miR-30a-5p靶向E2F7阻滞A549细胞周期并抑制其增殖

miR-30a-5p Targeted E2F7 to Block A549 Cell Cycle and Inhibit Its Proliferation

目的探究miR-30a-5p靶向E2F7调控非小细胞肺癌细胞周期和增殖的机制。方法通过荧光定量(qRT-PCR)验证miR-30a-5p在非小细胞肺癌A549细胞与人正常肺上皮细胞BEAS-2B中的相对表达水平。在线软件预测了miR-30a-5p的靶基因及其靶向结合位点,双荧光素酶报告基因实验进一步验证miR-30a-5p和E2F7 mRNA 3’UTR区域的靶向关系。其次在A549细胞中分别转染miR-30a-5p模拟物(miR-30a-5p mimics)和miR-30a-5p抑制剂(miR-30a-5p inhibitor),应用CCK-8细胞计数试剂盒检测A549细胞增殖情况,流式细胞术检测A549细胞凋亡和细胞周期。qRT-PCR和免疫印迹检测细胞周期相关基因和肺腺癌相关基因的mRNA和蛋白表达水平。结果miR-30a-5p在A549细胞中表达水平低于BEAS-2B,且miR-30a-5p靶向结合与E2F7 mRNA 3’UTR 514-520位点,抑制E2F7表达。转染miR-30a-5p mimics抑制A549细胞增殖,并促进A549凋亡。将A549细胞周期阻滞在G 1期。调控肺腺癌相关基因TFDP3、CDK1、E2F8、CCNA2和CDC6的mRNA和蛋白表达水平。结论非小细胞肺癌A549细胞中miR-30a-5p通过靶向负调控E2F7的表达,调控TFDP3、CDK1、E2F8、CCNA2和CDC6的mRNA和蛋白表达,抑制A549细胞增殖和细胞周期。

Objective To investigate the mechanism of miR-30a-5p targeting E2F7 in regulating cell cycle and proliferation of non-small cell lung cancer(NSCLC).Methods The relative expression levels of miR-30a-5p in non-small cell lung cancer A549 cells and human normal lung epithelial cells BEAS-2B were verified by quantitative real-time PCR(qRT-PCR).The target gene and binding site of miR-30a-5p were predicted by online software,and the targeting relationship between miR-30a-5p and E2F7 mRNA 3’UTR region was further verified by dual luciferase reporter gene assay.Secondly,A549 cells were transfected with miR-30a-5p mimics and miR-30a-5p inhibitor,and the proliferation of A549 cells was detected by CCK-8 cell counting kit.The apoptosis and cell cycle of A549 cells were detected by flow cytometry.qRT-PCR and Western blot were used to detect the mRNA and protein expression levels of cell cyclin-related genes and lung adenocarcinoma-related genes.Results The expression level of miR-30a-5p in A549 cells was significantly lower than that in BEAS-2B cells,and miR-30a-5p inhibited the expression of E2F7 by targeting the 514-520 site of E2F7 mRNA 3’UTR.Transfection of miR-30a-5pmimics significantly inhibited the proliferation and promoted the apoptosis of A549 cells.A549 cell cycle was arrested at G 1 phase.It also significantly regulated the mRNA and protein expression levels of lung adenocarcinoma related genes TFDP3,CDK1,E2F8,CCNA2 and CDC6.Conclusion miR-30a-5p inhibits the proliferation and cell cycle of non-small cell lung cancer A549 cells by negatively regulating the expression of E2F7 and the mRNA and protein expressions of TFDP3,CDK1,E2F8,CCNA2 and CDC6.