miR-513b-5p表达变化对结肠癌细胞增殖、迁移的影响及机制探讨

Effects of miR-513b-5p on proliferation and migration of colon cancer cells and its mechanism

目的观察结肠癌(CC)组织、细胞中微小RNA-513b-5p(miR-513b-5p)的表达变化,以及miR-513b-5p对CC细胞增殖和迁移的影响,并探讨相关机制。方法采用qRT-PCR法检测CC组织、癌旁组织和CC细胞株(HCT116、RKO、SW480、LoVo、SW620)、人结肠成纤维细胞(CCD-112CoN)中的miR-513b-5p。将HCT116细胞分为miR-con组(转染miR-con)、miR-513b-5p抑制剂组(转染miR-513b-5p inhibitors);采用CCK-8试剂盒和Transwell实验检测细胞增殖和迁移能力。通过生物信息预测miR-513b-5p在信号传导及转录激活因子3(STAT3)上的结合位点;分别将野生型STAT3(STAT3-WT)质粒、突变型STAT3(STAT3-MUT)质粒和miR-513b-5p mimic、miR-con转染HCT116细胞;用荧光素酶检测试剂盒检测荧光素酶活性,采用Western blotting法检测STAT3蛋白。取部分HCT116细胞,分为miR-con组(转染miR-con)、miR-513b-5p模拟物组(转染miR-513b-5p mimics)、STAT3过表达组(共转染STAT3过表达质粒和miR-513b-5p mimics);采用CCK-8法和Transwell实验检测细胞增殖和迁移能力。结果CC组织和细胞中miR-513b-5p表达分别低于正常组织和CCD-112CoN细胞(P均<0.01)。miR-513b-5p抑制剂组细胞增殖活力和迁移数量均高于miR-con组(P均<0.05)。StarBase在线分析数据库显示,STAT3与miR-513b-5p存在结合位点;miR-513b-5p mimics可抑制STAT3-WT组荧光素酶活性(P<0.05),而对STAT3-MUT组荧光素酶活性无显著影响;miR-513b-5p mimics组STAT3蛋白相对表达量低于miR-con组、miR-513b-5p inhibitors组STAT3蛋白相对表达量高于miR-con组(P均<0.01)。miR-513b-5p模拟物组细胞增殖活力和迁移数量均低于miR-con组,STAT3过表达组细胞增殖活力和迁移数量均高于miR-513b-5p模拟物组(P均<0.01)。结论miR-513b-5p在CC组织和细胞中低表达,下调miR-513b-5p可促进细胞增殖和迁移;miR-513b-5p通过靶向STAT3起到抑制CC细胞增殖和迁移的作用。

Objective To investigate the effects of miR-513b-5p on the proliferation and migration of colon cancer(CC)cells and its mechanism.Methods The qRT-PCR was used to detect miR-513b-5p in CC tissues,paracancerous tissues,CC cell lines(HCT116,RKO,SW480,LoVo,and SW620)and human colon fibroblasts(CCD-112CoN).HCT116 cells were divided into the miR-con group(transfected with miR-con)and miR-513b-5p inhibitors group(transfected with miR-513b-5p inhibitors).CCK-8 kit and Transwell assay were used to detect cell proliferation and migration.The binding sites of miR-513b-5p on STAT3 were predicted by biological information;wild-type STAT3(STAT3-WT),mutant STAT3(STAT3-mut),miR-513b-5p mimics and miR-con were transfected into HCT116 cells,respectively;luciferase detection reagent kit was used to detect the luciferase activity.Western blotting was used to detect the expression of STAT3 protein.Part of HCT116 cells was divided into the miR-con group(transfected with miR-con),miR-513b-5p mimics group(transfected with miR-513b-5p mimics),STAT3 overexpression+miR-513b-5p mimics group(co transfected with STAT3 overexpression plasmid and miR-513b-5p mimics);CCK-8 assay and Transwell assay were used to detect cell proliferation and migration.Results The expression of miR-513b-5p in the CC tissues and cells was lower than that in the normal tissues and CCD-112CoN cells(P<0.01).The cell proliferation and migration abilities in the miR-513b-5p inhibitor group were higher than those in the miR-con group(both P<0.05).StarBase online analysis database showed that there was a binding site between STAT3 and miR-513b-5p.The miR-513b-5p mimics could inhibit the luciferase activity of STAT3-WT group,but had no significant effect on luciferase activity of the STAT3-MUT group(both P<0.05).The relative protein expression of STAT3 in the miR-513b-5p mimics group was lower than that in the miR-con group,while the relative protein expression of STAT3 in the miR-513b-5p inhibitors group was higher than that in the miR-con group(both P<0.01).The cell proliferation and migration abilities in the miR-513b-5p group were lower than those in the miR-con group,and the cell proliferation and migration abilities in the STAT3 overexpression+miR-513b-5p group were higher than those in the miR-513b-5p group(all P<0.01).Conclusion The miR-513b-5p is low expressed in CC tissues and cells,down-regulation of miR-513b-5p can promote cell proliferation and migration,and miR-513b-5p can inhibit CC cell proliferation and migration by targeting STAT3.