关键词: 结直肠癌, microRNA-7, 耐药, 5氟尿嘧啶, mTOR

Abstract: Objective The aim of this study was to investigate the effects of microRNA-7(miR-7)combined with 5-fluorouracil(5-FU)on proliferation and apoptosis of 5-FU resistant colorectal cancer(SW620/5-FU)cells, and to explore the regulatory mechanism of miR-7 expression in sensitivity of colorectal cancer to 5-FU. Methods Colorectal cancer tissues and adjacent tissues surgically removed from May 2019 to July 2021 were collected;Colorectal cancer HCT116, SW1116, DLD1, and SW620 cell lines, normal colon epithelial NCM460 cell line, and 5-FU resistant SW620 cell line(SW620/5-FU)were cultured as routine methods. The expression of miR-7 in cells and tissues was detected using RT-qPCR. Both SW620 cells and SW620/5-FU cells were transiently transfected with miR-7 mimic, miR-7 inhibitor, and vector, respectively. They were divided into the miR-7 vector group(vector group), miR-7 mimic group(overexpression group), miR-7 inhibitor group(inhibition group), miR-7 mimic+5-FU group combined with 5-FU, and control group. The cell proliferation and the 50% inhibitory concentration(IC₅₀)of 5-FU were detected using the CCK-8 method. The cell apoptosis was detected by flow cytometry in each group, and Western blot was used to detect the expression of cleaved-caspase-3, Bid, mTOR, and p-mTOR proteins in each group. Results The expression of miR-7 in colorectal cancer tissues was significantly lower than that in adjacent tissues(2.1±1.28 vs. 8.4±2.49, P＜0.05). The expression of miR-7 in colorectal cancer cell lines was significantly lower than that in NCM460 cells(P＜0.05). The expression of miR-7 in SW620/5-FU cells was lower than that in SW620 cells(0.43±0.13 vs. 0.99±0.01, P＜0.05). After 48 hours of transfection, the expression of miR-7 in the mimic group was higher than that in the control group(5.14 ±1.18 vs. 0.96±0.04, P＜0.05), while the expression in the inhibitory group was lower than that in the control group(0.43±0.11 vs. 0.96±0.04, P＜0.05). Compared with the control group, the proliferation ability of SW620 cells in the overexpression group was significantly reduced, and the apoptosis rate increased(n=3, t=15.301, P=0.001), while the results in the inhibition group were opposite(n=3, t=17.610, P=0.002). The expression of cleaved-caspase-3 and Bid proteins in SW620 cells of the overexpression group were higher than those in the control group(n=3, t=14.367, P=0.008), while the expression of cleaved-caspase-3 and Bid in the inhibition group were lower than those in the control group(n=3, t=13.245, P=0.003). The IC₅₀ values of 5-FU on SW620/5-FU cells were higher than SW620 cells(1 210 μg/mL vs. 220.6 μg/mL, P＜0.05). Compared with the control group and overexpression group, the viability of SW620 cells and SW620/5-FU cells in the miR-7 mimic+5-FU group significantly decreased by(36.33±3.85% vs. 99.30±0.75%;55.43±4.65% vs. 99.30±0.75%)(P＜0.05), and the apoptosis rate increased. The level of p-mTOR protein in SW620/5-FU cells in the miR-7 mimic+5-FU group decreased(0.23±0.04 vs. 0.82±0.05, P＜0.05). Conclusion Overexpression of miR-7 can increase the drug sensitivity of SW620/5-FU cells to 5-FU, and its mechanism may be related to the inhibition of p-mTOR.

Key words: Colorectal carcinoma, microRNA-7, Drug resistance, 5-Fluorouracil, mTOR