reverse 5ʹ-GTCACCTTCACCGTTCCAGT-3ʹ Open in new tab Western blot analysis Western blot analysis was performed as previously reported [18]. The lysis buffer that contains protease inhibitors (Promega, Madison, USA) was used to extract total protein. Bicinchonninic acid (BCA) method was used to measure the protein concentration. In order to determine the core protein of SDC2, samples underwent deglycosylation using 0.1 unit Heparinase I and III (Sigma-Aldrich), 0.1 unit Chondroitinase ABC (Sigma-Aldrich), 0.1 unit Keratanase (Seikagaku, Joetsu, Japan), and 0.001 unit Keratanase II (Seikagaku) per 1 µg non-deglycosylated proteins at 37°C for 4 h [19]. Next, cell lysates (50 μg proteins) were separated by gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and proteins were transferred to polyvinylidene fluoride (PVDF) membranes. Membranes were blocked with 5% skim milk in TBST buffer (10 mM Tris-HCl, pH 8.0, 150 mM NaCl, and 0.1% Tween 20) for 1 h at room temperature. Subsequently, the membranes were incubated with primary antibodies of SDC2, MDR1, MRP5, LRP5, Bax, Bcl-2, Survivin, p-ERK1/2, ERK1/2, p-JNK, JNK, and β-actin (Abcam, Cambridge, USA) overnight at 4°C. Thereafter, the membranes were washed with TBST buffer three times and incubated for another 2 h with horseradish peroxidase (HRP)-conjugated secondary antibodies (Abcam). After an extensive wash, the membranes were visualized using an ECL kit (EMD Millipore, USA). ImageJ software was used to analyze the brand intensity. β-Actin was used as an internal control. BrdU cell proliferation assay CRC cells in the logarithmic phase were divided into different groups, including si-NC group, si-SDC2 group, miR-NC group, miR-20b-5p mimics group, pcDNA3.1 group, and miR-20b-5p mimics + pcDNA3.1-SDC2 group, treated with 10 μM of BrdU (Thermo Fisher Scientific) for 48 h. Then, CRC cells were collected and incubated with 4-6-diamidino-2-phenylindole (DAPI) solution (Sigma-Aldrich). The DAPI-stained cells were imaged and examined under a DMI3000B fluorescent microscope (Leica, Wetzlar, Germany). The BrdU-positive cell ratio was calculated in five random fields. Wound healing assay The monolayer of transfected cells in different groups was scratched using a 200-μl micropipette tip. Then, a BX53M inverted microscope (Olympus, Tokyo, Japan) was used to collect the images at 12 h after scratch. The wound healing process was evaluated by ImageJ using the three random microscopic fields. Cell invasion assay SW480/WT and SW480/5-FU cells were seeded into Matrigel-coated transwell inserts (BD Biosciences, San Jose, USA) with 4×105 cells/well. The lower chamber was filled with medium containing 10% FBS. After 24 h of culture, the cells from the top of the filter were removed, and the invaded cells were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet. The BX53M inverted microscope was used to record the number of cells in five randomly selected fields. Bioinformatics analysis and luciferase activity assay Targetscan (http://www.targetscan.org/vert_71/), PicTar (https://pictar.mdc-berlin.de/), and miRanda (http://www.bioinformatics.com.cn/local_miranda_miRNA_target_prediction_120) databases were utilized for bioinformatics analysis to seek for the interacted miRNAs of SDC2. To verify the prediction results, luciferase reporter assay was performed. Wild-type (WT) or mutant (MUT) SDC2-bound miR-20b-5p was inserted into a pmirloGLO dual luciferase plasmid (Promega) to establish SDC2/WT or SDC2/MUT reporter vector. SW480 cells were incubated for 48 h after transfection with SDC2/WT, SDC2/MUT, and/or miR-20b-5p mimics or miR-NC. The luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Animal experiment All animals were raised and maintained in a pathogen-free environment at the Nantong University. The animal experiment protocols were approved by the ethics committee of Affiliated Hospital of Nantong University. To explore the role of SDC2 in 5-FU resistance on BALB/c nude mice (Nantong University) with CRC, SW480/5-FU cells transfected with si-SDC2 or si-NC (1×106 cells) were injected into the mammary fat pads of nude mice to construct CRC animal model. When the tumors