microRNA-1304通过靶向血红素氧合酶-1对人肺癌细胞的抑制作用

miR-1304 inhibits non-small cell lung cancer cell invasion and proliferation in vitro via targeting heme oxygenase-1

目的:探讨miR-1304对肺癌细胞增殖和侵袭的作用及机制。方法:应用实时定量PCR检测miR-1304在不同转移能力肺癌A549和NCI-H1975细胞中的表达水平;脂质体2000介导分别转染miR-1304类似物和抑制物入A549和NCI-H1975细胞;应用MTT法和细胞集落形成实验分别检测miR-1304对肺癌细胞增殖活性的作用及生长抑制作用;细胞凋亡和细胞周期分别采用膜联蛋白V-PE/7-AAD和PI染色分析进行检测。双荧光素酶报告基因验证miR-1304是否作用于血红素氧合酶(heme oxygenase-1,HO-1) mRNA的3'UTR区预测靶位;Western blot检测HO-1蛋白水平。结果:miR-1304高表达可显著抑制肺癌细胞形成的数量和生存能力,以及诱导细胞凋亡和G_0/G_1期细胞周期阻滞。体外侵袭实验及细胞增殖实验结果显示,miR-1304可抑制肺癌细胞侵袭和增殖;经双荧光素酶报告基因验证HO-1是miR-1304的靶基因。结论:miR-1304在肺癌中表达,通过直接作用于HO-1可抑制肺癌细胞的侵袭和增殖,miR-1304是肺癌的一个潜在治疗靶点。

AIM :To investigate the direct target of miR-1304 and its function in NSCLC in vitro . METHODS : Real-time quantitative PCR was used to detect the expression of miR-1304 in lung cancer cell A549 and NCI-H1975 .Transwell assay was used to test the role of miR-1304 on regulating invasion and migration of cells. The cell proliferation and survival were investigated via cell counting, MTT and colony-formation assays. Cell apoptosis and cell cycle were examined using annexin V-PE/7-AAD and PI staining assays, respectively. Dual luciferase reporter gene was used to analyze the binding between miR-1304 and 3'UTR of heme oxygenase-1, Western blot detected the HO-1 protein levels. RESULTS :MiR-1304 significantly decreased the number and viability of NSCLC cells and colony formation, and induced cell apoptosis and G 0/G 1 phase cell cycle arrest. HO-1 was demonstrated to be a direct target of miR-1304 in NSCLC cells. Furthermore, altered expression of miR-1304 by transfection of pre-miR-1304 mimics and inhibitor signifcantly affected the ability of invasion and proliferation of lung cancer cells. Altered expression of miR-1304 markedly down-regulated the HO-1 protein levels of lung cancer cells. In addition, dual luciferase reporter gene assay indicated that miR-1304 regulated HO-1 expression by binding to the 3'UTR of HO-1 mRNA. CONCLUSION : MicroRNA-1304 is a tumor suppressor and HO-1 is its direct target in NSCLC. The results suggest the potential for miR-1304 as a therapeutic target for NSCLC.