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目的 :探讨miR-21对人唾液腺腺样囊性癌细胞株(SACC-LM)增殖、凋亡的影响及其相关作用机制。方法 :将SACC-LM细胞分为3组,采用Lipofectamine ~(TM)2000做瞬时转染。干扰组转染miR-21 inhibitor,阴性对照组转染无关核苷酸序列,空白对照组不转染。采用实时荧光定量PCR检测转染后细胞中miR-21和PDCD4、Bcl-2 mRNA的表达水平。以CCK8法检测转染24、48、72、96 h后细胞的增殖活性,Annexin-V/PI双染色流式细胞术检测细胞凋亡率,Western免疫印迹检测转染后靶蛋白PDCD4、Bcl-2的表达量。采用SPSS 16.0软件包对所得数据进行统计学分析。结果:转染miR-21 inhibitor的细胞中miR-21、Bcl-2表达下调(P<0.01),而PDCD4基因表达上调(P<0.05)。CCK8检测结果表明,miR-21表达下调可以明显抑制SACC-LM细胞的增殖活性,并呈时间依赖性(P<0.05)。流式细胞术检测结果表明,干扰组细胞凋亡率显著高于阴性对照组和空白组(P<0.05);Western免疫印迹结果显示,PDCD4蛋白的表达较对照组显著升高(P<0.01),Bcl-2蛋白表达显著降低(P<0.01)。结论:下调miR-21的表达可抑制SACC-LM细胞的增殖活性,诱导细胞凋亡,其机制可能与细胞内PDCD4表达上调,Bcl-2表达下调有关。
Objective: To investigate the effect of miR-21 on proliferation and apoptosis of human salivary adenoid cystic carcinoma cell line (SACC-LM) and its related mechanism. Methods: SACC-LM cells were divided into three groups and transiently transfected with LipofectamineTM 2000. MiR-21 inhibitor was transfected into the interference group, unrelated nucleotide sequence was transfected into the negative control group, and the blank control group was not transfected. The expression of miR-21, PDCD4 and Bcl-2 mRNA in transfected cells was detected by real-time fluorescence quantitative PCR. Cell proliferation was detected by CCK8 assay at 24,48,72,96 h after transfection. Cell apoptosis was detected by flow cytometry with Annexin-V / PI double staining. Western blotting was used to detect the expression of PDCD4, Bcl- 2 expression level. The data were statistically analyzed using SPSS 16.0 software package. Results: The expression of miR-21, Bcl-2 was down-regulated (P <0.01), while the expression of PDCD4 was up-regulated in miR-21 inhibitor transfected cells (P <0.05). The results of CCK8 showed that the down-regulation of miR-21 could significantly inhibit the proliferation of SACC-LM cells in a time-dependent manner (P <0.05). The results of flow cytometry showed that the apoptosis rate in the interference group was significantly higher than that in the negative control group and the blank group (P <0.05). Western blot results showed that the expression of PDCD4 protein was significantly higher than that in the control group (P <0.01) , Bcl-2 protein expression was significantly lower (P <0.01). Conclusion: Down-regulating the expression of miR-21 can inhibit the proliferation and induce the apoptosis of SACC-LM cells, which may be related to the upregulation of PDCD4 and the down-regulation of Bcl-2 in the cells.