MAP4K4促进肺腺癌恶性生物学行为的实验研究

Experimental study on MAP4K4 in promoting malignant biological behavior of lung adenocarcinoma

目的 探究丝裂原激活蛋白激酶4(MAP4K4)对肺腺癌恶性生物学行为的影响。方法 使用四甲基偶氮唑盐比色法(MTT)分别检测肺腺癌患者和健康志愿者肺组织中MAP4K4的表达情况;通过Westernblot方法确定MAP4K4蛋白在肺腺癌患者中的表达情况,并筛选MAP4K4高表达细胞系作为研究对象,通过慢病毒转染沉默/上调细胞MAP4K4蛋白表达,分析MAP4K4基因对肺腺癌细胞的增殖作用;采用transwell小室实验,检测MAP4K4基因对肺腺癌细胞的侵袭能力的影响;利用Westernblot方法检测上调/沉默MAP4K4蛋白表达,对下游信号蛋白c-Jun氨基末端激酶/磷酸化c-Jun氨基末端激酶(JNK/p-JNK)、蛋白激酶/磷酸化蛋白激酶(Erk/p-Erk)、人基质金属蛋白酶2(MMP-2)、人基质金属蛋白酶9(MMP-9)表达的影响。结果 肺腺癌患者的癌变组织样本MAP4K4蛋白表达量高于健康人群组织样本(P <0. 05);对多种肺腺癌细胞系分析发现H1795细胞的MAP4K4表达量最高;MTT结果显示上调MAP4K4表达,H1795细胞增殖能力升高,同时细胞的侵袭能力明显提升,MMP-2、MMP-9、p-Erk和p-JNK蛋白表达显著增加;而沉默MAP4K4基因能获得相反的结果。结论 MAP4K4通过调控JNK和Erk信号通路蛋白,并上调MMP-2、MMP-9蛋白表达量,促进肺腺癌的恶性生物学行为。

Objective To explore effects of MAP4K4 on promoting malignant biological behavior of lung adenocarcinoma.Methods The expression of MAP4K4 in lung tissues of patients with lung adenocarcinoma and healthy volunteers was detected by MTT.The expression of MAP4K4 protein in patients with lung adenocarcinoma was determined by western blot method.The MAP4K4 high expression cell lines were screened as research objects.The expression of MAP4K4 protein was silenced/up-regulated by lentiviral transfection.The proliferation effect of MAP4K4 gene on lung adenocarcinoma cells was analyzed by MTT method.The transwell chamber and scratch assay were applied to detect effects of MAP4K4 gene on invasion and migration of lung adenocarcinoma cells.Western blot method was applied to detect effects of up-regulated/silent MAP4K4 protein expression on downstream signaling protein c-Jun NH2-terminalprotein kinase/p-c-Jun N-terminal kinase(JNK/p-JNK),protein kinase/phosphorylated protein kinases(Erk/p-Erk),matrix metalloproteinase-2(MMP-2)and matrix metalloproteinase-9(MMP-9).Results The MAP4K4 protein content in cancerous tissue samples of patients with lung adenocarcinoma was higher than that in tissue samples of healthy people(P<0.05).The expression of MAP4K4 in H1795 cells was the highest in various lung adenocarcinoma cell lines.MTT results showed that the proliferation ability of H1795 cells was related to MAP4K4 expression,and the higher the MAP4K4 protein expression,the stronger the cell proliferation ability.At the same time,the invasion ability and migration ability of the cells were significantly improved.Western blot analysis showed that up-regulation of MAP4K4 protein expression could significantly increase expression of MMP-2,MMP-9,p-Erk and p-JNK protein.Silencing the MAP4K4 gene could achieve the opposite effect.Conclusion MAP4K4 protein promotes the malignant biological behavior of lung adenocarcinoma by regulating JNK and Erk signaling pathways and up-regulating expression of MMP-2 and MMP-9 proteins.