miR-1246通过靶向GSK3β促进食管癌转移的机制

目的:探讨miRNA-1246(miR-1246)促进食管癌细胞转移的作用及其机制.方法:Realtime-PCR法检测miR-1246在癌旁组织、食管癌组织、正常食管上皮和食管癌细胞系中的表达差异.Transwell侵袭实验观察转染miR-1246 mimics或inhibitors对食管癌细胞EC109转移能力的影响;裸鼠尾静脉转移实验观察稳定表达miR-1246对食管癌转移能力的影响;生物信息学分析miR-1246的候选靶基因为GSK3β,荧光素酶报告基因实验检测过表达miR-1246后EC109细胞中野生型和突变型GSK3β的荧光素酶活性.Westernblot检测miR-1246对GSK3β蛋白表达的影响.结果:食管癌组织中的miR-1246表达显著高于癌旁组织（P<0.05)；多个食管癌细胞中miR-1246的表达比正常食管上皮细胞Het-1A明显增高（P<0.05).与阴性对照相比,miR-1246 mimics显著促进EC109细胞的侵袭能力（P<0.05).反之,miR-1246 inhibitors明显抑制EC109细胞的侵袭能力（P<0.05)；体内实验发现稳定过表达miR-1246明显升高EC109细胞的肺转移能力.荧光素酶报告基因结果证实miR-1246能够抑制GSK3β的3'-UTR区荧光素酶活性；转染miR-1246后,EC109细胞中的GSK3β蛋白水平明显低于对照组；miR-1246过表达显著抑制GSK3β-wt,而不能抑制GSK3β-mut的蛋白表达.结论:miR-1246能够通过靶向GSK3β促进食管癌转移,抑制miR-1246的表达或增加GSK3β的表达可能是抑制食管癌转移的有效手段.

miR-1246 promotes esophageal cancer metastasis by targeting GSK3β

Objective:To investigate the role and mechanisms of miRNA-1246(miR-1246) in promoting esophageal cancer metastasis.Methods:Realtime-PCR was used to evaluate miR-1246 expression between esophageal cancer tissues and paired adjacent normal tissues,esophageal cancer cell lines and normal esophageal epithelial cells.After transfection of miR-1246 mimics or inhibitors,transwell invasion assay was applied to the effect of miR-1246 in esophageal cancer cell metastasis.Metastasis model in nude mice was used to observe the effect of miR-1246 overexpression in metastasis of esophageal cancer in vivo.Bioinformatics analysis revealed GSK3β as a candidate target genes of miR-1246.Luciferase activity of the luciferase reporter assay was used to detect the effect of miR-1246 on wild-type and mutant GSK3β in EC109 cells.Western blot was used to evaluate whether miR-1246 could affect protein expression of GSK3β and its mutant.Results:The expression of miR-1246 in esophageal cancer tissues was significantly elevated,compared with paired adjacent normal tissues(P<0.05).miR-1246 expression level in the esophageal cancer cells was significantly elevated,compared with normal esophageal epithelial cell Het-1A(P<0.05).miR-1246 mimics significantly increased invasion of EC109 cells,compared with the negative control(P<0.05).While miR-1246 inhibitors significantly inhibited invasion of EC109 cells(P<0.05).Stable expression of miR-1246 in EC109 cells demonstrated an increased invasion capacity in vivo.Luciferase reporter assay showed that miR-1246 inhibited luciferase activity of GSK3β by targeting its 3'-UTR region.Western blot assay showed EC109 cells transfected with miR-1246 significantly decreased GSK3β expression,compared with the negative control.Western blotting showed that overexpression of miR-1246 significantly inhibited the expression of GSK3β-wt,but not GSK3β-mut.Conclusion:MiR-1246 promotes esophageal cancer metastasis by targeting GSK3β.Inhibition of miR-1246 expression or GSK3β restoration may be effective for the treatment of esophageal cancer metastasis.