miR-486-3p通过直接靶向BIK调控结直肠癌细胞增殖、凋亡、迁移和侵袭

miR-486-3p regulates proliferation,apoptosis,migration and invasion of colorectal cancer cells by directly targeting BIK

目的:探讨miR-486-3p对结直肠癌细胞增殖、凋亡、迁移和侵袭的调节作用。方法:采用q-PCR法测定结直肠癌细胞株SW620和正常结肠细胞系NCM-460中miR-486-3p和BIK表达,荧光素酶报告试验测定miR-486-3p的直接作用靶点,将结直肠癌细胞株SW620分为NC组、miR-486-3p模拟物组、miR-486-3p抑制剂组和BIK siRNA组,各组细胞转染24 h后,MTT法测定各组细胞增殖情况,划痕实验和Transwell实验测定细胞迁移和侵袭能力,流式细胞术测定细胞凋亡情况,RT-PCR和Western blot法测定BIK、APAF1、p-Erk1/2、Erk1/2、Cyt C、Caspase-9和Caspase-3 mRNA和蛋白表达。结果:结直肠癌细胞SW620 miR-486-3p表达水平较正常结直肠细胞NCM-460显著提高(P<0.05),BIK mRNA和蛋白水平均较正常结直肠细胞显著降低(P<0.05)。双荧光素酶报告试验结果显示BIK3′-UTR是miR-486-3p的直接靶标;与NC组相比,miR-486-3p抑制剂组24 h、48 h和72 h结肠癌细胞光密度显著降低(P<0.05或P<0.01),划痕宽度显著增加(P<0.01),穿膜细胞数显著减少(P<0.01),细胞凋亡数显著增加(P<0.01),Cyt C、APAF1、Caspase-9和Caspase-3 mRNA和蛋白表达显著增加,p-Erk1/2 mRNA和蛋白表达显著降低(P<0.01);miR-486-3p模拟剂组和BIK siRNA组24 h、48 h和72 h结肠癌细胞光密度显著提高(P<0.05或P<0.01),细胞划痕宽度显著降低(P<0.05),穿膜细胞数显著增加(P<0.05),细胞凋亡数显著减少(P<0.01),Cyt C、APAF1、Caspase-9和Caspase-3 mRNA和蛋白表达显著降低,p-Erk1/2 mRNA和蛋白水表达显著增加(P<0.01),4组细胞Erk1/2表达差异无统计学意义(P>0.05)。结论:miR-486-3p是结直肠癌的致癌基因,可通过直接靶向BIK调节结直肠细胞增殖、凋亡、迁移和侵袭。

Objective:To investigate regulatory effect of miR-486-3p on colorectal cancer cell proliferation,apoptosis,migration and invasion.Methods:Expressions of miR-486-3p and BIK in colorectal cancer cell line SW620 and normal colonic cell line NCM-460 was determined by q-PCR.Direct target of miR-486-3p was determined by luciferase report test.Colorectal cancer cell lines SW620 were divided into NC group,miR-486-3p mimic group,miR-486-3p antagonist group and BIK siRNA group.After cell transfection for 24 h,cell proliferation was determined by MTT method,cell migration and invasion were determined by scratch assay and Transwell assay,apoptosis was determined by flow cytometry,and mRNA and protein expressions of BIK,APAF1,p-ERk1/2,ERk1/2,Cyt C,Caspase-9 and Caspase-3 were determined by RT-PCR and Western blot.Results:Expression of miR-486-3p in colorectal cancer cell line SW620 was significantly higher than that in normal colorectal cell line NCM-460(P<0.05),and expressions of BIK mRNA and protein were significantly lower than that in normal colorectal cell line NCM-460(P<0.05).Results of dual-luciferase reporter assay showed that BIK 3’-UTR was a direct target of miR-486-3p.Compared with NC group,optical density of colon cancer cells in miR-486-3p inhibitor group at 24 h,48 h and 72 h was significantly decreased,(P<0.05 or P<0.01),cell scratch widths were significantly increased(P<0.01),number of membrane-penetrating cells was significantly reduced(P<0.01),number of apoptosis cells was significantly increased(P<0.01),mRNA and protein levels of Cyt C,APAF1,Caspase-9 and Caspase-3 were significantly increased,mRNA and protein levels of p-Erk1/2 was significantly decreased(P<0.01);optical density of colon cancer cells in miR-486-3p mimics group and BIK siRNA group at 24 h,48 h and 72 h was significantly increased(P<0.05 or P<0.01),cell scratch widths were significantly decreaseD(P<0.05),number of membrane-penetrating cells was significantly increased(P<0.05),number of apoptosis cells was significantly reduced(P<0.01),mRNA and protein levels of Cyt C,APAF1,Caspase-9 and Caspase-3 were significantly decreased,mRNA and protein levels of p-Erk1/2 were significantly increased(P<0.01).There were no significant difference in mRNA and protein levels of erk1/2 in four groups(P>0.05).Conclusion:miR-486-3p is an oncogene of colorectal cancer,which plays a regulatory role in colorectal cell proliferation,apoptosis,migration and invasion by directly targeting BIK.