Bai Jinghui, Zhu Xiangyu, Ma Jianqi, Wang Wanting
Intensive Care Unit, Liaoning Cancer Hospital Xiaoheyan Road, 92, Dadong District, Shenyang 110042, Liaoning, P.R. China.
Int J Clin Exp Pathol. 2015 Feb 1;8(2):1175-83. eCollection 2015.
miR-205 is an epithelial-specific miRNA and has been shown to orchestrate some cellular processes such as epithelial mesenchymal transition (EMT) and differentiation fate of stem cells in mammary gland. miR-205 play a part of a tumor suppressor in human cancers. However, the role of miR-205 in lung cancer is unclear. In this study, we detected the expression level of miR-205 in 46 cases clinical lung cancer specimens and adjacent normal tissues by stem-loop RT-PCR. We found that the expression of miR-205 was significantly increased in lung cancer specimens compared to adjacent normal tissues (P < 0.01). Furthermore, we observed the expressions of PTEN protein and mRNA in lung cancer tissues and adjacent normal tissues by methods of western blot and Real time PCR respectively. We found that the expressions of PTEN protein and mRNA was significantly decreased in lung cancer specimens compared to adjacent normal tissues. And then, we found there is a negative relationship between the expression of miR-205 and PTEN mRNA in lung cancer by analyzed. To validate whether PTEN was direct targets of miR-205, a dual-luciferase reporter assay was employed, the result showed that PTEN is a target gene of MiR-205. In subsequent experiments, we examined the expressions of PTEN protein and mRNA after transfection of miR-205 mimics or inhibitor into A549 cells, and A549 cell proliferation was measured by CCK-8 tests. We found that the expression of PTEN protein and mRNA in A549 cells were significantly down-regulated or up-regulated after miR-205 mimics and miR-205 inhibitors transfected into, and miR-205 could inhibits A549 cells proliferation. These results indicate that miR-205 might inhibitor the proliferation of A549 cells by regulating the expression of PTEN.
miR-205是一种上皮特异性微小RNA,已被证明可调控一些细胞过程,如上皮-间质转化(EMT)以及乳腺中干细胞的分化命运。miR-205在人类癌症中发挥肿瘤抑制作用。然而,miR-205在肺癌中的作用尚不清楚。在本研究中,我们通过茎环逆转录-聚合酶链反应(stem-loop RT-PCR)检测了46例临床肺癌标本及癌旁正常组织中miR-205的表达水平。我们发现,与癌旁正常组织相比,肺癌标本中miR-205的表达显著增加(P < 0.01)。此外,我们分别通过蛋白质免疫印迹法和实时定量聚合酶链反应检测了肺癌组织及癌旁正常组织中PTEN蛋白和mRNA的表达。我们发现,与癌旁正常组织相比,肺癌标本中PTEN蛋白和mRNA的表达显著降低。然后,通过分析我们发现肺癌中miR-205的表达与PTEN mRNA之间存在负相关关系。为验证PTEN是否为miR-205的直接靶点,我们进行了双荧光素酶报告基因检测,结果表明PTEN是MiR-205的靶基因。在后续实验中,我们将miR-205模拟物或抑制剂转染至A549细胞后,检测了PTEN蛋白和mRNA的表达,并通过CCK-8实验检测了A549细胞的增殖情况。我们发现,将miR-205模拟物和miR-205抑制剂转染至A549细胞后,A549细胞中PTEN蛋白和mRNA的表达分别显著下调或上调,并且miR-205可抑制A549细胞的增殖。这些结果表明,miR-205可能通过调节PTEN的表达来抑制A549细胞的增殖。