were palpable, mice were administered with 5-FU (5 mg/kg) every 4 days through intraperitoneal injection. Next, the volumes of the tumor on nude mice were measured every 4 days. After 20 days of observation, all mice were sacrificed, and the CRC tumor tissues were isolated and analyzed. Statistical analysis GraphpadPrism 7.0 and SPSS 21.0 statistical softwares were utilized to analyze the data. Differences between two groups were compared using Student’s t-test. Additionally, one-way analysis of variance was applied to analyze comparisons between multiple groups. Data are shown as the mean±SD. P<0.05 means statistically significant difference. Results Elevated SDC2 expression level was observed in SW480/5-FU cells To study the mechanism of chemo-resistance, the 5-FU resistant CRC cell line (SW480/5-FU) was developed. Next, MTT assay was used to assess the resistance of cells to 5-FU. WT and 5-FU-resistant SW480 cells were incubated with different 5-FU concentrations ranging from 0.25 to 500 μg/ml. The 50% inhibitory concentration (IC50) of WT SW480 cells was 1.8±0.27 μg/ml, while the IC50 of SW480 5-FU-resistant cells was 54.3±8.2 μg/ml (Fig. 1A,B), which indicated that the 5-FU-resistant SW480 cell line was successfully established. Then, the role of SDC2 in 5-FU resistance of SW480 cells was explored. First, we determined the SDC2 mRNA expression level in WT or 5-FU-resistant SW480 cells. Interestingly, both the mRNA level and protein expression level of SDC2 were remarkably increased in 5-FU-resistant cells compared with in SW480/WT cells (P<0.001; Fig. 1C,D). Therefore, SDC2 was knocked down using small interfering RNAs (siRNAs) for subsequent studies. The successful knockdown of SDC-2 was achieved, especially by using si-SDC2-1 that showed a more significant knockdown efficiency (Fig. 1E,F). Therefore, si-SDC2-1 was selected for the subsequent assays. Figure 1. Open in new tabDownload slide DC2 is abnormally expressed in 5-FU-resistant SW480 cells (A) Inhibition rates and (B) IC50 values of 5-FU in SW480/WT cells and SW480/5-FU cells. (C) SDC2 mRNA expression and (D) SDC2 protein expression levels in SW480/WT and SW480/5-FU cells. (E, F) qRT-PCR and western blot analysis were used to check the transfection efficiency of si-SDC2 in SW480/5-FU cells. **P<0.01, ***P<0.001 vs SW480/WT group or si-NC group. Figure 1. Open in new tabDownload slide DC2 is abnormally expressed in 5-FU-resistant SW480 cells (A) Inhibition rates and (B) IC50 values of 5-FU in SW480/WT cells and SW480/5-FU cells. (C) SDC2 mRNA expression and (D) SDC2 protein expression levels in SW480/WT and SW480/5-FU cells. (E, F) qRT-PCR and western blot analysis were used to check the transfection efficiency of si-SDC2 in SW480/5-FU cells. **P<0.01, ***P<0.001 vs SW480/WT group or si-NC group. SDC2 knockdown restrained SW480/5-FU cell proliferation, migration, and invasion in vitro and in vivo Next, in order to explore the role of SDC2 in SW480/5-FU cells in vitro and in vivo, the MTT, BrdU, transwell invasion, wound healing assay, and animal studies were carried out. According to the MTT results, transfection of si-SDC2 led to a decrease in IC50 value in SW480/5-FU cells down to 13.6±4.3 μg/ml, which was remarkably reduced compared with the si-NC SW480/5-FU group (Fig. 2A). The BrdU assay also revealed that 5-FU-resistant cells had a higher proliferation rate compared with normal cells, whereas silencing SDC2 resulted in a lower proliferation rate compared with the si-NC SW480/5-FU group (Fig. 2B). The wound healing assay also revealed a similar trend, as si-SDC2 significantly inhibited the migratory ability of SW480/5-FU cells (Fig. 2C). In the transwell assay, a lower capacity of 12-h invasion of SW480/5-FU cells was observed in the si-SDC2 group compared with in the si-NC SW480/5-FU group (Fig. 2D). Further investigation of si-SDC2 in vivo was conducted using the BALB/c nude mice loaded with SW480/5-FU cells transfected with si-SDC2 or si-NC. The volumes of the tumor were recorded for 20 days. Figure 2E,F indicated that down-regulation of SDC2 in SW480/5-FU cells inhibited tumor growth in vivo, since the tumor volume remained small