Background and objective: MiR-145 functions as a protective miRNA identified in tumor tissues of lung adenocarcinoma patients. The aim of this study is to investigate the relationship between miR-145 and proliferation of lung cancer stem cells and involved molecular mechanisms in human lung adenocarcinoma A549 cell line.
Methods: MicroRNA microarray technology was conducted to compare miRNA signature between tumor and adjacent normal tissue of lung adenocarcinoma. The potential target gene of miR-145 was predicted by online bioinformatic softwares. Pre-miR-145 mimics and anti-miR-145 inhibitor were transfected into A549 cell line by lipofectamine 2000. miR-145 expression in each group was detected by real time PCR. The OCT4 protein level was analyzed by Western blot. The predicted miR-145 binding site in OCT4 3'-untranslated region (UTR) was validated by dual-luciferase reporter gene assay. CCK-8 assay was employed to observe the proliferation activity of A549 cells. The ratio of CD133 positive cells in each group was analyzed by flow cytometry.
Results: miR-145 expression was significantly down-regulated in lung adenocarcinoma compared with ajacent normal tissue. OCT4 is a potential target gene of miR-145 predicted by miRanda. Compared with control group, miR-145 was significantly up-regulated and down-regulated in the pre-miR-145 mimics and anti-miR-145 inhibitor groups respectively. Overexpression of miR-145 inhibited the proliferation of A549 cells. Both the OCT4 protein level and CD133 positive ratio were remarkably decreased in the pre-miR-145 mimics group, whereas significantly increased in the anti-miR-145 inhibitor group. Dual-luciferase reporter gene assay validated the predicted miR-145 binding site of OCT4 3'UTR.
Conclusions: MiR-145 can inhibit the proliferation of lung cancer stem cells in A549 cell line via down-regulating OCT4 expression. MiR-145 is a potential protective miRNA of lung cancer.
背景与目的: miR-145是通过miRNA芯片及qPCR验证筛选出的一种潜在肺癌“保护性”miRNA。本研究旨在探讨miR-145与肺癌干细胞之间的关系及分子机制。
方法: miRNA芯片对肺腺癌患者瘤旁和正常组织进行表达谱分析；生物信息学软件预测miR-145潜在的靶基因；脂质体2000介导转染miR-145模拟物和阻遏物进入A549细胞株；实时定量PCR检测miR-145表达水平；Western blot检测OCT4蛋白水平；双荧光素酶报告基因验证miR-145是否作用于OCT4 mRNA的3’UTR区预测靶位；细胞增殖实验检测miR-145对于A549细胞生长的作用；流式细胞术检测干细胞表型CD133+的表达。
结果: 在肺腺癌组织中miR-145表达明显低于瘤旁正常组织；miRanda软件预测OCT4是miR-145潜在靶基因；与对照组相比，miR-145模拟物组和阻遏物组miR-145表达分别明显上调和下调；miR-145对A549细胞的生长有双向调节作用，过表达miR-145抑制细胞生长；过表达miR-145可明显降低OCT4蛋白水平及干细胞表型CD133百分比，而抑制miR-145表达则明显增加OCT4蛋白水平及CD133百分比。双荧光素酶报告基因检测证明miR-145可作用于OCT4 mRNA的3’UTR区预测靶位。
结论: miR-145可通过下调OCT4基因表达抑制A549肺腺癌细胞株中干细胞的增殖，是一种潜在的肺癌“保护性”miRNA。