RG108调控TFPI-2甲基化/TMPRSS4表达对NSCLC细胞增殖和凋亡的影响

RG108 affects the proliferation and apoptosis of NSCLCcells by regulating TFPI-2 methylation and TMPRSS4 expression

目的:探讨RG108对人非小细胞肺癌(non-small cell lung cancer,NSCLC)A549和H1299细胞增殖、凋亡的影响及其可能的作用机制。方法:体外培养A549和H1299细胞,经不同浓度RG108处理后,采用MTT法、流式细胞术分别检测细胞增殖率、细胞周期和凋亡水平;qPCR和Western blotting(WB)法检测细胞内TFPI-2 mRNA和蛋白的表达及TMPRSS4的表达量,以甲基化特异性PCR和比色法检测细胞中TFPI-2启动子区域甲基化的状态和程度;分别采用siRNA-TFPI-2和pcDNA3.0-TMPRSS4质粒敲减TFPI-2或过表达TMPRSS4,然后检测细胞增殖率及凋亡率的变化。结果:RG108处理后,A549和H1299细胞的增殖率显著降低(均P<0.05)、细胞周期阻滞于G1/S期(均P<0.05)而凋亡率显著增加(均P<0.01),细胞中TFPI-2 mRNA及蛋白表达水平均显著升高(P<0.01和P<0.05),同时细胞中TFPI-2启动子区域甲基化程度显著降低(均P<0.05)、TMPRSS4的表达也明显减少(P<0.05)。沉默TFPI-2表达后,A549和H1299细胞的增殖水平显著增加(均P<0.05),而转染pcDNA3.0-TMPRSS4质粒则显著降低细胞的凋亡率(均P<0.05)。结论:RG108能够通过抑制TFPI-2甲基化负向调控TMPRSS4表达进而抑制A549和H1299细胞的增殖,并促进其凋亡。

Objective:To investigate the effect of RG108 on the proliferation and apoptosis of human non-small cell lung cancer(NSCLC)cell lines(A549,H1299)and explore its molecular mechanism.Methods:A549 and H1299 cells were cultured in vitro and treated with different concentrations of RG108.The cell proliferation,cell cycle and apoptosis were detected by MTT assay and Flow cytometry,respectively.qPCR and Western blotting(WB)were used to detect the TFPI-2 mRNA and protein expressions as well as the expression of TMPRSS4 in cells.Meanwhile,the methylation status and degree of TFPI-2 promoter in cells were detected with Methylation-specific PCR(MSP)and colorimetry.Finally,siRNA-TFPI-2 and pcDNA3.0-TMPRSS4 plasmids were used to silence TFPI-2 or overexpress TMPRSS4,and then the changes in cell proliferation and apoptosis were detected.Results:After treatment with RG108,the proliferation rate of A549 and H1299 cells were significantly decreased(all P<0.05),while the apoptosis rate were significantly increased(P<0.05),the cell cycle were arrested in G1/S phase(P<0.05),and the intracellular mRNA and protein expressions of TFPI-2 were significantly increased(P<0.01 or P<0.05).Meanwhile,the methylase degree in TFPI-2 promoter region and the expression of TMPRSS4 in cells were all significanly decreased(all P<0.05).After TFPI-2 silence,the proliferation levels of A549 and H1299 cells were significantly increased(all P<0.05);however,the apoptosis rate of A549 and H1299 cells were significantly reduced after transfection with pcDNA3.0-TMPRSS4(all P<0.05).Conclusion:RG108 can inhibit proliferation of A549 and H1299 cells and promote apoptosis by inhibiting the methylation of TFPI-2 and negatively regulates the expression of TMPRSS4.