miR-96-5p靶向叉头状转录因子O1对Ishikawa细胞的作用

Effect of miR-96-5p on Ishikawa Cells by Targeting Forkhead Box Transcription Factor O1

【目的】探讨微小RNA(miR)-96-5p通过靶向叉头状转录因子O1(FOXO1)基因对子宫内膜癌细胞增殖和侵袭的影响。【方法】qRT-PCR检测正常组织和子宫内膜癌组织中miR-96-5p和FOXO1 mRNA的表达,West⁃ern blot法检测FOXO1蛋白表达。分析miR-96-5p及FOXO1表达与子宫内膜癌临床病理特征的关系。转染pcD⁃NA、pcDNA-FOXO1、inhibitor NC、miR-96-5p inhibitor、miR-96-5p mimic、miR-96-5p+FOXO1到子宫内膜癌Ishi⁃kawa细胞,分别记为pcDNA组、FOXO1组、inhibitor NC组、miR-96-5p inhibitor组、miR-96-5p组、miR-96-5p+FOXO1组,对照组不进行转染。取稳定转染的各组细胞,CCK-8和Transwell小室检测细胞活力和侵袭能力,West⁃ern blot检测细胞周期蛋白D1(Cyclin D1)、活化多聚ADP核糖聚合酶(cleaved PARP)、p21及波形蛋白的表达水平。TargetScan网站预测miR-96-5p与FOXO1的靶向关系,双荧光素酶报告基因检测细胞荧光素酶活性。Ishikawa细胞分为mimic NC组、miR-96-5p组、inhibitor NC组、miR-96-5p inhibitor组,qRT-PCR检测各转染组miR-96-5p和FOXO1 mRNA的表达,Western blot法检测FOXO1蛋白表达。【结果】子宫内膜癌组织中miR-96-5p的相对表达量较正常组织上调,相反,FOXO1 mRNA和蛋白相对表达量下调(P<0.01)。子宫内膜癌患者中miR-96-5p高表达与病理分级和临床分期呈正相关(P=0.034,P=0.010),与年龄和是否绝经无明显相关性(P=0.370,P=0.166);FOXO1低表达与病理分级和临床分期呈负相关(P=0.023,P=0.007),与年龄和是否绝经无明显相关性(P=0.344,P=0.144)。过表达FOXO1或抑制miR-96-5p表达后,Ishikawa细胞活力明显降低(P<0.05)、侵袭细胞数减少(P<0.01),cyclin D1、Vimentin蛋白表达水平明显降低,而cleaved PARP、p21蛋白表达水平明显升高(P<0.01)。与对照组比较,miR-96-5p组Ishikawa细胞活力明显升高(P<0.05)、侵袭细胞数增加(P<0.01),Cyclin D1、Vimentin蛋白表达水平明显升高,而cleaved PARP、p21蛋白表达水平明显降低(P<0.01)。与miR-96-5p组比较,过表达FOXO1可明显逆转miR-96-5p对Ishikawa细胞活力和侵袭的促进作用(P<0.05)。miR-96-5p靶向并负调控FOXO1的表达(P<0.05)。【结论】miR-96-5p可通过靶向负调控FOXO1促进子宫内膜癌细胞的增殖和侵袭。

【Objective】To study the effect of microRNA-96-5p(miR-96-5p)on the proliferation and invasion of en⁃dometrial cancer cells by targeting forkhead box transcription factor O1(FOXO1)genes.【Methods】The expressions of miR-96-5p and FOXO1 mRNA in normal and endometrial carcinoma tissues were detected by qRT-PCR,and FOXO1 protein expression was detected by Western blot.The relationship between miR-96-5p and FOXO1 expression and clinico⁃pathological features of endometrial carcinoma was analyzed.pcDNA,pcDNA-FOXO1,inhibitor NC,miR-96-5p inhibi⁃tor,miR-96-5p mimic and miR-96-5p+FOXO1 were transfected into endometrial carcinoma Ishikawa cells,which were recorded as pcDNA group,FOXO1 group,inhibitor NC group,miR-96-5p inhibitor group,miR-96-5p group and miR-96-5p+FOXO1 group,respectively.Cells in stable transfection groups were taken,CCK-8 and Transwell chamber were employed to detect cell viability and invasion,and the expression levels of cyclin D1,cleaved PARP,p21 and Vi⁃mentin were measured by Western blot.TargetScan website was used to predict the targeting relationship between miR-96-5p and FOXO1,and dual luciferase reporter gene was used to detect cell luciferase activity.Ishikawa cells were divided in⁃to mimic NC group,miR-96-5p group,inhibitor NC group and miR-96-5p inhibitor group.The expressions of miR-96-5p and FOXO1 mRNA were detected by qRT-PCR,and the protein expression of FOXO1 was detected by Western blot.【Results】The relative expression level of miR-96-5p in endometrial carcinoma tissues was up-regulated compared with that in normal tissues.On the contrary,the relative expression levels of FOXO1 mRNA and protein were down-regulated(P<0.01).The high expression of miR-96-5p was positively correlated with pathological grade and clinical stage(P=0.034,P=0.010),but not significantly correlated with age and menopause in endometrial carcinoma patients(P=0.370,P=0.166).The low expression of FOXO1 was negatively correlated with pathological grade and clinical stage(P=0.023,P=0.007),but not significantly correlated with the age and menopause in endometrial cancer patients(P=0.344,P=0.144).After FOXO1 overexpression or inhibition of miR-96-5p expression,the viability of Ishikawa cells decreased significantly(P<0.05),the number of invasive cells decreased(P<0.01),the expression levels of cyclin D1 and Vimentin proteins decreased significantly,and the expression levels of cleaved PARP and p21 proteins increased significantly(P<0.01).Compared with the control group,the viability of Ishikawa cells was significantly increased(P<0.05),the number of in⁃vasive cells was increased(P<0.01),the expression levels of cyclin D1 and Vimentin were significantly increased,and the expression levels of cleaved PARP and p21 were significantly decreased in miR-96-5p group(P<0.01).Compared with the miR-96-5p group,overexpression of FOXO1 could significantly reverse the promotion of miR-96-5p on the via⁃bility and invasion of Ishikawa cells(P<0.05).miR-96-5p targeted and negatively regulated FOXO1 expression(P<0.05)【.Conclusion】miR-96-5p promotes the proliferation and invasion of endometrial cancer cells by targeting and nega⁃tively regulating FOXO1.