GATA4基因甲基化及GATA4对非小细胞肺癌生长的影响及其作用机制

目的 探讨GATA4在非小细胞肺癌（non-small cell lung cancer，NSCLC）组织中的甲基化状态和GATA4对NSCLC细胞生长的作用及其可能机制。方法 收集2017年7月—2018年6月于新疆医科大学第一附属医院接受肺癌根治术的78例NSCLC患者癌组织及其癌旁组织标本，以及人肺癌细胞株A549、HCC827、NCI-H1299和人正常肺上皮细胞BEAS-2B，用MSP和qMSP检测GATA4基因甲基化状态，RT-PCR和Western blot检测GATA4表达。将sh-GATA4-1（sh-GATA4组）、sh-NC、GATA4过表达载体（pLV-GATA4组）及其空载体对照慢病毒液（pLV-NC组）分别转染至肺癌A549细胞中，同时设置空白对照组（Blank组），用Western blot检测GATA4、p-ERK、ERK、p-p38和p38蛋白表达，CCK-8试剂盒检测细胞活力，细胞克隆形成实验检测细胞增殖，流式细胞术检测细胞凋亡。结果 NSCLC组织中GATA4基因甲基化率以及p-ERK、p-p38和Bcl-2蛋白表达水平均高于癌旁组织（P＜0.001），GATA4 mRNA表达、GATA4和Bax蛋白表达均低于癌旁组织（P＜0.001）。GATA4基因甲基化程度与p-ERK、p-p38和Bcl-2蛋白表达水平呈正相关（P＜0.05）；与GATA4 mRNA表达水平，GATA4、Bax蛋白表达水平呈负相关（P＜0.05）。NSCLC细胞中GATA4呈甲基化状态，人正常肺上皮细胞BEAS-2B表现为去甲基化状态，GATA4 mRNA和蛋白表达低于BEAS-2B细胞（P＜0.01）。与pLV-NC组比较，pLV-GATA4组细胞中GATA4 mRNA和蛋白表达水平、细胞凋亡率升高（P＜0.001），细胞活力、细胞克隆数、p-ERK/ERK和p-p38/p38蛋白表达水平降低（P＜0.05）；sh-GATA4组细胞中GATA4 mRNA、蛋白表达和细胞凋亡率低于sh-NC组（P＜0.001），细胞活力、细胞克隆数、p-ERK/ERK和p-p38/p38蛋白表达高于sh-NC组（P＜0.05）。结论 GATA4基因在非小细胞肺癌中呈高甲基化状态并诱导GATA4基因表达降低，GATA4过表达可抑制肺癌细胞增殖并诱导细胞凋亡，可能通过调控MAPK通路实现。

Effect of GATA4 gene methylation on the growth of non-small cell lung cancer and underlying mechanism

Objective To investigate the methylation status of GATA4 in non-small cell lung cancer(NSCLC), and its effect on NSCLC cells and possible mechanism. Methods Cancer tissues and paracancerous tissues, as well as human lung cancer cell lines A549, HCC827, NCI-H1299 and human normal lung epithelial cells BEAS-2B, of 78 patients with NSCLC who received radical surgery of lung cancer in First Affiliated Hospital of Xinjiang Medical University from July 2017 to June 2018 were collected. The methylation status of GATA4 gene was detected by MSP and qMSP, the expression of GATA4 was detected by RT-PCR and Western blot. The sh-GATA4-1(SH-GATA4 group), sh-NC, GATA4 overexpression vector(pLV-GATA4 group) and empty vector control lentiviral vector(pLV-NC group) were transfected into lung cancer A549 cells, respectively, and a blank control group(Blank group) was set up. The protein expressions of GATA4, p-ERK, ERK, p-p38 and p38 were detected by Western blot; cell viability was detected by CCK-8; cell proliferation was detected by cell clone formation assay; cell apoptosis was detected by flow cytometry. Results The methylation rate of GATA4 gene, expression levels of p-ERK, p-p38 and Bcl-2 proteins in NSCLC tissues were higher than those in paracancerous tissues(P<0.001), while the expression of GATA4 mRNA, GATA4  and Bax proteins were lower(P＜0.001). The methylation level of GATA4 gene was positively correlated with the expression of p-ERK, p-p38 and Bcl-2 proteins(P<0.05), and negatively correlated with the expression of GATA4 mRNA, GATA4 and Bax proteins(P<0.05). GATA4 was in a methylated state in NSCLC cells, while human normal lung epithelial cells BEAS-2B was in a demethylated state, and the GATA4 mRNA and protein expressions were lower than those of BEAS-2B cells(P<0.01). Compared with the pLV-NC group, the GATA4 mRNA and protein expression and apoptosis rate were increased in pLV-GATA4 group(P<0.001), while the cell viability, cell clone number, p-ERK/ERK and p-p38/p38 protein expression were decreased(P<0.05); GATA4 mRNA and protein expression and cell apoptosis rate in sh-GATA4 group were lower than those in sh-NC group(P<0.001), while cell viability, cell clone number, p-ERK/ERK and p-p38/p38 protein expression were higher(P<0.05). Conclusion GATA4 gene is hypermethylated in non-small cell lung cancer and in-duces a decrease in GATA4 gene expression. Overexpression of GATA4 may inhibit the proliferation and induce apoptosis of lung cancer cells by regulating MAPK pathway.