miR-760靶向c-Myc对人食管鳞状细胞癌TE-10细胞生物学特性的影响

Effects of miR-760 targeting c-Myc on biological characteristics of human esophageal squamous cell carcinoma TE-10 cells

目的·观察miR-760对人食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)TE-10细胞增殖、凋亡、迁移和侵袭的影响并初步探究其机制。方法·应用反转录–定量聚合酶链反应(RT-qPCR)和Western blotting检测miR-760和c-Myc在5个ESCC细胞株(正常食管鳞状上皮细胞作为对照)和14例ESCC组织(癌旁组织作为对照)标本中的mRNA与蛋白表达水平。使用miR-760的模拟物、抑制剂(miR-mimics组、miR-inhibitor组)及模拟物阴性对照、抑制剂阴性对照(mimics-NC组、inhibitor-NC组)转染TE-10细胞,通过RT-qPCR验证miR-760的过表达和抑制效率以及c-Myc在mRNA水平的表达变化。CCK-8和集落形成实验检测miR-760对TE-10细胞增殖能力的影响;流式细胞术检测细胞周期分布及凋亡比例的变化;Western blotting分析c-Myc蛋白及细胞周期、凋亡、迁移和侵袭等相关蛋白表达水平的变化。通过双荧光素酶实验验证miR-760与c-Myc的靶向结合作用。结果·5个ESCC细胞株的miR-760表达水平均低于正常食管鳞状上皮细胞,而c-Myc mRNA和蛋白的表达水平高于正常食管鳞状上皮细胞;在癌组织中miR-760的表达水平较癌旁组织降低,而c-Myc mRNA和蛋白的表达水平较癌旁组织升高。与mimics-NC组相比,miRmimics组的细胞增殖能力降低,细胞形成的集落数目减少,阻滞在G1期的细胞和凋亡细胞的比例均明显增加,迁移和侵袭细胞的数量减少;miR-mimics组周期蛋白D1、B细胞淋巴瘤2蛋白、基质金属蛋白酶2和波形蛋白的表达水平降低,而裂解型半胱氨酸蛋白酶3、上皮钙黏素和β-连环蛋白的表达水平升高;miR-inhibitor组则呈现与miR-mimics组相反的结果。双荧光素酶实验证实了miR-760与c-Myc 3'非翻译区具有靶向结合作用。结论·miR-760可能通过靶向c-Myc抑制TE-10细胞的增殖、迁移和侵袭,并诱导其凋亡。

Objective·To detect the effects of miR-760 on proliferation,apoptosis,migration and invasion of human esophageal squamous cell carcinoma(ESCC)TE-10 cells and to analyse the underlying mechanism.Methods·The mRNA and protein expression levels of miR-760 and c-Myc in five ESCC cell lines(normal esophageal epithelial cells as control)and 14 pieces of ESCC tissue specimens(paracancerous tissues as control)were detected by using reverse transcription quantitative PCR(RT-qPCR)and Western blotting.TE-10 cells were transfected with miR-760 mimics,miR-760 inhibitor(miRmimics/miR-inhibitor group)and corresponding negative controls(mimics-NC/inhibitor-NC group),in which the overexpression and inhibition efficiency of miR-760 and the expression of c-Myc were verified by RT-qPCR.The effect of miR-760 on the proliferation of TE-10 cells was assessed by CCK-8 and colony formation assay.Changes of cell cycle distribution and proportion of apoptotic cells were measured by flow cytometry.Expression levels of cell cycle-,apoptosis-,migration-,and invasion-associated proteins as well as c-Myc were analyzed by Western blotting.The targeting relationship between miR-760 and c-Myc was verified by using the dual luciferase reporter assay.Results·The expressions of miR-760 were down-regulated and the expressions of c-Myc mRNA and protein were up-regulated in five ESCC cell lines compared with those in the normal esophageal epithelial cells.In 14 cases of ESCC tissue specimens,the expressions of miR-760 were down-regulated but the expressions of c-Myc were up-regulated compared with those in the cancer-adjacent tissues.The proliferation ability of TE-10 cells in the miR-mimics group was markedly attenuated,and colony numbers were also decreased.Flow cytometry assay showed that the proportion of cells in G1 phase was notably augmented,and the proportion of apoptotic cells was also increased.The miR-mimics group cells had weaker migration and invasion potential compared with mimics-NC group.Western blotting analysis conformed that expression levels of cyclin D1,B cell lymphoma 2,matrix metalloproteinase 2 and vimentin were decreased,but expression levels of cleaved-caspase3,E-cadherin andβ-catenin were elevated.The miR-inhibitor group showed opposite results compared with the miR-mimics group.The dual luciferase reporter assay validated the direct targeted binding of miR-760 to the 3'UTR of c-Myc.Conclusion·miR-760 can suppress proliferation,migration and invasion,and induce apoptosis of TE-10 cells by directly targeting c-Myc.