Tang Yunlan, Lin Yi, Li Chuang, Hu Xunwu, Liu Yi, He Mingyang, Luo Jun, Sun Guihong, Wang Tao, Li Wenxin, Guo Mingxiong
College of Life Sciences and State Key Laboratory of Virology, Wuhan University, 430072 Wuhan, People's Republic of China.
Department of Pathology, Zhongnan Hospital, Wuhan University, 430071 Wuhan, People's Republic of China.
Cell Biosci. 2015 Sep 25;5:56. doi: 10.1186/s13578-015-0047-5. eCollection 2015.
MicroRNA-720 (miR-720), a nonclassical miRNA, is involved in the initiation and progression of several tumors. In our previous studies, miR-720 was shown to be significantly upregulated in cervical cancer tissues compared with normal cervical tissues. However, the precise biological functions of miR-720, and its molecular mechanisms of action, are still unknown.
Microarray expression profiles, luciferase reporter assays, and western blot assays were used to validate Rab35 as a target gene of miR-720 in HEK293T and HeLa cells. The regulation of Rab35 expression by miR-720 was assessed using qRT-PCR and western blot assays, and the effects of exogenous miR-720 and Rab35 on cell migration were evaluated in vitro using Transwell(®) assay, wound healing assay, and real-time analyses in HeLa cells. The influences of exogenous miR-720 on cell proliferation were evaluated in vitro by the MTT assay in HeLa cells. In addition, expression of E-cadherin and vimentin associated with epithelial-mesenchymal transition were also assessed using western blot analyses after transfection of miR-720 mimics and Rab35 expression vectors. The results showed that the small GTPase, Rab35, is a direct functional target of miR-720 in cervical cancer HeLa cells. By targeting Rab35, overexpression of miR-720 resulted in a decrease in E-cadherin expression and an increase in vimentin expression and finally led to promotion of HeLa cell migration. Furthermore, reintroduction of Rab35 3'-UTR(-) markedly reversed the induction of cell migration in miR-720-expressing HeLa cells.
The miR-720 promotes cell migration of HeLa cells by downregulating Rab35. The results show that miR-720 is a novel cell migration-associated gene in cervical cancer cells.
微小RNA-720（miR-720）是一种非经典微小RNA，参与多种肿瘤的发生和发展。在我们之前的研究中，与正常宫颈组织相比，miR-720在宫颈癌组织中显著上调。然而，miR-720的确切生物学功能及其分子作用机制仍不清楚。
利用基因芯片表达谱、荧光素酶报告基因检测和蛋白质印迹分析，在人胚肾293T细胞（HEK293T）和人宫颈癌细胞（HeLa）中验证Rab35是miR-720的靶基因。采用定量逆转录聚合酶链反应（qRT-PCR）和蛋白质印迹分析评估miR-720对Rab35表达的调控，并在体外利用Transwell（®）实验、划痕愈合实验以及HeLa细胞实时分析评估外源性miR-720和Rab35对细胞迁移的影响。通过MTT实验在体外评估外源性miR-720对HeLa细胞增殖的影响。此外，在转染miR-720模拟物和Rab35表达载体后，还利用蛋白质印迹分析评估了与上皮-间质转化相关的E-钙黏蛋白和波形蛋白的表达。结果表明，小GTP酶Rab35是宫颈癌HeLa细胞中miR-720的直接功能靶标。通过靶向Rab35，miR-720的过表达导致E-钙黏蛋白表达降低和波形蛋白表达增加，最终促进HeLa细胞迁移。此外，重新导入Rab35 3'-UTR（-）可显著逆转miR-720表达的HeLa细胞中细胞迁移的诱导。
miR-720通过下调Rab35促进HeLa细胞迁移。结果表明，miR-720是宫颈癌细胞中一个新的与细胞迁移相关的基因。
