miR-15a-5p Regulates Oxaliplatin Resistance in Colorectal Cancer through the Wnt/β-catenin Pathway

Yanjie HUANG, Tong XU, Luwen WEN, Qi FENG, Jining ZHENG

Chengde Medical College, Chengde 067000, China

Abstract [Objectives] To explore the effects and mechanism of miR-15a-5p on oxaliplatin resistance in colorectal cancer HCT116/L cells. [Methods] The expression of miR-15a-5p in colorectal cancer sensitive cells HCT116 and resistant cells HCT116/L was detected by RT-qPCR method; the effect of oxaliplatin on the proliferation of HCT116 and HCT116/L cells was detected by MTT, and its IC50 and drug resistance fold of HCT116/L cells were calculated; the expressions of Wnt3a, β-Catenin and P-gp in HCT116 and HCT116/L cells were detected by Western Blot method. HCT-116/L cells were divided into 5 groups: blank control group HCT116/L, control group 1 transfected with miR-15a-5p mimics NC, experimental group 1 transfected with miR-15a-5p mimics, control group 2 transfected with miR-15a-5p inhibitor NC, and experimental group 2 transfected with miR-15a-5p inhibitor. The expression of miR-15a-5p in each group was detected by RT-qPCR method and the transfection efficiency was detected. The effect of different concentrations of oxaliplatin on the proliferation of cells in each group after transfection was detected by MTT and the half inhibitory concentration (50% inhibiting concentration, IC50) was calculated. The expressions of Wnt3a, β-catenin and P-gp in each group after transfection were detected by Western Blot method, and the expressions of Wnt3a, β-catenin and MDR1 mRNA in each group after transfection were detected by RT-qPCR method. [Results] (i) RT-qPCR results showed that the expression of miR-15a-5p in HCT116/L cells was (0.16±0.05) significantly lower than that in HCT116 cells (P<0.05). (ii) The IC50 of oxaliplatin for HCT-116 cells and HCT116/L cells detected by MTT method were (13.51±2.62 ) and (103.08±12.29) μg/mL, respectively. The drug resistance index of HCT116/L was 7.63. (iii) Western Blot results showed that the expressions of Wnt3a, β-catenin and P-gp in HCT116/L cells were significantly higher than those in HCT-116 cells (P<0.05). (iv) After successful transfection, the IC50 of miR-15a-5p mimics group to oxaliplatin decreased to (40.78±2.47) μg/mL by MTT method, and its sensitivity to the drug was significantly improved compared with the control group. Western Blot results showed that the relative expressions of P-gp, Wnt3a and β-catenin were significantly down-regulated (all P<0.05); RT-qPCR results showed that the relative expressions of MDR1, Wnt3a and β-catenin mRNA were significantly down-regulated (all P<0.05). (v) After successful transfection, the expression of miR-15a-5p in the miR-15a-5p inhibitor transfection group was (0.38±0.04); MTT results showed that its IC50 for oxaliplatin was up-regulated to (132.77±7.97) μg/mL, and its sensitivity to chemotherapy drugs was significantly lower than that of the control group; Western Blot results showed that the relative expressions of P-gp, Wnt3a and β-catenin were significantly up-regulated (all P<0.05); RT-qPCR results showed that the relative expressions of MDR1, Wnt3a and β-catenin mRNA were significantly up-regulated (all P<0.05). [Conclusions] Up-regulation of miR-15a-5p expression can reverse the resistance of HCT116/L cell line to oxaliplatin. Up-regulation or down-regulation of miR-15a-5p will affect the expression of P-gp and Wnt/β-catenin signaling pathway-related proteins, suggesting that the mechanism of miR-15a-5p reversal of drug resistance may be related to the inhibition of Wnt/β-catenin pathway, thereby down-regulating the expression of P-gp.

