敲除TBRG4对肺癌H1299细胞增殖的影响及其机制研究

Inhibitory effect of transfected transforming growth factor β regulator 4 knockdown on the proliferation of lung cancer H1299 cells

目的探讨敲除转化生长因子β调节因子4(TBRG4)对肺癌H1299细胞增殖的影响及其机制。方法培养肺癌H1299细胞、TBRG4敲除阴性对照及TBRG4敲除H1299细胞,分别命名为对照组、阴性对照组、TBRG4敲除组。采用细胞计数(CCK)-8试剂盒检测各组细胞的增殖情况。采用还原型谷胱甘肽(GSH)检测试剂盒检测各组细胞GSH的含量。采用实时荧光定量聚合酶链反应(qRT-PCR)检测各组细胞TBRG4、沉默信息调节因子2相关酶4(SIRT4)、c-Myc和谷氨酰胺酶1(GLS1)mRNA的表达。采用Western blot检测各组细胞TBRG4、SIRT4、c-Myc和GLS1蛋白的表达水平。结果CCK-8和GSH含量检测结果显示,对照组、阴性对照组、TBRG4敲除组H1299细胞的吸光度值分别为1.025±0.021、1.032±0.019、0.726±0.018,GSH的含量分别为(20.836±0.367)、(21.167±0.460)、(15.091±0.241)μmol/L,与对照组和阴性对照组相比,TBRG4敲除组的吸光度值、GSH含量均降低,差异均有统计学意义(P值均<0.001)。与对照组和阴性对照组相比,TBRG4敲除组TBRG4、c-Myc、GLS1基因mRNA和蛋白的相对表达水平均减少,SIRT4基因mRNA和蛋白的相对表达水平均增加,差异均有统计学意义(P值均<0.001)。结论敲除TBRG4可抑制肺癌H1299细胞增殖,其作用机制可能与促进SIRT4的表达、抑制c-Myc和GLS1的表达、阻断谷氨酰胺代谢有关。

Objective To investigate the effect of transforming growth factorβregulator 4(TBRG4)knockdown on glutamine metabolism and lung cancer H1299 cell proliferation and to elucidate the underlying mechanism.Methods H1299 cells,TBRG4 knockout negative control,and TBRG4 knockout H1299 cells were cultured and set as the control group,the negative control group,and the TBRG4 knockout group,respectively.The proliferation of cells was measured using cell counting kit(CCK)-8 assay,and the content of glutathione(GSH)was evaluated using GSH assay.The mRNA expression levels of TBRG4,silencing information regulator 2-related enzyme 4(SIRT4),c-Myc,and glutaminase 1(GLS1)genes were detected using quantitative real-time polymerase chain reaction.The protein expression levels of TBRG4,SIRT4,c-Myc,and GLS1 were measured using western blot.Results CCK-8 and GSH assays showed that the optical density values of H1299 cells in the control group,negative control group,and TBRG4 knockout group were 1.025±0.021,1.032±0.019,and 0.726±0.018,respectively,and the GSH contents were(20.836±0.367),(21.167±0.46),and(15.091±0.241)μmol/L,respectively.Compared with the those of the control group and the negative control group,the OD value and GSH content of the TBRG4 knockout group significantly decreased(all P values<0.001).Compared with the control group and the negative control group,the TBRG4 knockout group showed significantly lower mRNA and protein expression levels of TBRG4,c-myc,and GLS1 and significantly higher mRNA and protein expression levels of SIRT4(all P values<0.001).Conclusion TBRG4 knockout inhibits the proliferation of lung cancer H1299 cells possibly by promoting the expression of SIRT4 and inhibiting the expression of c-Myc and GLS1 to block glutamine metabolism.