miRNA-21对肺癌细胞增殖和侵袭能力的影响及作用机制的研究

Effects of miRNA-21 on the growth,migrating and invasive potential of lung cancer cell and its action mechanism

目的探讨微小RNA-21(miR-21)对肺癌细胞A549增殖、迁移和侵袭能力的影响,并对相应的机制进行探究。方法构建空白组、miR-21高表达质粒空载对照组、miR-21高表达组、miR-21敲减质粒空载对照组、miR-21敲减组。采用荧光定量qRT-PCR检测细胞中miR-21mRNA表达情况,蛋白质印迹法分析相关通路蛋白表达水平,确定miR-21转染效率。CCK-8试剂盒检测细胞增殖和生长能力。采用Transwell小室模型检测miR-21上调和下调以对A549细胞迁移和侵袭能力的影响。流式细胞仪检测细胞周期变化和细胞凋亡情况。结果荧光定量qRT-PCR提示,miR-21高表达组miR-21表达水平显著高于空白组和miR-21高表达质粒空载对照组(t=4856、4525,P值均<001)。CCK-8检测显示miR-21转染48h及72h,miR-21高表达组细胞增殖均较其他组强(F=43061、74862,P值均<001)。Transwell迁移和侵袭实验提示,miR-21高表达组细胞迁移和侯袭能力均较空白组和miR-21高表达质粒空载对照组强(P值均<005)。结论miRNA-21促进肺癌细胞增殖并抑制肺癌细胞凋亡。miRNA-21对人肺癌细胞株的增殖、侵袭、转移能力的影响与AKT/P-AKT/Cleaved-Caspsae3/MMP-2/MMP-9细胞信号通路有关。

Objective To demonstrate the effects of m icroRNA-21(miR-21)on the growth,migrating and invasive potential of lung cancer cell A 549 in vitro and the possible mechanism.Methods Five groups were built:blank control group,miR-21 mimic control group,miR-21 mimic group,miR-21 inhibitor control group,miR-21 inhibitor group.Real-time qRT-PCR was used to measure the m RNA expression levels of miR-21.Protein expression level of related pathway was analyzed by Western blot.CCK-8 assay was used to assess the effect of miR-21 on cell proliferation.Transwell chamber assay was used to investigate the effects of up-and down-regulation of miR-21 on migration and invasion of A 549 cells.Flow cytometer was used to assess the effect of miR-21 on A 549 cell cycle and apoptosis.Results Real time qRT-PCR showed that miR-21 mimics group expressed higher level of miR-21 compared to the blank control group and miR-21 mimic control group(t=48.56,45.25,both P<0.01).CCK-8 cell proliferation test showed that,48 h and 72 h after the miR-21 transfection,the miR-21 mimic group presents an increasing trend compared with other groups(F=43.061,74.862,both P<0.01).Transwell chamber assay showed that miR-21 mimic group had stronger ability of migration and invasion than the blank control group and miR-21 mimic control group(all P<0.05).Conclusions m iRNA-21 promotes lung cancer cell proliferation and inhibits lung cancer cell apoptosis.The effect of m iRNA-21 on proliferation,invasion and metastasis of human lung cancer cell lines is related to the signal pathway of AKT/PAKT/Cleaned-Caspsae3/MMP-2/MMP-9 cells.