Hsa-miR-218靶向调控LASP1对宫颈癌HeLa细胞生长的影响

发布时间：2018-11-18 06:31

【摘要】：目的:探讨人微小RNA-218(Homo sapiens microRNA-218,hsa-miR-218)对宫颈癌HeLa细胞生长的影响及分子机制。方法:构建hsa-miR-218的重组慢病毒表达载体pmiR-218,并将pmiR-218感染至HeLa细胞,台盼蓝拒染法检测细胞数量的变化,WST-8法检测细胞活力,构建萤光素酶报告基因载体验证miR-218与LIM和SH3蛋白1(LASP1)的相互结合作用,real-time PCR检测LASP1的mRNA相对表达水平,Western blot检测LASP1蛋白的表达水平。结果:经DNA测序证实与设计完全一致,测序结果显示成功构建了重组慢病毒表达载体pmiR-218。pmiR-218转染HeLa细胞72 h后HeLa细胞存活数量为176±9,对HeLa细胞活力的抑制率具有时间效应关系,较空白对照组比较,差异显著(P0.05);萤光素酶活性结果显示,克隆LASP1-3’UTR的质粒与miR-218 mimics共转染293T细胞,引起萤光素酶活性的减低;real-time PCR结果显示转染pmiR-218后,miR-218表达水平增加;过表达miR-128能下调LASP1 mRNA及蛋白的相对表达水平,与空白对照组及阴性对照组比较,差异显著(P0.01)。结论:pmiR-218有效抑制HeLa细胞的生长,并具有时间-效应关系,其机制可能是miR-218通过靶向结合LASP1的3’UTR,从而下调其在HeLa细胞的表达有关。
[Abstract]:Objective: to investigate the effect of human minimal RNA-218 (Homo sapiens microRNA-218,hsa-miR-218 on the growth of cervical cancer HeLa cells and its molecular mechanism. Methods: the recombinant lentivirus expression vector pmiR-218, of hsa-miR-218 was constructed and pmiR-218 was infected into HeLa cells. Trypan blue exclusion assay was used to detect cell number and WST-8 method was used to detect cell viability. The luciferase reporter gene vector was constructed to verify the interaction of miR-218 with LIM and SH3 protein 1 (LASP1). The mRNA relative expression level of LASP1 was detected by real-time PCR and the expression level of LASP1 protein was detected by, Western blot. Results: the results of DNA sequencing showed that the recombinant lentivirus expression vector pmiR-218.pmiR-218 was successfully constructed, and the survival number of HeLa cells was 176 卤9 after 72 h transfection. There was a time-effect relationship on the inhibition rate of HeLa cell viability, compared with the blank control group, the difference was significant (P0.05). The results of luciferase activity showed that the plasmids of LASP1-3'UTR cloned into 293T cells were cotransfected with miR-218 mimics, which resulted in the decrease of luciferase activity, and real-time PCR showed that the expression level of miR-218 increased after transfection of pmiR-218. Overexpression of miR-128 could down-regulate the relative expression of LASP1 mRNA and protein, compared with the control group and negative control group, the difference was significant (P0.01). Conclusion: pmiR-218 can effectively inhibit the growth of HeLa cells and has a time-effect relationship. The mechanism may be that miR-218 can down-regulate the expression of miR-218 in HeLa cells by targeting LASP1.
【作者单位】： 吉安市妇幼保健院妇产科;广州市南吉生物技术有限公司;广州市番禺区中心医院检验科;
【基金】：广州市番禺区科技局珠江科技新星项目(No.2013-专-15-6.09)
【分类号】：R737.33

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9 徐t，

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