miR-451通过靶向ROCK1对卵巢癌细胞侵袭和迁移能力的调控作用

Regulation of miR-451 on invasion and migration of ovarian cancer cells by targeting ROCK1

目的:探讨miR-451对卵巢癌细胞侵袭和迁移能力的作用及机制。方法:通过实时荧光定量PCR(qPCR)检测卵巢癌组织及卵巢癌细胞、癌旁组织及正常卵巢上皮细胞中miR-451与Rho的卷曲螺旋激酶1(ROCK1)mRNA的表达;将卵巢癌CaOV-3细胞分为6组:Control组(未转染组)、miR-NC组(用miR-NC无序序列转染)、miR-451 mimic组(用过表达miR-451的模拟物转染)、miR-451 inhibitor组(用抑制miR-451表达的质粒转染)、pc-ROCK1组(用过表达ROCK1的质粒转染)和miR-451+pc-ROCK1组(miR-451的模拟物和过表达ROCK1的质粒联合转染);通过划痕实验检测细胞迁移能力;通过Transwell法检测细胞侵袭能力,通过蛋白印迹法检测E-钙粘附蛋白(E-carderin)、N-钙粘附蛋白(N-carderin)、细胞基质金属蛋白酶2(MMP2)、ROCK1蛋白表达水平;通过TargetScan数据库预测miR-451靶基因;通过双荧光素酶报告检测miR-451与ROCK1靶向关系。结果:与癌旁组织相比,在大多数卵巢组织中miR-451表达下调(57/70,81.43%);低表达的miR-451患者卵巢癌肿瘤较大,分化程度较差,多属于FIGO晚期,并存在明显的腹膜转移(P<0.05)。与对照组相比,miR-451 mimic组卵巢癌CaOV-3细胞划痕闭合率明显降低、侵袭细胞数目明显减少、E-cadherin明显上调、N-cadherin和MMP2表达明显下调(P<0.01),miR-451 inhibitor组和卵巢癌CaOV-3细胞划痕闭合率明显升高、侵袭细胞数目明显增多、E-cadherin明显下调、N-cadherin和MMP2表达明显上调(P<0.01)。miR-451靶向下调ROCK1。共转染pc-ROCK1逆转miR-451 mimic对卵巢癌CaOV-3细胞侵袭和迁移的影响(P<0.01)。结论:miR-451通过靶向下调RCOK1抑制卵巢癌CaOV-3细胞迁移和侵袭能力。

Objective:To investigate the effect and mechanism of miR-451 on invasion and migration of ovarian cancer cells.Methods:The expressions of miR-451 and ROCK1 mRNA in ovarian cancer tissues and cells,paracancerous tissues and normal ovarian epithelialcells were detected by RTqPCR.Ovarian cancer CaOV-3 cells were divided into 6 groups:Control group(non-transfection group),miR-NC group(transfected with miR-NC disordered sequence),miR-451 mimic group(transfected with over-expressed miR-451 mimics),miR-451 inhibitor group(transfected with plasmids inhibiting miR-451 expression),pc-ROCK1 group(transfected with plasmids overexpressing ROCK1)and miR-451+pc-ROCK1 group(co-transfected with miR-451 mimics and plasmids overexpressing ROCK1).The ability of cell migration was detected by scratch test.The invasive ability of cells was detected by Transwell method.The protein expression levels of E-cadherin,N-cadherin,MMP2 and ROCK1 were detected by Western blot.The target gene of miR-451 was predicted by TargetScan database.The targeting relationship between miR-451 and ROCK1 was detected by double luciferase report.Results:Compared with paracancerous tissues,miR-451 expression was down-regulated in most ovarian tissues(57/70,81.43%).Patients with low expression of miR-451 had larger ovarian cancer tumors,poorer differentiation,mostly belonged to the late stage of FIGO,and had obvious peritoneal metastasis(P<0.05).Compared with the Control group,the scratch closure rate of ovarian cancer CaOV-3 cells in the miR-451 mimic group was significantly reduced,the number of invading cells was significantly reduced,E-cadherin was significantly up-regulated,and N-cadherin and MMP2 expression were significantly down-regulated(P<0.01).Compared with the Control group,the scratch closure rate of ovarian cancer CaOV-3 cells in miR-451 inhibitor group was significantly increased,the number of invading cells was significantly increased,E-cadherin was significantly down-regulated,and N-cadherin and MMP2 expression were significantly up-regulated(P<0.01).miR-451 down-regulated ROCK1.Co-transfection of pc-ROCK1 reversed the effect of miR-451 mimic on the invasion and migration of ovarian cancer CaOV-3 cells(P<0.01).Conclusion:MiR-451 inhibits the migration and invasion of ovarian cancer CaOV-3 cells by targeting down-regulation of RCOK1.