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目的通过抑制miRNA-21的表达观察对胆管癌细胞株增殖、凋亡的影响,并探讨miRNA-21可能调控的靶基因。方法以人胆管癌细胞RBE细胞为研究对象,根据处理方法不同,细胞分为四组:正常细胞组、脂质体组(仅予脂质体)、阴性对照组(转染相关阴性对照试剂)、miRNA-21抑制组(转染miRNA-21抑制剂)。应用MTT方法检测细胞增殖活性的变化,应用流式细胞仪检测细胞凋亡的变化,通过荧光定量PCR技术及蛋白质免疫印迹法(Western blot)检测RBE细胞中maspin基因表达的改变。结果 miRNA-21在经转染miRNA-21抑制剂后在RBE细胞中的表达得到有效抑制,荧光定量PCR结果显示与正常细胞相比,其表达下降了0.02倍。MTT结果显示RBE细胞在抑制miRNA-21表达后增殖活性明显受到抑制(P<0.05),流式细胞仪结果显示各组细胞凋亡率分别为2.29%、2.43%、2.89%、19.35%,miRNA-21的表达降低后,RBE细胞的凋亡率明显增高(P<0.05),并且荧光定量PCR及western blot结果显示maspin mRNA及其蛋白的表达明显上调(P<0.05)。结论通过抑制miRNA-21的表达可抑制RBE细胞的增殖,促进RBE细胞的凋亡,miRNA-21可通过靶向抑制maspin的表达发挥生物学作用。
Objective To observe the effect of miRNA-21 on the proliferation and apoptosis of cholangiocarcinoma cell lines and to explore the possible target genes regulated by miRNA-21. Methods The RBE cells of human cholangiocarcinoma cells were used as the research objects. According to the different treatment methods, the cells were divided into four groups: normal cell group, liposome group (liposome only), negative control group (transfection-related negative control reagent) , MiRNA-21 inhibition group (transfected with miRNA-21 inhibitor). The changes of cell proliferation were detected by MTT assay. The changes of apoptosis were detected by flow cytometry. The changes of maspin gene expression in RBE cells were detected by fluorescence quantitative PCR and Western blot. Results The expression of miRNA-21 in RBE cells was inhibited after transfected with miRNA-21 inhibitor. The results of quantitative real-time PCR showed that the expression of miRNA-21 was decreased by 0.02-fold compared with normal cells. The results of MTT showed that the proliferation of RBE cells was significantly inhibited after the miRNA-21 was inhibited (P <0.05). The results of flow cytometry showed that the apoptosis rates of RBE cells were 2.29%, 2.43%, 2.89%, 19.35% (P <0.05), and the expression of maspin mRNA and protein was significantly up-regulated by fluorescence quantitative PCR and western blot (P <0.05). Conclusion Inhibition of miRNA-21 expression can inhibit the proliferation of RBE cells and promote the apoptosis of RBE cells. MiRNA-21 can play a biological role by targeting the inhibition of maspin expression.