miR-10a-5p通过靶向RORA促进胃癌细胞侵袭、迁移

The miR-10a-5p promotes invasion and migration of gastric cancer cells by targeting RORA

目的探讨miR-10a-5p是否通过靶向维甲酸受体相关孤儿受体A(RAR-related orphan receptor A,RORA)对胃癌细胞侵袭、迁移产生影响及其作用机制。方法qRT-PCR检测人正常胃黏膜细胞GES-1与三株胃癌细胞株AGS、SGC-7901、BGC-823中miR-10a-5p的表达水平。以miR-10a-5p高表达的胃癌细胞株SGC-7901作为后续实验研究胃癌细胞株,分为control组(只转染Lipofectamine 2000)、NC组(转染Lipofectamine 2000和NC序列,即转染无关序列)和miR-10a-5p inhibitor组(转染Lipofectamine 2000和miR-10a-5p inhibitor),qRT-PCR检测转染后各组细胞株中miR-10a-5p的表达水平。生物信息学网站Target Scan预测miR-10a-5p与RORA的靶向结合位点。qRT-PCR、Western blot实验分析miR-10a-5p与RORA的靶向调控关系。集落克隆和Transwell实验分别检测下调miR-10a-5p对SGC-7901细胞增殖和侵袭迁移能力的影响。为探究RORA基因对胃癌细胞侵袭、迁移能力的影响,实验分为control组(只转染Lipofectamine 2000)、RORA NC转染组(转染Lipofectamine 2000及NC序列)、RORA过表达组(转染Lipofectamine 2000及RORA过表达序列),Transwell实验检测上调RORA对SGC-7901细胞侵袭迁移能力的影响。Western blot法检测侵袭、迁移相关蛋白E-cadherin、N-cadherin、Snail的相对表达水平。结果miR-10a-5p在胃癌细胞株中相对于正常胃黏膜细胞中高表达(P<0.05)。生物信息学软件Target Scan预测结果显示,RORA 3′UTR上存在潜在与miR-10a-5p靶向结合的位点(P<0.05),miR-10a-5p能够负向调控RORA基因表达。下调miR-10a-5p后SGC-7901细胞的增殖、侵袭和迁移显著被抑制(P<0.05)。过表达RORA能抑制SGC-7901细胞侵袭、迁移能力(P<0.05)。过表达RORA后N-cadherin、Snail蛋白表达降低,E-cadherin表达增加(P<0.05)。结论miR-10a-5p通过靶向RORA促进胃癌细胞侵袭、迁移,侵袭、迁移机制可能与上皮间质转化有关。

Objective To explore whether miR-10a-5p affects the invasion and migration of gastric cancer cells by targeting the retinoic acid receptor-related orphan receptor A(RORA).Methods The qRT-PCR was used to detect the expression levels of miR-10a-5p in human normal gastric mucosa cells GES-1 and different gastric cancer cell lines.The gastric cancer cell line SGC-7901 with high expression of miR-10a-5p was used as the following experiment.SGC-7901 cells were divided into control group(only transfected with Lipofectamine 2000),NC group(transfected with Lipofectamine 2000 and NC sequence)and miR-10a-5p inhibitor group(transfected with Lipofectamine 2000 and miR-10a-5p inhibitor),and then qRT-PCR was used to detect the expression level of miR-10a-5p in each group after transfection.The bioinformatics website Target Scan was used to predict the targeted binding site of miR-10a-5p and RORA.Western blot and qRT-PCR were used to analyze the relationship between miR-10a-5p and RORA.Colony cloning and Transwell experiments were used to detect the effects of down-regulation of miR-10a-5p on the proliferation,invasion and migration abilities of SGC7901 cells.SGC-7901 cells were divided into control group(only transfected with Lipofectamine 2000),RORA NC transfection group(transfected with Lipofectamine 2000 and NC sequence),RORA overexpressing group(transfected with Lipofectamine 2000 and overexpressing sequence),and then Transwell test was used to observe the effect of upregulation of RORA on SGC-7901 cell invasion and migration abilities.Western blot was used to detect the relative expression levels of invasion-and migration-related proteins E-cadherin,N-cadherin and Snail.Results The expression of miR-10a-5p was higher in gastric cancer cell lines than in normal gastric mucosal cells(P<0.05).Bioinformatics software Target Scan predicted that RORA 3′UTR had potential sites for miR-10a-5p targeted binding(P<0.05),and that miR-10a-5p was able to negatively regulate RORA gene expression.After down-regulation of miR-10a-5p,the proliferation,the invasion and the migration of SGC-7901 cells were significantly inhibited(P<0.05).After overexpression of RORA,the invasion and migration abilities of SGC-7901 cells were inhibited(P<0.05),N-cadherin and Snail protein expression was decreased(P<0.05),while E-cadherin expression was increased(P<0.05).Conclusion The miR-10a-5p can promote the invasion and the migration of gastric cancer cells by targeting RORA,which may be related to the epithelial-mesenchymal transition.