Hsa-miR202通过下调IL-10的表达抑制肺癌A549细胞的增殖
Hsa-mir-202 inhibit the proliferation of lung cancer A549 cells by reducing expression of IL-10
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摘要:目的 探讨Hsa-miR202(人微小非编码RNA-202)对肺癌A549细胞增殖的影响及分子机制。方法 将Hsa-miR-202序列与ppG/miR/eGFP/Blasitidin质粒定向连接, 构建靶向Hsa-miR-202基因的真核表达载体pmiR-202, 运用脂质体将pmiR-202转染至A549细胞, WST-8法检测细胞增殖率, RT-PCR检测IL-10、miR-202的相对表达水平, Western blot与ELISA检测IL-10蛋白的表达水平, 构建荧光素酶报告基因载体验证miR-202与IL-10的相互结合作用。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miR-202的真核表达载体(pmiR-202), pmiR-202对A549细胞的增殖抑制率(24、48、72 h)分别为12%、38%、52%, 并具有时间效应关系, 较空白对照组及阴性对照组比较, 差异有统计学意义(P < 0.05);RT-PCR结果显示, 转染pmiR-202后, miR-202的表达水平增加; 过表达miR-202能下调IL-10基因及蛋白的相对表达水平, 其相对水平分别为25%、0.75, IL-10活性的相对表达量为3.26±0.43 pg/mL, 较空白对照组及阴性对照组比较, 差异有统计学意义(P < 0.05):荧光素酶活性结果显示, 克隆IL-10-3'UTR的质粒与miR-202 mimics共转染293T细胞, 引起荧光素活性的减低。结论 pmiR-202有效抑制A549细胞的增殖, 并具有时间-效应关系, 其机制可能是miR-202通过靶向结合IL-10的3'UTR从而下调其在A549细胞的表达有关。Abstract:Objective To study the effects of the overexpression of hsa-miR-202 on the proliferation and molecular mechanism of lung cancer A549 cells.Methods A sequence of hsa-miR-202 with ppG/miR/eGFP/Blasitidin pasmid was directionally connected and a eukaryotic expression vector pmiR-202 of the target hsa-miR-202 gene was constructed.pmiR-202 was transtected to A549 cell with Lipofectamine 2000.The WST assay was used to detect the cell proliferation rate, and RT-PCR was used to detect the relative gene expression levels.Western blot analysis was used to detect the IL-10 protein expression levels.The interaction between miR-202 and IL-10 was examined using a luciferase reporter assay.Results The design from the DNA sequencing results shows that a eukaryotic expression vector of miR-202 was successfully constructed.The proliferation inhibition rates of A549 cells by Pmir-202 were 12%, 38%, and 52%.The differences in the treatment group compared with the blank control and negative control groups were statistically significant.The RT-PCR results showed that the relative expression levels of miR-202 increased after transfection with pmiR-202.Overexpression of miR-202 can downregulate the relative gene and protein expression levels of IL-10, and the relative levels were 25% and 0.75, respectively.Compared with the blank control and the negative control groups, the difference was statistically significant (P < 0.05).The fluorescent activity was reduced when transfection was performed with miR-202 mimics, and IL-10-3'UTR plasmid was cloned.Conclusion pmiR-202 effectively inhibited the proliferation of A549 cells and exhibited a time-effect relationship with miR-202 by targeted combination with IL-10 3'UTR to downregulate IL-10 expression in A549 cells.
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