Key words Colorectal cancer, miR-15a-5p, Wnt/β-catenin, P-gp

1 Introduction

Colorectal cancer (CRC) is a common malignant tumor of the digestive tract, annually causing about 900 000 deaths[1]. Combined chemotherapy with platinum drugs represented by oxaliplation is the standard chemotherapy regimen for adjuvant treatment of colorectal cancer. However, as chemotherapy proceeds, colorectal cancer cells are gradually insensitive to oxaliplatin, and drug resistance leads to chemotherapy failure. Therefore, it is of great significance to study to improve the sensitivity of colorectal cancer cells to oxaliplatin. The microRNA is a non-coding single-stranded RNA with a length of 21-25 bp. It base-pairs with the 3’-end non-coding region (3′UTR) of the mRNA of the target gene, and inhibits the translation and expression of the target gene through post-transcriptional regulation. In recent years, more and more evidences have proven that miRNA is closely related to the drug resistance of colorectal cancer, and to a certain extent influences the chemotherapy effect and prognosis of colorectal cancer[2]. miR-15a-5p is a member of the miR-15 family and encoded by a gene located in the region of chromosome 13q14. Studies have found that up-regulation of miR-15a can inhibit the growth of colorectal cancer cells, thereby suppressing the tumor[3]. However, there are few reports on the mechanism of miR-15a resistance to platinum-based drugs in colorectal cancer cells. In this study, we explored the regulatory role of miR-15a-5p in oxaliplatin resistance in colorectal cancer cells and its possible mechanism.

2 Materials and methods

2.1 CellsColorectal cancer HCT-116 cells were purchased from GuangZhou Jennio Biotech Co., Ltd., and colorectal cancer oxaliplatin-resistant cells HCT116/L were purchased from Shanghai Bogoo Biotechnology. Co., Ltd.

2.2 Main reagentsRPMI 1640 medium was purchased from GIBCO company, fetal bovine serum was purchased from BI company, trypsin was purchased from Sigma company, RIPA lysate and MTT kit were purchased from Beijing Solarbio Science & Technology Co., Ltd. The miRcute miRNA isolation kit, Tiangen miRcute Plus miRNA First-Strand cDNA Kit, miRcute Plus miRNA qPCR Kit, Tiangen FastQuant cDNA kit, SuperReal PreMix Plus kit were purchased from Tiangen Biotech (Beijing) Co., Ltd. Wnt3a and P-gp antibodies were purchased from Beijing Biosynthesis Biotechnology Co., Ltd. β-actin antibody was purchased from ABclonal Company, β-catenin antibody was purchased from CST Company, miR-15a-5p mimics, miR-15a-5p mimics NC, miR-15a-5p inhibitor, miR-15a-5p inhibitor NC were purchased from Shanghai GenePharma Co., Ltd. β-catenin and MDR1 mRNA primers were designed and synthesized by Dalian Takara Bio Inc., and Wnt3a primers were synthesized by Sangon Bitech (Shanghai) Co., Ltd. Secondary antibody (rabbit antibody) was purchased from Bioword Company. Lipofectamine 2000 was purchased from Thermo Fisher Scientific Inc.

2.3 Major methods

2.3.1Cell culture. HCT116 and HCT116/L cells were cultured in RPMI 1640 medium containing 10% fetal bovine serum. The concentration of oxaliplatin in HCT116/L cell culture medium was gradually increased from 1 000 ng/mL to 6 000 ng/mL to maintain cell drug resistance.

2.3.2Drug resistance detection of drug-resistant cell line HCT116/L. HCT-116 and HCT116/L cells were digested to prepare a cell suspension and inoculated into a 96-well plate at a density of 8.0×103per well. After the cells had adhered, discarded the medium. Set the blank group: only containing the same volume of culture medium; the control group: added the culture medium without oxaliplatin to the wells inoculated with cells; experimental group: added 10, 20, 40, 80, 160, 320 μg/mL oxaliplatin culture medium to the wells inoculated with cells, and three replicate wells were set for each concentration. After dosing, incubated in a carbon dioxide incubator for 48 h, then added 20 μL of MTT (5 mg/mL) to each well, incubated at 37 ℃ for 4 h in the dark, aspirated the medium with a disposable syringe, added 150 μL of dimethyl sulfoxide (DMSO), and shook for 5 min in the dark. Measured the optical density value of each well with a microplate reader at a wavelength of 490 nm. Calculated the half inhibitory concentration (50% inhibiting concentration,IC50) of oxaliplatin, resistance index (RI)=IC50of resistant cell group/IC50of sensitive cell group.

