益气除痰方下调MALAT-1表达对肺癌细胞发生上皮间质转化的影响

Effect of Yiqi Chutan Formula on Inhibiting of Epithelial-Mesenchymal Transition in Lung Cancer Cells by Down-Regulating MALAT-1

目的:研究益气除痰方对肺癌细胞发生上皮间质转化(EMT)的抑制作用及可能的分子机制。方法:聚合酶链式反应(PCR)检测并筛选出MALAT-1明显上调的肺癌细胞系H1229,及相对表达偏低的A549,用含益气除痰方的培养基分别进行干预。通过CCK8(Cell Counting Kit-8)试剂盒比色法检测肿瘤细胞的增殖能力;细胞划痕实验以及Transwell侵袭小室实验检测细胞的侵袭转移能力;用蛋白质免疫印记法(Western-blot)检测上皮间质转化相关蛋白转录是否上调;聚合酶链式反应(PCR)检测MALAT-1表达情况;并用蛋白质印记法检测TGF诱导的相关通路蛋白Smad2/3磷酸化水平。结果:与空白对照组相比,益气除痰方可抑制不同肺癌细胞株的增殖,抑制作用与MALAT-1表达上调与否关系不大,但是可在较低浓度即可对侵袭转移能力有明显抑制,抑制作用对MALAT-1上调的细胞株更明显,提示抑制水平与MALAT-1的表达下调有关,进一步检测发现益气除痰方可以下调TGF诱导的Smad2/3蛋白磷酸化。结论:益气除痰方抑制肺癌细胞侵袭转移的机制可能是通过下调MALAT-1,抑制Smad2/3磷酸化,从而抑制肺癌细胞发生上皮间质转化的进程。

Objective:To study the effect and mechanism of Yiqi Chutan Formula(YQCTF)on inhibiting of epithelial-mesenchymal transition in lung cancer cells. Methods:Lung cancer cell lines with significantly up-regulated and normal expression of metastasis-associated lung adenocarcinoma transcript 1(MALAT-1) were used to intervene with serum containing YQCTF. Cell proliferation was detected by cell counting Kit-8(CCK-8). Wound healing assay and Transwell assay were used to examine the invasive and metastatic ability of lung cancer cells.EMT biomarkers(including E-cadherin, Vimentin, Snail and GAPDH protein) were examined by Western blotting. The expression of MALAT-1 in different cells was detected by PCR. Protein phosphorylation of Smad2/3 was examined by Western blotting. Results: Compared with the blank control group, YQCTF inhibited the proliferation of lung cancer cell lines at higher drug concentrations, and had nothing to do with MALAT-1 expression. But it significantly inhibited invasion and metastasis at lower drug concentrations. Inhibition was more pronounced in cell lines upregulated by MALAT-1. Inhibition levels were associated with down-regulation of MALAT-1 expression. Further test found that the Smad2/3 protein phosphorylation induced by TGF-β was inhibited. Conclusions:YQCTF can reverse the EMT induced by transforming growth factor(TGF) in lung cancer cells by down-regulating MALAT-1. The mechanism is partially correlated with inhibition of Smad2/3 protein phosphorylation.