compared with the control group. Taken together, SDC2 knockdown restrained the proliferation, migration and invasion abilities of SW480/5-FU cells. Figure 2. Open in new tabDownload slide SDC2 knockdown restrained proliferation, migration, and invasion of SW480/5-FU cells in vitro and in vivo (A) IC50 values of SW480/5-FU cells in different transfection groups. (B) BrdU assay. (C) Wound healing assay was used to assess cell migration. (D) Transwell assay was used to evaluate cell migration and invasion. (E) The tumor was smaller in the si-SDC2 group than in the si-NC group after 20 days of treatment. (F) Tumor growth was slower in the si-SDC2 group than in the si-NC group. *P<0.05, **P<0.01, ***P<0.001 vs SW480/5-FU group. Figure 2. Open in new tabDownload slide SDC2 knockdown restrained proliferation, migration, and invasion of SW480/5-FU cells in vitro and in vivo (A) IC50 values of SW480/5-FU cells in different transfection groups. (B) BrdU assay. (C) Wound healing assay was used to assess cell migration. (D) Transwell assay was used to evaluate cell migration and invasion. (E) The tumor was smaller in the si-SDC2 group than in the si-NC group after 20 days of treatment. (F) Tumor growth was slower in the si-SDC2 group than in the si-NC group. *P<0.05, **P<0.01, ***P<0.001 vs SW480/5-FU group. SDC2 knockdown inhibited the expressions of drug-resistance-related proteins and regulated the expressions of apoptosis-related proteins As drug resistance is associated with drug-resistance-related and apoptosis-related proteins, we utilized PCR and western blot analysis to test the influence of SDC2 on these proteins. The drug-resistance-related proteins that we evaluated included MDR1, MRP5, and LRP5, and the apoptosis-related proteins included Bax, Bcl-2, and survivin. As expected, MDR1, MRP5, and LRP5 were significantly up-regulated at both the mRNA and protein levels in SW480/5-FU cells compared with in SW480/WT cells (Fig. 3A,B). Hence, si-SDC2 significantly impaired this up-regulation. Figure 3. Open in new tabDownload slide SDC2 knockdown inhibited the expressions of drug-resistance-related proteins and regulated the expressions of apoptosis-related proteins The expressions of drug-resistance-related proteins (MDR1, MRP5, and LRP5) determined by qRT-PCR (A) and western blot analysis (B). The expressions of apoptosis-related proteins (Bax, Bcl-2, and survivin) determined by qRT-PCR (C) and western blot analysis (D). *P<0.05, **P<0.01, ***P<0.001 vs SW480/5-FU group. Figure 3. Open in new tabDownload slide SDC2 knockdown inhibited the expressions of drug-resistance-related proteins and regulated the expressions of apoptosis-related proteins The expressions of drug-resistance-related proteins (MDR1, MRP5, and LRP5) determined by qRT-PCR (A) and western blot analysis (B). The expressions of apoptosis-related proteins (Bax, Bcl-2, and survivin) determined by qRT-PCR (C) and western blot analysis (D). *P<0.05, **P<0.01, ***P<0.001 vs SW480/5-FU group. With regard to apoptosis-related proteins, survivin and Bcl-2 were significantly up-regulated in SW480/5-FU cells compared with those in SW480/WT cells, while Bax expression was significantly reduced (Fig. 3C,D). Additionally, their expressions were remarkably reversed after transfection with si-SDC2. Taken together, SDC2 regulates apoptosis-related proteins in SW480/5-FU cells. miR-20b-5p is a binding target of SDC2 Targetscan, PicTar, and miRanda databases were utilized for bioinformatics analysis, results of which suggested that miR-20b-5p has a binding domain for SDC2 (Fig. 4A). This was confirmed by luciferase reporter assay in SW480/5-FU cells. The luciferase activity in Fig. 4B was distinctly decreased in the SDC2/WT group, but no alterations in the SDC2/MUT group, which indicated that miR-20b-5p is a binding target of SDC2. Figure 4. Open in new tabDownload slide miR-20b-5p is a binding target of SDC2 (A) Prediction of binding site between SDC2 and miR-20b-5p. (B) Direct interaction between miR-20b-5p and SDC2 was determined by dual-luciferase reporter assay. (C) miR-20b-5p levels in SW480/WT cells and SW480/5-FU cells. qRT-PCR was used to examine miR-20b-5p