2.3.3miR-15a-5pcell transfection. One day before transfection, placed HCT116/L cells into 6-well plates at a density of 1×105cells per well. After their adherent growth, added 1.5 mL of serum-free medium to each well. Take miR-15a-5p mimics, miR-15a-5p mimics NC, miR-15a-5p inhibitor and miR-15a-5p inhibitor NC with 150 pmol and 5 μL liposome Lipofectamine 2000 and mixed well. Finally, transfected HCT-116/oxaliplatin cells. Taking untransfected HCT116/L cells as blank control, after 6 h of incubation in a carbon dioxide incubator, replaced the medium containing 10% fetal bovine serum to continue the culture for later use. At 24 h after after transfection, extracted the RNA; 48 h later, extracted the protein for subsequent experiment.

2.3.4RNA extraction and RT-qPCR. Extracted the total RNA of log-phase cells using miRcute miRNA isolation kit, detected using Nanodrop 2000 spectrophotometer, and took samples withA260/A280of 1.8 to 2.1 for subsequent experiment. Used a 20 μL reverse transcription reaction loading system for reverse transcription to obtain cDNA, and then performed real-time fluorescence quantitative detection. The relative expression level of the target gene was expressed as 2-△△Ct. Set three replicate wells for each sample, and the difference inCtbetween the replicate wells was ≤0.5, otherwise repeated the sample experiment (Table 1).

Table 1 Real-time fluorescent quantitative PCR primers

2.3.5Effects of transfection of miR-15a-5p mimics and miR-15a-5p inhibitor on the proliferation of HCT116/L cells detected by MTT. At 24 h after blank control group HCT116/L cells, HCT116/L cells transfected with miR-15a-5p mimics (experimental group 1), miR-15a-5p mimics NC (control group 1), and miR-15a-5p inhibitor (experimental group 2) and miR-15a-5p inhibitor NC (control group 2), Cells in each group were inoculated into 96-well plates at a density of 8×103per well, with a final volume of 200 μL per well. Different concentrations of oxaliplatin (10, 20, 40, 80, 160, 320 μg/mL) were used to treat cells in each group. The MTT experimental operation is the same as before, and measured the optical density value (OD value) using a microplate reader at the wavelength of 490 nm. Growth inhibition rate=(ODvalue of positive control group-ODvalue of experimental group)/(ODvalue of positive control group-ODvalue of blank control group) × 100%, calculated the half inhibitory concentration of oxaliplatin (IC50). Resistance index (RI)=IC50of resistant cell group/IC50of sensitive cell group.

2.3.6Western blot detection of drug resistance-related proteins and pathway-related proteins. Cells in each group were lysed with RIPA lysis buffer, and the protein concentration of the samples was calculated by BCA method. Prepared 40 μg/15 μL equal volume and equal concentration of protein samples. Performed SDS-PAGE gel electrophoresis. After electrophoresis, transferred the protein to PVDF membrane, blocked with 5% nonfat milk powder for 2 h, and washed with TBST for 3 times, 10 min each time. Separately added Wnt3a (1∶1 000), β-catenin (1∶1 000), P-gp (1∶1 000) and β-actin (1∶100 000), incubated for 2 h at room temperature, overnight at 4 ℃, and washed 3 times with TBST, 10 min each time, added the corresponding secondary antibody (1∶1 000), incubated at room temperature for 1.5 h, washed the membrane with TBST and developed. With the aid of Image J software, we conducted scanning and analysis. Relative expression=Target protein/β-actin gray ratio, and repeated the experiment 3 times independently.

3 Results and analysis

3.1 MTT assay detectionWe treated HCT116 cells and HCT116/L cells with different concentrations of oxaliplatin (0, 10, 20, 40, 80, 160, 320 μg/mL) for 48 h. MTT results showed that theIC50of oxaliplatin on HCT116 cells and HCT116/L cells were (13.51±2.62) and (103.08±12.29) μg/mL, respectively. The resistance index (RI) was 7.63 (103.08/13.51), Indicating that the HCT116/L cell line was successfully constructed, as shown in Fig.1.

