siRNA-SRF对人肺癌A549增殖和侵袭的影响

The effects of siRNA-SRF on proliferation and invasion in NSCLC A549 cells in vitro

目的研究siRNA-SRF对肺癌A549增殖和侵袭能力的影响。方法应用RNAi技术基因沉默SRF,观察其对肺癌A549细胞增殖和侵袭的影响。设计并合成4条siRNA(SRF-900,SRF-901,SRF-1107和SRF-1649)。采用Realtime PCR筛选沉默效率最高的siRNA用于后续研究,采用免疫细胞化学染色和western blot进行验证。采用CCK-8法检测细胞增殖,流式细胞仪检测细胞周期,transwell小室检测细胞侵袭能力。结果设计合成的4条针对SRF的siRNA(900、901、1107、1649)均能有效下调A549细胞SRF mRNA的表达,分别为空白对照组的56.4%、36.4%、25.0%和53.6%,差异有统计学意义(P<0.05)。免疫细胞化学染色显示SRF定位于细胞核,应用siRNA后SRF阳性染色显著减少,Western blot结果显示转染siRNA后SRF蛋白下调为空白对照组的40.1%,差异有统计学意义(P<0.05)。与空白对照组、阴性对照组和转染对照组相比,siRNA-1107转染组细胞增殖明显受抑,抑制率为64.24%,细胞周期S期比例明显下调,侵袭细胞数为空白对照组的41.7%,差异有统计学意义(P<0.05)。结论采用RNA干扰技术基因沉默SRF能够显著抑制A549细胞增殖和侵袭能力。

Objective To investigate the effects of siRNA-SRF on proliferation and invasion in NSCLC A549 cells in vitro.Methods RNAi gene silencing techniue was used to silence SRF,and its effects on the proliferation and invasion of NSCLC A549 cells were observed in vitro,and four siRNAs(SRF-900,SRF-901,SRF-1107 and SRF-1649)were constructed.The transfection efficiency of plasmid vector was evaluated by calculating the ratio of fluorescent cells to total cells by Laser confocol scan microscopy.The best silencing rate of siRNA-SRF was selected by Realtime PCR.Moreover the cell proliferation ability,cell cycle and cell invasion ability of A549 cells were detected by CCK-8,flow cytometry and transwell analysis,respectively.Results The synthetical four siRNAs(SRF-900,SRF-901,SRF-1107 and SRF-1649)could effectively down-regulate the expression levels of SRF mRNA of A549 cells by 56.4%,36.4%,25.0%,53.6%,respectively,as compared with those in control group(P<0.05).Immunocytochemical assay showed that that the positive expressions of SRF were observed in nuclear of A549 cells,which were markedly reduced after treated by siRNA-1107,as compared with those in control group.Western Blot analysis results showed that after siRNA transfection,the SRF protein levels were decreased by 40.1%,as compared with those in control group(P<0.05).As compared with that in blank control group,negative control group and transfection control group,the cell proliferation of siRNA-SRF were significantly inhibited in siRNA-1107 transfection group,with the inhibiting rate being 64.24%,and the cell counts at S stage were significantly decreased,and the invasive cell counts were decreased by 41.77%,as compared with those in blank control group(P<0.05).Conclusion SRF gene silenced by RNAi interference technique can significantly inhibit the proliferation and invasion of A549 cells.