level (D) and SDC2 level (E) in SW480/5-FU cells transfected with different plasmids. *P<0.05, **P<0.01, ***P<0.001, #P<0.05, ##P<0.01. Figure 4. Open in new tabDownload slide miR-20b-5p is a binding target of SDC2 (A) Prediction of binding site between SDC2 and miR-20b-5p. (B) Direct interaction between miR-20b-5p and SDC2 was determined by dual-luciferase reporter assay. (C) miR-20b-5p levels in SW480/WT cells and SW480/5-FU cells. qRT-PCR was used to examine miR-20b-5p level (D) and SDC2 level (E) in SW480/5-FU cells transfected with different plasmids. *P<0.05, **P<0.01, ***P<0.001, #P<0.05, ##P<0.01. According to qRT-PCR data, miR-20b-5p exhibited a remarkable decrease in 5-FU-resistant SW480 cells compared with in SW480/WT cells (Fig. 4C). Further, qRT-PCR was conducted to detect miR-20b-5p level, which reflects the transfection efficiency of miR-20b-5p mimics or SDC2 in SW480/5-FU cells. The data showed that miR-20b-5p level was remarkably up-regulated in miR-20b-5p mimics-transfected resistant cells (Fig. 4D); when co-transfected with full-length SDC2, the miR-20b-5p level was decreased significantly compared with in the miR-20b-5p mimics group, while full-length SDC2 rescued the SDC2 expression that was markedly reduced by miR-20b-5p mimics (Fig. 4E). We also demonstrated the negative association between miR-20b-5p and SDC2. Taken together, our results demonstrate that miR-20b-5p is a binding target of SDC2, and miR-20b-5p is a negative regulator of SDC2. SDC2 reversed miR-20b-5p function in SW480/5-FU cells It has been shown that miR-20b-5p can negatively regulate SDC2. Next, we wanted to verify the mechanism of the biological functions of miR-20b-5p in 5-FU-resistant cells. Previous evidence supported that miR-20b-5p can increase 5-FU-induced inhibition of SW480/5-FU cells, leading to a reduced IC50 value (Fig. 5A). Whereas co-transfected with SDC2, reversal resistance effect led to a significant decrease compared with the miR-20b-5p mimics group. The BrdU result revealed that the cell viability of SW480/5-FU cells was significantly decreased after transfection with miR-20b-5p mimics (Fig. 5B), whereas the cells co-transfected with SDC2 showed increased proliferation ability compared with the cells in the miR-20b-5p mimics group. Wound healing and cell invasion assays also revealed a similar trend (Fig. 5C,D). Figure 5. Open in new tabDownload slide SDC2 reversed miR-20b-5p function in SW480/5-FU cells (A) IC50 values in SW480/5-FU cells in different transfection groups determined by MTT assay. (B) BrdU assay. (C) Wound healing assay was used to assess cell migration. (D) Transwell assay was used to evaluate cell migration and invasion. **P<0.01, ***P<0.001 vs respective control; #P<0.05, ##P<0.01, ###P<0.001 vs miR-20b-5p mimics group. Figure 5. Open in new tabDownload slide SDC2 reversed miR-20b-5p function in SW480/5-FU cells (A) IC50 values in SW480/5-FU cells in different transfection groups determined by MTT assay. (B) BrdU assay. (C) Wound healing assay was used to assess cell migration. (D) Transwell assay was used to evaluate cell migration and invasion. **P<0.01, ***P<0.001 vs respective control; #P<0.05, ##P<0.01, ###P<0.001 vs miR-20b-5p mimics group. SDC2/miR-20b-5p interaction influenced drug-resistance-related proteins and apoptosis-related proteins via ERK/JNK signaling Next, we detected the expression levels of drug-resistance-related and apoptosis-related genes. Our results indicated that the drug-resistance-related proteins, including MRP5, MDR1, and LRP5, were down-expressed in the miR-20b-5p mimics group at both mRNA and protein levels (Fig. 6A–C). On the other hand, transfection with SDC2 reversed the results in gene expression caused by miR-20b-5p mimics. The expressions of apoptosis-related proteins including Bcl-2 and survivin, were remarkably lower at both mRNA and protein levels in the miR-20b-5p mimics group compared with the miR-NC group. The opposite effect was indicated in the Bax expression. Upon transfection with SDC2, the relative expressions of these genes were significantly reversed. These results further confirmed that miR-20b-5p negatively regulated the