MiR-186-5p通过靶向调控PTTG1抑制肺腺癌细胞的上皮-间质转化

先前研究表明，miR-186-5p在人类许多恶性肿瘤中扮演抑癌基因的作用，但其在肺腺癌上皮-间质转化（epithelial-mesenchymal transition，EMT）中的作用并不明确。本研究旨在证明，miR-186-5p可通过靶向调控PTTG1抑制肺腺癌细胞的上皮-间质转化。我们首先分析了miR-186-5p在人肺癌细胞中的表达。荧光定量PCR（QRT-PCR）结果显示，与人正常肺上皮细胞BEAS-2B相比，肺腺癌细胞SPC-A1、A549中的miR-186-5p表达量明显降低。为研究miR-186-5p在肺腺癌细胞中的功能，利用GV369-miR-186-5p表达载体，实现了在A549细胞中的过表达。基因转染结合Transwell侵袭结果显示，与对照质粒转染的A549细胞相比，过表达miR-186-5p的A549细胞的体外侵袭能力明显下降。Western印迹检测细胞中EMT相关标志物揭示，GV369-miR-186-5p转染的A549细胞中的上皮-钙黏着蛋白（E-cadherin）表达明显上调，而神经-钙黏着蛋白（N-cadherin）和波形蛋白（vimentin）表达明显下调。同时，GV369-miR-186-5p转染引起其靶基因--垂体肿瘤转化基因1（pituitary tumor-transforming gene 1，PTTG1）编码蛋白在A549细胞中明显降低。重要的是，过表达miR-186-5p与敲减PTTG1均可导致上皮-钙黏着蛋白表达上调，而神经-钙黏着蛋白和波形蛋白下调|而miR-186-5p和PTTG1表达载体共转染后，3种EMT相关标志物在A549的表达与对照细胞的表达无明显差异，提示过表达PTTG1可抵消miR-186-5p对EMT相关标志物表达的影响。综上所述，miR-186-5p可通过靶向调控PTTG1抑制EMT的发生，进而抑制肺腺癌细胞的侵袭转移。

miR-186-5p Inhibits Epithelial Mesenchymal Transition in Lung Adenocarcinoma Cells Through Modulating PTTG1

Previous studies have shown that miR-186-5p plays an suppressor role in many human cancers, but the function of miR-186-5p in epithelial mesenchymal transition (EMT) of lung adenocarcinoma reminded unclear. This study aims at demonstrating the inhibition of EMT in lung adenocarcinoma cells by miR-186-5p-targeted PTTG1. We first analyzed miR-186-5p expression in lung adenocarcinoma cells using quantitative real-time PCR (qRT-PCR) and showed that the levels of miR-186-5p were markedly decreased in lung adenocarcinoma SPC-A1 and A549 cells, compared with normal epithelial BEAS-2B cells. To investigate the role of miR-186-5p in lung cancer cells, we used the GV369-miR-186-5p expression vector that was over-expressed in transfected A549 cells. The combination of gene transfection with transwell experiment revealed that the invasion ability of miR-186-5p-transfected A549 cells was declined significantly, compared with scrambled RNA-transfected (control) A549. Western blotting for EMT marker proteins showed that the E-cadherin was up-regulated, while N-cadherin and vimentin were down-regulated in GV369-miR-186-5p-transfected A549. Moreover, the level of pituitary tumor-transforming gene 1 (PTTG1) protein was much lower in GV369-miR-186-5p-transfected A549 than that in control cells. Importantly, over-expression of miR-186-5p and knock-down of PTTG1 both led to up-regulation of E-cadherin and down-regulation of N-cadherin and vimentin. Co-transfection of miR-186-5p and PTTG1 expression vectors, however, did not change the expression of three EMT related proteins in transfected cells, as control cells did so, indicating that over-expressing PTTG1 may counteract the effects of miR-186-5p on EMT related protein expression. Taken together, these data suggested that miR-186-5p could inhibit EMT occurrence by targeting PTTG1, therefore inhibiting the invasion and metastasis of lung adenocarcinoma cells.