微小RNA-30a-5p调控喉鳞状细胞癌Hep-2细胞增殖和凋亡的机制探讨

Mechanism of microRNA-30a-5p regulating proliferation and apoptosis of laryngeal squamous cell carcinoma Hep-2 cells

目的探讨miR-30a-5p对喉鳞状细胞癌Hep-2细胞增殖凋亡的影响及其机制。方法用实时荧光定量PCR检测喉癌及癌旁组织中miR-30a-5p的表达;将Hep-2细胞分为NC组(转染miR-30a-5p mimics阴性对照)、miR-30a-5p组(转染miR-30a-5p mimics)、anti-NC组(转染miR-30a-5p inhibitor阴性对照)和anti-30a-5p组(转染miR-30a-5p inhibitor),MTT实验、平板克隆形成实验和Annexin V-FITC/PI双染实验分别检测细胞增殖、克隆形成和细胞凋亡情况。双荧光素酶报告基因实验检测miR-30a-5p和SOX9的靶向关系。结果与癌旁组织表达量(1.00±0.10)相比,喉癌组织中miR-30a-5p的表达水平(0.37±0.03)显著降低(P<0.05)。与NC组相比,miR-30a-5p组细胞活性(48小时:0.42±0.03 vs 0.58±0.04)降低,细胞克隆数(75.36±6.85 vs 136.25±10.50)降低,细胞凋亡率[(23.86±2.21)%vs(9.95±1.16)%]升高(P<0.05)。双荧光素酶报告实验结果显示,转染SOX9-WT质粒的Hep-2细胞荧光素酶活性明显降低(P<0.05),而转染SOX9-MUT质粒的Hep-2细胞荧光素酶活性无显著变化。anti-miR-30a-5p组对Hep-2细胞的作用与miR-30a-5p组相反。结论miR-30a-5p可能通过靶向下调SOX9抑制喉鳞状细胞癌Hep-2细胞增殖并促进细胞凋亡。

OBJECTIVE To investigate the effect of microRNA-30a-5p on proliferation and apoptosis of laryngeal squamous cell carcinoma Hep-2 cells and its mechanism.METHODS Real-time fluorescence quantitative PCR was used to detect the expression of miR-30a-5p in laryngeal carcinoma and adjacent tissues;Hep-2 cells were divided into NC group,miR-30a-5p group,anti-NC group and anti-miR-30a-5p group.MTT experiment,plate clone formation experiment and Annexin V-FITC/PI double staining experiment were used to detect cell proliferation,clone formation and apoptosis,respectively.The dual luciferase reporter gene experiment detected the targeting relationship between miR-30a-5p and SOX9.RESULTS Compared with the adjacent tissues(1.00±0.10),the expression of miR-30a-5p(0.37±0.03)in laryngeal carcinoma tissues was significantly reduced(P<0.05).Compared with the NC group,the cell activity(48h:0.42±0.03 vs 0.58±0.04)of miR-30a-5p group was decreased,the number of cell clones(75.36±6.85 vs 136.25±10.50)was decreased,and the apoptosis rate[(23.86±2.21)%vs(9.95±1.16)%]was increased(P<0.05).The results of the dual luciferase report experiment showed that the luciferase activity of Hep-2 cells transfected with SOX9-WT plasmid was significantly reduced(P<0.05),while the luciferase activity of Hep-2 cells transfected with SOX9-MUT plasmid was not significantly changed.The effect of anti-miR-30a-5p group on Hep-2 cells was opposite to that of miR-30a-5p group.CONCLUSIONmiR-30a-5p may inhibit the proliferation of laryngeal squamous cell carcinoma Hep-2 cells and promote apoptosis by targeting down-regulation of SOX9.