proliferation, migration, and invasion caused by SDC2 in SW480/5-FU cells. Figure 6. Open in new tabDownload slide SDC2/miR-20b-5p interaction influenced drug-resistance-related proteins and apoptosis-related proteins via ERK/JNK signaling (A) qRT-PCR was used to examine the levels of drug-resistance-related proteins (MDR1, MRP5, and LRP5). (B) qRT-PCR was used to assess the expression levels of apoptosis-related proteins (Bax, Bcl-2, and survivin). (C) Western blot analysis was used to evaluate the relative expressions of drug-resistance-related and apoptosis-related proteins. (D–F) SP600125 significantly inhibited proliferation, migration, and invasion of SW480/5-FU cells compared with NC group. (G) The relative levels of p-JNK, JNK, p-ERK, and ERK determined by densitometry analysis using ImageJ. *P<0.05, **P<0.01, ***P<0.001 vs respective control; #P<0.05, ##P<0.01, ###P<0.001 vs miR-20b-5p mimics group. Figure 6. Open in new tabDownload slide SDC2/miR-20b-5p interaction influenced drug-resistance-related proteins and apoptosis-related proteins via ERK/JNK signaling (A) qRT-PCR was used to examine the levels of drug-resistance-related proteins (MDR1, MRP5, and LRP5). (B) qRT-PCR was used to assess the expression levels of apoptosis-related proteins (Bax, Bcl-2, and survivin). (C) Western blot analysis was used to evaluate the relative expressions of drug-resistance-related and apoptosis-related proteins. (D–F) SP600125 significantly inhibited proliferation, migration, and invasion of SW480/5-FU cells compared with NC group. (G) The relative levels of p-JNK, JNK, p-ERK, and ERK determined by densitometry analysis using ImageJ. *P<0.05, **P<0.01, ***P<0.001 vs respective control; #P<0.05, ##P<0.01, ###P<0.001 vs miR-20b-5p mimics group. As reported previously, the MAPK signal pathway is closely associated with 5-FU resistance in renal carcinoma cells [17]. Therefore, the effect of the MAPK signal pathway was also explored in SW480/5-FU cells using a JNK inhibitor SP600125. As shown in Fig. 6D–F, 10 μM SP600125 significantly inhibited the proliferation, migration, and invasion of SW480/5-FU cells compared with NC group, suggesting that the MAPK signal pathway plays a role in 5-FU resistance in CRC. Whether the miR-20b-5p/SDC2 axis affects the MAPK signal pathway was investigated. Our results showed that the phosphorylation of both JNK and ERK was remarkably attenuated in the miR-20b-5p mimics group when compared with in the miR-NC group, and SDC2 reversed this function (Fig. 6G). Hence, the above evidence supports that the SDC2/miR-20b-5p interaction participates in the JNK/ERK pathway to modulate 5-FU-induced resistance in SW480/5-FU cells. Discussion Although anticancer drugs, such as 5-FU, have been widely used in CRC treatment, chemoresistance is still a major problem that limits the efficacy of treatment and the associated molecular mechanisms remain unclear [18,19]. More and more studies have revealed that aberrant SDC2 levels are often accompanied with complex biological behaviors of human tumors, which attracted much attention in the cancer research field. SDC2, a cell surface HSPG, has been identified to be mainly expressed on mesenchymal cells. SDC2 is associated with the development of numerous diseases [12,13]. SDC2 exerts different functions in different cell types. For instance, SDC2 can be used as a common factor for fibroblast growth factor, a receptor for Wnt protein, or a controller of cell adhesion, proliferation, differentiation, and apoptosis [8]. Overexpression of SDC2 has been shown to participate in the occurrence and prognosis of a variety of epithelial-derived malignancies, such as pancreatic cancer, non-small cell lung cancer, renal carcinoma, and glioblastoma [20]. In this study, we found that SDC2 was highly up-regulated in SW480/5-FU cells relative to the normal SW480/WT cells. Additionally, micro RNAs tend to play a crucial role in cancer and other complex diseases. They have been proved to inhibit gene expression at the post-transcription level, thus regulating cell cycle progression, differentiation, and apoptosis [21]. Using the luciferase reporter assay, we validated