miRNA-541-5p inhibits proliferation and migration of colon cancer cells by negatively regulating cyclin D1 / 肿瘤研究与临床

ABSTRACT

Objective:To investigate the effect of cyclin D1 (CCND1) negatively regulated by miRNA-541 (miR-541-5p) on the proliferation and migration of colon cancer cells as well as its related mechanism.Methods:Expression levels of miR-541-5p in colon cancer cell lines HT29, SW480, SW620, HCT116 and enterocyte line HIEC of the normal people as well as cancer tissues and pericarcinomatous normal tissues of 112 patients undergoing the colon cancer surgery from the First Affiliated Hospital of Hebei North University between April 2017 and March 2020 were detected by using quantitative real-time polymerase chain reaction(qRT-PCR). The potential target gene of miR-541-5p was predicted by using TargetScan, and was verified by using dual luciferase reporter gene assay, qRT-PCR and Western blot. Expression level of CCND1 was detected in colon cancer cell lines and tissues. Cells with the lowest expression level of miR-541-5p were divided into miR-NC group (the transfected control plasmid), miR-541-5p group (the transfected miR-541-5p mimics), miR-541-5p+CCND1 group (the co-transfected miR-541-5p mimics and CCND1). Effect of miR-541-5p and CCND1 on proliferation and migration ability of colon cancer cells was detected by using cell counting kit-8 (CCK8) and Transwell method. The xenograft model of colon cancer in nude mice was constructed to observe the effect of miR-541-5p on tumor growth.Results:The relative expression level of miR-541-5p in colon cancer tissues was lower than that in pericarcinomatous normal tissues (0.45±0.06 vs. 1.00±0.12, t = 43.385, P < 0.01). The relative expression level of miR-541-5p was 0.46±0.03, 0.67±0.04, 0.57±0.06, 0.17±0.02, 1.00±0.15, respectively in colon cancer cell lines HT29, SW480, SW620, HCT116 and enterocyte line HIEC of the normal people, and the difference was statistiacally significant ( F = 5.621, P < 0.01); the relative expression level of miR-541-5p in all colon cancer cell lines was lower than that in enterocyte line HIEC of the normal people. HCT116 cells were selected to make the subsequent experiments. The predicted results of TargetScan showed that 3'UTR of CCND1 might have sites complementary to those of miR-541-5p. Dual luciferase reporter gene assay showed that CCND1 was the target gene of miR-541-5p, and miR-541-5p negatively regulated the expression of CCND1. CCK-8 method showed that cell proliferation rate of HCT116 was (2.00±0.16)%, (0.89±0.08)%, (2.56±0.23)%, respectively in miR-NC group, miR-541-5p group, miR-541-5p+CCND1 group, and the difference was statistically significant ( F = 6.715, P < 0.01); among HCT116 cells with the overexpression of miR-541-5p, the transfected CCND1 chould reverse the inhibitory effect of miR-541-5p on cell proliferation. Transwell results showed that the overexpression of miR-541-5p inhibited the cell migration ability of HCT116, while the co-transfection of miR-541-5p mimics and CCND1 could reverse the inhibitory effect. In the colon cancer nude mice xenograft model, the tumor mass and size of nude mice in miR-541-5p group was decreased compared with that in the control group (all P < 0.05). Conclusions:miR-541-5p inhibits cell proliferation and migration of colon cancer cells via negatively regulating CCND1, and inhibits tumor growth in xenograft model of colon cancer in nude mice, thereby acting as a tumor suppressor in colon cancer.