Fig.1 Inhibitory effect of oxaliplatin on proliferation of HCT-116 cells and HCT116/L cells by MTT assay detection

The results of MTT assay detection showed that through different concentrations of oxaliplatin (0, 10, 20, 40, 80, 160, 320 μg/mL) treated cells in each group, theIC50of oxaliplatin on HCT116/L in blank control group, miR-15a-5p mimics NC group, miR-15a-5p mimic group, miR-15a-5p inhibitor NC group and miR-15a-5p inhibitor group were (99.98±2.63), (100.35±10.57), (40.78±2.47), (102.08±5.95), and (132.77±7.97) μg/mL, respectively, (P<0.01). Compared with the blank control group HCT116/L, the miR-15a-5p mimics NC group and miR-15a-5p inhibitor NC group showed no significant difference in the inhibition rate of cell proliferation (allP>0.05), as shown in Fig.2.

Fig.2 Changes in sensitivity of HCT116/L cells to oxaliplatin after transfection with miR-15a-5p detected by MTT assay

3.2 RT-qPCR detection

3.2.1Relative expressions of miR-15a-5p in HCT116 and HCT116/L by RT-qPCR detection. RT-qPCR results showed that the relative expression levels of miR-15a-5p in HCT116 and HCT116/L were (1.00±0.00) and (0.16±0.05), respectively, and the difference was statistically significant (P<0.05), as shown in Fig.3.

Note: *P<0.05.Fig.3 Expression of miR-15a-5p in HCT-116 cells and HCT116/L cells

3.2.2RT-qPCR detection of expressions of miR-15a-5p, MDR1 and wnt/β-catenin mRNA in each group of cells after transfection. The relative expression levels of miR-15a-5p in blank control group HCT116/L, control group 1 transfected with miR-15a-5p mimics NC, experimental group 1 transfected with miR-15a-5p mimics, control group 2 transfected with miR-15a-5p inhibitor NC, and experimental group 2 transfected with miR-15a-5p inhibitor were (1.00±0.00), (1.08±0.10), (277.01±20.81), (1.07±0.12) and (0.38±0.04), respectively. The relative expression of miR-15a-5p in the transfected miR-15a-5p mimics group was significantly increased (F=527.82,P<0.001), and the relative expression of miR-15a-5p in the transfected miR-15a-5p inhibitor group was significantly higher decreased (F=79.93,P<0.001), indicating that the transfection was successful. The results of RT-qPCR showed that the expressions of Wnt3a, β-catenin and MDR1 mRNA in the transfected miR-15a-5p mimics group were lower than that in the miR-15a-5p mimics transfected NC group (P<0.05). Compared with the miR-15a-5p inhibitor transfected group, the expressions of Wnt3a, β-catenin and MDR1 mRNA in the miR-15a-5p inhibitor NC transfected group were all increased (P<0.05). Compared with the blank control group, the expressions of Wnt3a, β-catenin and MDR1 mRNA in transfected miR-15a-5p mimics NC group and miR-15a-5p inhibitor NC group were not significantly different (P>0.05), as shown in Fig.4.

Fig.4 RT-qPCR detection of expressions of Wnt3a, β-catenin and MDR1 mRNA after transfection with miR-15a-5p mimics and miR-15a-5p inhibitor

3.3 Western blot detection of expressions of Wnt3a/β-catenin signaling pathway protein and P-gp protein in HCT116/L cellsThe results of Western blot detection showed that the relative expressions of Wnt3a, β-catenin and P-gp in HCT-116/L-OHP cells were higher than those in HCT-116 cells, and the difference was statistically significant (P<0.01), as shown in Fig.5.

Fig.5 Western blot detection of expressions of Wnt3a/β-catenin signaling pathway protein and P-gp protein in HCT116 cells and HCT116/L cells

Through Western blot experiment, we found that the expressions of Wnt3a, β-catenin and P-gp proteins in the miR-15a-5p mimics transfected group was lower than that in the miR-15a-5p mimics NC transfected group (P<0.05); compared with the miR-15a-5p inhibitor transfected group, the expressions of Wnt3a, β-catenin and P-gp in miR-15a-5p inhibitor NC transfected group were all increased (P<0.05); compared with the blank control group, the miR-15a-5p mimics NC transfected group and miR-15a-5p inhibitor NC group had no significant differences in the protein expressions of Wnt3a, β-catenin and P-gp (P>0.05), as shown in Fig.6.

