MiR-21对肺癌细胞增殖与凋亡的影响

发布时间：2018-08-25 11:55

【摘要】：目的探讨miR-21对肺癌细胞增殖与凋亡的影响。方法培养人肺癌细胞HBE、H460、A549和H446,将实验分为miR-21 inhibitor组、miRNA scramble组、空白对照组。荧光定量检测肺癌细胞H460、A549和H446 miR-21的表达;荧光定量PCR检测将miR-21 inhibitor和miRNA scramble转染到培养至对数生长期的人肺癌细胞H460后,对照组不转染的各组细胞的表达量;MTT实验检测转染miR-21 inhibitor后对细胞增殖的影响;流式细胞仪检测转染miR-21 inhibitor后对细胞凋亡的影响;Western印迹检测与凋亡相关蛋白的活性。结果肺癌细胞H460、A549和H446miR-21的表达显著高于正常肺癌细胞HBE(P0.01),由于肺癌细胞株H460的miR-21表达在3个细胞株中高表达,因此选择肺癌细胞株H460做后续研究;将miR-21 inhibitor组和miRNA scramble组转染到肺癌细胞株H460后,荧光定量PCR显示,miR-21 inhibitor组的miR-21的相对表达量为显著低于空白对照组(P0.01),miRNA scramble组的相对表达量与空白对照组无差异(P0.05);MTT结果显示,转染4 d后,miRNA scramble组与空白对照组细胞增殖率无差异(P0.05),而miR-21 inhibitor组显著低于空白对照组(P0.05),且随时间延长,细胞增殖率逐渐下降;流式结果显示,与空白对照组和miRNA scramble组比较,miR-21 inhibitor组细胞的凋亡率显著升高(P0.01);Western印迹显示,miR-21 inhibitor组Cleaved-caspase-3和Bax蛋白的表达显著高于miRNA scramble组和空白对照组,而miR-21 inhibitor组Bcl-2蛋白的表达显著低于miRNA Scramble组和空白对照组(P0.05)。结论下调miR-21的表达,可抑制肺癌细胞H460的生长,增加细胞的凋亡,其作用机制可能与Bcl-2家族的调节有关。
[Abstract]:Objective to investigate the effect of miR-21 on proliferation and apoptosis of lung cancer cells. Methods Human lung cancer cells HBE,H460,A549 and H446 were cultured and divided into miR-21 inhibitor group and blank control group. The expression of H460A549 and H446 miR-21 in lung cancer cells was detected by fluorescence quantitative PCR, and miR-21 inhibitor and miRNA scramble were transfected into human lung cancer cells H460 at logarithmic growth stage by fluorescence quantitative PCR. The effect of miR-21 inhibitor transfection on cell proliferation and apoptosis after transfection of miR-21 inhibitor were detected by flow cytometry and the activity of apoptosis-related protein was detected by Western blot. Results the expression of H460 A549 and H446miR-21 in lung cancer cell line was significantly higher than that in normal lung cancer cell line HBE (P0.01). Because the miR-21 expression of lung cancer cell line H460 was highly expressed in three cell lines, lung cancer cell line H460 was selected for follow-up study. After transfection of miR-21 inhibitor group and miRNA scramble group into lung cancer cell line H460, fluorescence quantitative PCR showed that the relative expression of miR-21 in the inhibitor group was significantly lower than that in the control group (P0.01) and there was no difference between the control group and the control group (P0.05). After 4 days of transfection, there was no difference in cell proliferation rate between scramble group and blank control group (P0.05), but the proliferation rate of miR-21 inhibitor group was significantly lower than that of blank control group (P0.05). Compared with the control group and the miRNA scramble group, the apoptosis rate of the miR-21 inhibitor group was significantly higher than that of the control group (P0.01). Western blot showed that the expression of Cleaved-caspase-3 and Bax protein in the miR-21 inhibitor group was significantly higher than that in the miRNA scramble group and the blank control group. The expression of Bcl-2 protein in miR-21 inhibitor group was significantly lower than that in miRNA Scramble group and blank control group (P0.05). Conclusion the down-regulated expression of miR-21 can inhibit the growth of H460 cells and increase the apoptosis of H460 cells. The mechanism may be related to the regulation of Bcl-2 family.
【作者单位】：湖北科技学院附属第一医院心胸外科;
【基金】：湖北省自然科学基金(No.2014CFB307)
【分类号】：R734.2

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