miR-20b-5p as a binding target of SDC2. miR-20b-5p was proved that it could weaken hypoxia-induced apoptosis in cardiomyocytes via the hypoxia inducible factor-1α (HIF-1α)/nuclear factor kappa-B (NF-κB) pathway and inhibit mitochondrial dysfunction-mediated apoptosis in hyperoxia-induced acute lung injury [22,23]. In this study, SDC2 and miR-20b-5p were demonstrated to be negatively correlated in SW480/5-FU cells. SDC2 level was highly down-regulated after transfection with miR-20b-5p mimics in SW480/5-FU cells. SDC2 knockdown was able to suppress the invasion ability of SW480/5-FU cells. Additionally, SDC2 weakened the effect of miR-20b-5p mimics on 5-FU-resistance in SW480/5-FU cells, indicating that the effect relies on SDC2 expression. JNK and ERK, two major members of the MAPK pathway, play a key role in cell proliferation, apoptosis, and differentiation [24]. MAPK is an intracellular serine/threonine protein kinase found in most cells [25,26]. The MAPK signaling pathway enables eukaryotic cells to transduce extracellular signals into the cells, causing cellular responses and affecting biological behaviors, such as cell proliferation, differentiation, transformation, and apoptosis, by affecting transcription and regulation of genes. After stimulation with external stimuli, JNK is activated to be transferred into the nucleus, which phosphorylates the amino terminal kinase protein of the downstream substrate and participates in a variety of external stimuli [27]. It is closely related to cell proliferation and apoptosis. ERK is widely distributed across various tissues. Upon activation, the signal can be transduced into the nucleus, and the increase in the activity and duration of the reaction helps determine different forms of response to stimulation, which have important regulatory effects on cell proliferation, growth, and differentiation [26]. miR-20b-5p can reduce p-JNK and p-ERK expression and specifically inhibit the phosphorylation of these proteins, which can effectively inhibit SDC2 expression in CRC cells. In summary, in this study, we revealed the role of SDC2 in 5-FU resistance and its underlying potential mechanisms in CRC. Our results revealed that upregulated expression of SDC2 and miR-20b-5p in SW480/5-FU cells compared with in SW480/WT cells. Luciferase reporter assay verified the direct interaction between SDC2 and miR-20b-5p. SDC2 knockdown suppressed proliferation, migration, and invasion and promoted the apoptosis of SW480/5-FU cells, while increased levels of miR-20b-5p reversed 5-FU resistance. After co-transfection with SDC2, the 5-FU reversing activity caused by miR-20b-5p was impaired, indicating that SDC2 and miR-20b-5p reciprocally negatively regulate each other. In addition, SDC2 could regulate JNK and ERK phosphorylation (Fig. 7). This study fully identified and characterized a new molecular target that has the potential to treat 5-FU resistance in CRC therapy. Figure 7. Open in new tabDownload slide A schematic diagram of miR-20b-5p/SDC2 axis in 5-FU resistance in CRC The interaction between miR-20b-5p and SDC2 influences SW480/5-FU cell proliferation, migration, and invasion via EMT progression, drug resistance proteins, and JNK/ERK activation. Figure 7. Open in new tabDownload slide A schematic diagram of miR-20b-5p/SDC2 axis in 5-FU resistance in CRC The interaction between miR-20b-5p and SDC2 influences SW480/5-FU cell proliferation, migration, and invasion via EMT progression, drug resistance proteins, and JNK/ERK activation. Funding This work was supported by the grant from the Social Development Foundation of Nantong city (No. JC2019074). Conflict of Interest The authors declare that they have no conflict of interest. References 1. Bray F , Ferlay J, Soerjomataram I, Siegel RL, Torre LA, Jemal A. Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries . 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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 SinicaOxford University Press

Published: Oct 1, 2021

Keywords: fluorouracil; colorectal cancer; syndecan; signal pathway; signal transduction pathways

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