miR-124-3p靶向FOXQ1抑制宫颈鳞状细胞癌细胞增殖和侵袭的机制研究

目的:探讨miR-124-3p在宫颈鳞状细胞癌（cervical squamous cell carcinoma,CSCC）中的表达水平及对细胞增殖、侵袭能力的影响，初步阐明miR-124-3p对癌基因叉头框蛋白Q1(forkhead box Q1,FOXQ1)的靶向调控机制。方法:收集2015年1月至2017年12月手术切除的CSCC组织标本61份,分别应用RT-qPCR和IHC检测癌组织和癌旁组织中miR-124-3p和FOXQ1蛋白的表达水平，分析miR-124-3p和FOXQ1 mRNA表达的相关性；RT-qPCR检测miR-124-3p和FOXQ1 mRNA在人CSCC细胞株（SiHa和CaSki）及人宫颈永生化鳞状细胞株（Ect1/E6E7）中的表达水平；应用miR-124-3p模拟物转染CaSki细胞，分别采用CCK-8法和Transwell小室检测miR-124-3p对细胞增殖和侵袭能力的影响；采用Western blot检测miR-124-3p对FOXQ1、E-cadherin和Vimentin蛋白表达水平的影响；最后，应用双荧光素酶报告基因验证miR-124-3p对FOXQ1的靶向调控作用。结果:miR-124-3p在CSCC组织的表达水平低于癌旁组织（P＜0.05），在人CSCC细胞株（SiHa和CaSki）中的表达水平也均低于人宫颈永生化鳞状细胞株（Ect1/E6E7）（P＜0.05）；与转染阴性对照细胞比较，转染miR-124-3p模拟物的CaSki细胞的增殖和侵袭能力明显受到抑制（P＜0.05）。FOXQ1 mRNA和蛋白在CSCC组织中的表达水平均显著高于癌旁组织(P＜0.05)，相关性分析显示，FOXQ1 mRNA的表达水平与miR-124-3p呈负相关(r=-0.882,P＜0.05)。在转染miR-124-3p模拟物后，CSCC细胞株CaSki中FOXQ1和Vimentin蛋白表达水平均降低，而E-cadherin蛋白表达增高。双荧光素酶报告基因试验确认miR-124-3p可通过与FOXQ1 mRNA的3' UTR直接结合，靶向调控FOXQ1的表达。结论:miR-124-3p在CSCC中表达下调，其通过靶向调控FOXQ1的表达影响细胞的上皮间质转化状态，从而影响CSCC细胞的增殖和侵袭。

miR-124-3p inhibits cell proliferation and invasion in cervical squamous cell carcinoma by targeting FOXQ1

Objective:To investigate the expression level of miR-124-3p in cervical squamous cell carcinoma (CSCC) and its effect on cell proliferation and invasion,and to preliminarily clarify the mechanism of miR-124-3p targeting the forkhead box Q1 (FOXQ1).Methods:A total of 61 CSCC specimens were collected from January 2015 to December 2017.Expressions of miR-124-3p and FOXQ1 protein in cancer tissues and paired adjacent tissues were detected by RT-qPCR and IHC,respectively,and the correlation between miR-124-3p and FOXQ1 mRNA expression was analyzed.RT-qPCR was used to detect the expression of miR-124-3p and FOXQ1 mRNA in human CSCC lines (SiHa and CaSki) and human immortalized squamous cell lines (Ect1/E6E7).CaSki cells were transfected by miR-124-3p mimics,and the effects of miR-124-3p on cell proliferation and invasion were detected by CCK-8 and Transwell assays,respectively.Western blot was used to detect the effect of miR-124-3p on FOXQ1,E-cadherin,and Vimentin protein expression levels.Finally,a dual luciferase reporter gene was used to verify the targeted regulation of miR-124-3p on FOXQ1.Results:The expression of miR-124-3p in CSCC tissues was lower than that in paired adjacent tissues (P＜0.05) and its expression in human CSCC cell lines (SiHa and CaSki) were also lower than those in human cervical immortalized squamous cell lines (Ect1/E6E7) (P＜0.05).Compared with transfected negative control cells,the proliferation and invasion of CaSki cells transfected with miR-124-3p mimics was significantly inhibited (P＜0.05).The expression of FOXQ1 mRNA and protein in CSCC tissues was significantly higher than that in adjacent tissues (P＜0.05),and further correlation analysis showed that the expression level of FOXQ1 mRNA was negatively correlated with miR-124-3p (r=-0.882,P＜0.05).After transfection of miR-124-3p mimics,the expression of FOXQ1 and Vimentin protein in CaSki cells was decreased while the expression of E-cadherin protein was increased.Dual luciferase reporter assays confirmed that miR-124-3p can target FOXQ1 expression through direct binding to the 3' UTR of FOXQ1 mRNA.Conclusion:miR-124-3p is down-regulated in CSCC,which influences the epithelial-mesenchymal transition state of cells through targeted regulation of FOXQ1 expression,thereby affecting the proliferation and invasion of CSCC cells.