Fig.6 Western blot detection of expressions of Wnt3a, β-catenin and P-gp after transfection with miR-15a-5p mimics and miR-15a-5p inhibitor

4 Discussion

MicroRNA is a kind of non-coding small molecule single-stranded RNA that inhibits the translation of target mRNA or promotes the degradation of target mRNA by targeting and binding to the 3′UTR of various coding gene mRNAs. It regulates genes after transcription and indirectly plays the role of oncogenes or tumor suppressor genes, thereby participating in the occurrence and development of tumors. Many studies have shown that miR-15a-5p can affect the sensitivity of tumor cells to chemotherapeutic drugs. It is also found that the expressions of miR-15a is significantly reduced in pancreatic ductal carcinoma, and it can inhibit tumor cell proliferation and epithelial-mesenchymal transition by downregulating drug resistance-related factor Bmi-1[4]. Besides, miR-15a is overexpressed in non-small cell lung cancer[5], which can increase the sensitivity of tumor cells to chemotherapy drugs. Dai Lixiaetal.[6]demonstrated that miR-15a plays a role as a tumor suppressor gene in the occurrence and development of colorectal cancer, but there is still no study about the relationship between miR-15a and colorectal cancer drug resistance. In this study, we compared the expression level of miR-15a-5p in HCT-116 cells and oxaliplatin-resistant cell line HCT116/L, and found that the expression level of miR-15a-5p in HCT116/L cells was significantly lower than that in HCT-116 cells, and the differences were statistically significant. Therefore, in this study, we further transfected miR-15a-5p mimics and miR-15a-5p inhibitor into HCT116/L cells by cell transfection method. After adding different concentrations of oxaliplatin, compared with the control group, the sensitivity of HCT116/L to oxaliplatin in the miR-15a-5p mimics transfection group was significantly improved, while the sensitivity of HCT116/L to oxaliplatin was significantly decreased in the miR-15a-5p inhibitor transfection group, suggesting that increasing the expression level of miR-15a-5p may reverse the resistance of colorectal cancer cells to oxaliplatin through some mechanisms.

A large number of studies have shown that the Wnt/β-catenin pathway is closely related to tumor multidrug resistance[7]. After this signaling pathway is activated, Wnt can be stabilized and lead to a large accumulation of β-catenin. Activated β-catenin enters the nucleus and can interact with TCF/LEF family transcription factors to promote the expression of downstream target genes[8]. It has foun that MDR1 is a direct target gene of the β-catenin/TCF4/LEF transcription complex, and inhibiting the Wnt/β-catenin signaling pathway can down-regulate the expression of MDR1[9]. As a member of the classic ABC transporter family, the transmembrane glycoprotein P-gp encoded by the MDR1 gene can pump chemotherapeutic drugs that enter tumor cells out of the cell. Its overexpression in tumor cells is one of the most important factors for tumor resistance to oxaliplatin. Studies have found that miR-15a-5p inhibits the Wnt/β-catenin signaling pathway by targeting Wnt3a in endometrial cancer, thereby inhibiting tumor cell proliferation[10]. In this study, we carried out Western blot experiments and RT-qPCR detection, and found that up-regulation or down-regulation of miR-15a-5p expression could significantly affect the expressions of Wnt3a, β-catenin and its downstream P-gp. Up-regulation of miR-15a-5p expression can significantly inhibit the expressions of key proteins in the Wnt/β-catenin signaling pathway, such as the expressions of Wnt3a, β-catenin and their downstream P-gp, while down-regulation of miR-15a-5p could significantly increase the expressions of Wnt3a, β-catenin and P-gp, suggesting that overexpression of miR-15a-5p may increase the sensitivity of colorectal cancer cells to oxaliplatin by negatively regulating the activity of Wnt/β-catenin pathway and inhibiting the expression of the transporter P-gp.

In summary, up-regulation of miR-15a-5p expression can significantly increase the sensitivity of HCT116/L cell line to oxaliplatin, and its mechanism may be related to negative regulation of the activity of Wnt/β-catenin pathway, thereby inhibiting the transporter P-gp expression. miR-15a-5p is expected to become a target for reversing chemotherapeutic drug resistance and provide new ideas for clinical individualized treatment.