微小RNA-206抑制恶性胸膜间皮瘤细胞增殖的作用  

MicroRNA-206 inhibits cell growth in malignant pleural mesothelioma

作　　者：石珍亮 张逊[2] 孙大强[2] Shi Zhenliang;Zhang Xun;Sun Daqiang(Department of Thoracic Surgery, Tianjin Chest Hospital, Tianjin 300222, China;Department of Thoracic Surgery, Tianjin Chest Hospital, Tianjin 300222, China)

机构地区：[1]天津医科大学研究生院,300070 [2]天津市胸科医院胸外科,300222

出　　处：《中华实验外科杂志》2019年第3期492-495,共4页Chinese Journal of Experimental Surgery

摘　　要：目的探讨微小RNA(miRNA,miR)-206对恶性胸膜间皮瘤细胞增殖的抑制作用及其机制。方法在H513、H2052和H2373等3种不同恶性胸膜间皮瘤(MPM)细胞株中通过转染引入一种miR-206的分子模拟物来过表达这种miRNA:(1)通过结晶紫染色实验分析转染后的MPM细胞的增殖变化;(2)通过实时定量反转录聚合酶链反应(RT-qPCR)检测与细胞周期稳态和凋亡相关的基因变化;(3)通过基因敲除实验和PCR分析miR-206抑制作用可能的分子机制。结果(1)转染3 d后,结晶紫实验量化分析提示在H513、H2052和H2373转染细胞组与对照组比较显著减少(570 nm吸光度值:0.406±0.196比0.682±0.042,0.192±0.181比0.587±0.264,0.178±0.056比0.794±0.276,P<0.05);(2)在转染48 h后,RT-qPCR检测结果显示促凋亡基因B细胞淋巴瘤/白血病-2相关X蛋白(bax)、p53和细胞周期阻滞基因p27的表达水平显著升高(平均差异倍数:1.489±0.355,1.298±0.336,1.746±0.389,P< 0.05),而细胞增殖基因增殖细胞核抗原(PCNA)和抗凋亡基因B细胞淋巴瘤/白血病-2(bcl-2)的表达水平显著减少(平均差异倍数:1.489±0.355,1.298±0.336,P<0.05);检测同时提示组蛋白去乙酰化转移酶(HDAC)基因活性受到抑制,miR-206过表达对HDAC4和HDAC6都有显著的负调控作用(平均差异倍数:0.984±0.271比0.442±0.149比0.520±0.193,P<0.05)。(3)在HDAC4或HDAC6基因被敲除后,3种MPM细胞株均显示出明显的结晶紫染色生长阻滞(570 nm吸光度值:0.971±0.205比0.427±0.152比0.520±0.191,P<0.05),这种表现与HDAC活性受到抑制一致,提示HDAC4和HDAC6基因亚型可能是miR-206转染过程中调控MPM细胞增殖的主导效应因子。结论miR-206过表达在恶性胸膜间皮瘤中起到抑制肿瘤细胞增殖的作用。Objective To investigate the suppressive effect of miR-206 on malignant pleural mesothelioma (MPM) cell lines and mechanism. Methods A mimic of miR-206 was introduced into MPM cell lines H513, H2052 and H2373 to overexpress this microRNA.(1) miRNA-induced changes in cell growth were evaluated by crystal violet staining.(2) Genes associated with cell cycle homeostasis and apoptosis were examined by real-time quantitative reverse transcriptase-polymerase chain reaction (RT-qPCR).(3) By gene knockdown experiment and RT-qPCR, the potential mechanism of the suppressive effect was analyzed. Results (1) Cellular growth in H513, H2052, H2373 were significantly inhibited after overexpression of miR-206 at day 3 (570 nm: 0.406±0.196 vs. 0.682±0.042, 0.192±0.181 vs. 0.587±0.264, 0.178±0.056 vs. 0.794±0.276, P<0.05).(2) Gene expression levels of B cell lymphoma/leukemia-2 associated X protein (bax), p53, and p27 were all significantly increased (average fold change: 1.489±0.355, 1.298±0.336, 1.746±0.389, P<0.05) in miR-206-transfected MPM cells. Coordinately, gene expression levels of proliferating cell nuclear antigen (PCNA) and B cell lymphoma/leukemia-2 (bcl-2) were significantly decreased (average fold change: 1.489±0.355, 1.298±0.336, P<0.05). Significant attenuation of histone deacetylase (HDAC) activity was observed (average fold change: 0.984±0.271 vs. 0.442±0.149 vs. 0.520±0.193, P<0.05), and suppression of HDAC activity mediated by negative regulation of HDAC4 and 6 genes is an effect unique to miR-206.(3) All MPM cell lines tested with either HDAC4 or HDAC6 knockdown exhibited significant growth arrest by crystal violet staining (570 nm: 0.971±0.205 vs. 0.427±0.152 vs. 0.520±0.191, P<0.05), which was consist with suppression of HDAC, indicating HDAC4 and HDAC6 were significant effector molecules for the suppressive effect. Conclusion Overexpression of miR-206 acts as a tumor suppressor in MPM.

关 键 词：恶性胸膜间皮瘤 微小RNA 脱噬作用 生长停滞 组蛋白去乙酰化转移酶 

分 类 号：R734.3[医药卫生—肿瘤]