抑制miRNA-21表达对胆管癌细胞凋亡、侵袭的影响及其靶基因探讨

Effects of suppressing the expression of miRNA-21 on the apoptosis and invasion abilities of cholangiocarcinoma cells and its target gene

目的探讨抑制miRNA-21表达对胆管癌细胞株凋亡、侵袭的影响及其可能调控的靶基因。方法通过RNAi技术抑制胆管癌细胞株RBE中miRNA-21的表达并加以验证。通过荧光定量PCR技术及蛋白质免疫印迹法（Westernblot）检测RBE细胞中RECK基因表达的改变。应用流式细胞仪检测细胞凋亡的变化。用体外侵袭实验分析胆管癌细胞侵袭能力的改变。结果miRNA-21在胆管癌细胞株RBE中的表达得到有效抑制，并且RECK基因及其蛋白的表达明显上调。流式细胞仪显示RBE细胞凋亡率明显增高，且抑制miRNA-21的表达后胆管癌细胞的侵袭性明显下降。结论抑制miRNA-21的表达可促进RBE细胞的凋亡，并降低其侵袭能力，RECK作为miRNA-21的靶基因参与调控过程。

Objective To discuss the effects of apoptosis and invasion of RBE cells caused by miRNA 21 suppression and further investigate the potential role of miRNA-21 plays on target mRNA regulation. Methods The RNAi technology was employed to suppress the expression of RBE ceils. The changes in RECK mRNA and protein expressions were detected by RT-PCR and Western blotting respectively. Changes occurred in apoptosis was closely monitored by flow cytometry （FCM）. The invasion of RBE cells was analyzed in vitro by invasion assay （transwell）. Results The expression of miRNA-21 was clearly suppressed while the RECK mRNA and protein were over-expressed. The rate of apoptosis was significantly accelerated and there was a dramatic decrease in RBE cells＇ ability to invade after miRNA-21 knockdown. Conclusion Through miRNA-21 suppression, the rate of apoptosis of RBE cells was accelerated whereas their invasion ability was greatly reduced. RECK was found to be the target gene of miRNA-21 which participates in the regulation process of regulation.