微小RNA-21对宫颈癌HeLa细胞程序性细胞死亡4蛋白表达及细胞增殖的影响

Effect of microRNA-21 transfection on PDCD4 expression and cell proliferation in cervical cancer cells

目的:探讨微小RNA-21(miR-21)对宫颈癌HeLa细胞中程序性细胞死亡4(programmed cell death,PDCD4)基因表达及细胞增殖的影响,为应用miR-21对宫颈癌进行基因治疗提供一定的依据。方法:用脂质体转染的方法,将pre-miR-21、pre-miR-21阴性对照、anti-miR-21、anti-miR-21阴性对照瞬时转染HeLa细胞。实时荧光定量PCR(qRT-PCR)法检测miR-21相对表达量;MTT法检测宫颈癌HeLa细胞增殖的变化;蛋白质印迹法检测PDCD4蛋白表达情况;荧光素酶报告基因法验证miR-21的靶位点。结果:pre-miR-21上调miR-21的表达,抑制PDCD4蛋白表达,促进HeLa细胞增殖;而anti-miR-21下调miR-21的表达,增加PDCD4蛋白的表达,抑制HeLa细胞增殖。pMIR-Luc-PDCD4-3'UTR与pre-miR-21共转染后,荧光素酶活性下降,而与anti-miR-21共转染后,荧光素酶活性升高。结论:PDCD4基因是miR-21的靶基因;miR-21通过调节PDCD4基因的表达调控细胞增殖。

Objective： To investigate the effect of miR-21 transfection on the expression of programmed cell death 4（PDCD4） and cell proliferation in cervical cancer cell line HeLa cells,and to provide experimental foundation for miR-21 gene therapy of cervical cancer.Methods： Lipid-mediated transfection was used to transfect pre-miR-21,pre-miR-21 negative control and anti-miR-21,anti-miR-21 negative control into HeLa cervical cancer cells.The miR-21 expression was measured by quantitative real-time PCR（qRTPCR）.The cellular growth activity was determined by MTT assay.The protein expression of PDCD4 was measured by western blotting.Detection of the target sites of miR-21 was analyzed by luciferase report gene assays.Results： In pre-miR-21 transfected cells,the cellular growth activity increased,and the expression of PDCD4 protein decreased.Conversely,in anti-miR-21 transfected cells,the cellular growth activity decreased,and the expression of PDCD4 protein increased.pMIR-Luc-PDCD4-3＇ UTR co-transfected with pre-miR-21,luciferase activity decreased;while it co-transfected with anti-miR-21,luciferase activity increased.Conclusion： PDCD4 gene was the target gene of miR-21,which suggested the potential possibility of miR-21 as a target gene for cervical cancer therapy.