hsa-miRNA-30a-5p促进肺癌细胞A549增殖的机制研究
投稿时间:2018-12-11  修订日期:2019-04-03  点此下载全文
引用本文:周静,吴杲,马福家.hsa-miRNA-30a-5p促进肺癌细胞A549增殖的机制研究[J].药学实践杂志,2019,37(5):433~439
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作者单位E-mail
周静 宁波市妇女儿童医院, 浙江 宁波, 315012  
吴杲 海军军医大学附属长海医院虹口院区, 上海 200081  
马福家 海军特色医学中心药剂科, 上海 200052 mfujia@126.com 
中文摘要:目的 研究hsa-miRNA-30a-5p促进肺癌细胞A549增殖的调控机制。方法 收集临床肺癌标本及癌旁组织5对,用荧光定量法(real time-PCR)和蛋白质印迹法(Western blotting)分别检测肿瘤及其癌旁组织中hsa-miRNA-30a-5p和SCARA5的蛋白含量;生物信息学预测hsa-miRNA-30a-5p与SCARA5基因3'UTR区结合位点,并通过荧光素酶报告基因法进行结合位点验证;构建SCARA5基因沉默表达载体pshRNA-SCARA5,脂质体瞬时转染pshRNA-SCARA5及hsa-miRNA-30a-5p阻遏物(miRNA-30a-5p inhibitor)到A549细胞,转染后48 h,real time-PCR检测细胞内hsa-miRNA-30a-5p含量,Western blotting检测细胞中SCARA5蛋白含量;MTT法检测转染后A549细胞对数期增殖活性。结果 肺癌组织中SCARA5蛋白表达量明显低于癌旁组织,组间差异显著(P<0.05);肺癌组织中hsa-miRNA-30a-5p含量则明显高于癌旁组织,组间差异显著(P<0.05)。荧光素酶实验显示,与报告基因表达载体单独转染组比较,miRNA-30a拟似物(miRNA-30a-5p mimics)和miRNA-30a-5p inhibitor与野生型荧光素酶报告基因共转染组可以明显抑制或增强荧光素酶活性(P<0.05);miRNA-30a-5p inhibitor转染细胞后48 h,与转染对照组比较,细胞内hsa-miRNA-30a-5p含量明显降低(P<0.05),阴性对照(miRNA-30a-5p NC)转染组和转染对照组细胞内hsa-miRNA-30a-5p含量无显著差异(P>0.05)。转染后24~72 h,miRNA-30a-5p inhibitor转染组A549细胞增值活性明显降低(P<0.05),而pshRNA-SCARA5转染能够逆转miRNA-30a-5p inhibitor增强A549细胞增殖活性的趋势,共转染组与miRNA-30a-5p inhibitor单独转染组及转染对照组比较,差异有统计学意义(P<0.05)。结论 Hsa-miRNA-30a-5p通过抑制SCARA5基因表达,增强A549细胞的增殖活性,转染miRNA-30a-5p inhibitor,可以抑制A549细胞增殖活性。
中文关键词:SCARA5  A549细胞  hsa-miRNA-30a-5p  增殖活性
 
Study on the mechanism of proliferation of human lung cancer A549 cells regulated by hsa-miRNA-30a-5p
Abstract:Objective To study the regulation and mechanism of hsa-miRNA-30a-5p on the proliferation of human lung cancer A549 cells.Methods Five pairs of lung cancer and para-carcinoma tissues were harvested in clinical and measured for hsa-miRNA-30a-5p and SCARA5 levels by real-time PCR and western blotting,respectively.The theoretical binding site of hsa-miRNA-30a-5p in SCARA5's 3'UTR was predicted by bioinformatics,and validated by luciferase report assay.A549 cells were transfected with pshRNA-SCARA5 and miRNA-30a-5p inhibitor and 48 h later,the proliferation of A549 cells after genetic intervention was assayed by the MTT method,and the cell growth curve was plotted to observe the effect of hsa-miRNA-30a-5p knockdown on cell proliferation.Results For all five pairs of samples tested,hsa-miRNA-30a-5p was higher in the cancer tissues than in the adjacent tissue(P<0.05),and SCARA5 protein was lower in the cancer tissues than in the adjacent tissue (P<0.05).The bioinformatics analysis showed that there was a theoretical binding site of hsa-miRNA-30a-5p in SCARA5's 3'UTR.The luciferase assay showed that miRNA-30a mimics inhibited the luciferase expressed by the luciferase reporter vector carrying a wild-type SCARA5's 3'UTR(pGL3-WT- SCARA5)(P<0.05,vs pGL3-WT-SCARA5 alone),and miRNA-30a-5p inhibitor enhanced the luciferase activity(P<0.05,vs pGL3-WT- SCARA5 alone).No change was observed when miRNA-30a-5p mimics or miRNA-30a-5p inhibitor was co-transfected with the luciferase reporter vector carrying a mutant SCARA5's 3'UTR(pGL3-MT- SCARA5)(P>0.05,vs pGL3-WT- SCARA5 alone).Hsa-miRNA-30a-5p level in A549 cells transfected with miRNA-30a inhibitor for 48 h was significantly decreased(P<0.05,vs cell control or NC),and there was no difference in hsa-miRNA-30a-5p between the negative control and the transfection control(P>0.05).The proliferation of A549 cells was suppressed by transfection with miRNA-30a-5p inhibitor 24-72 h after transfection(P<0.05,vs control or NC).Conclusion Hsa-miRNA-30a-5p could increase the proliferation in A549 cells via suppressing the expression of SCARA5,and transfection of miRNA-30a-5p inhibitor could inhibit the proliferation of A549 cells via up-regulating SCARA5 expression.
keywords:SCARA5  A549  hsa-miRNA-30a-5p  proliferation activity
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