乳酸对人非小细胞肺癌细胞A549生物学特性影响的研究

Effects of lactic acid on biological characteristics of human non-small cell lung cancer cell A549

背景肿瘤的重要特征之一是代谢异常,乳酸作为代谢产物也参与肿瘤代谢。目的探讨乳酸对非小细胞肺癌(non-small cell lung cancer,NSCLC)细胞A549糖酵解压力、增殖、迁移和周期等生物学特性的影响。方法培养人非小细胞肺癌细胞A549,分为对照组、5 mmol/L乳酸处理组和10 mmol/L乳酸处理组。对照组不作任何处理,其他两组乳酸处理24 h后,通过Seahorse细胞代谢分析仪分析乳酸处理后A549细胞的糖酵解压力和线粒体底物选择,qPCR检测糖酵解相关基因(HK3、LDHA、LDHB、PKM)和细胞周期及迁移相关基因(CCNE1、CCND1、CCNB1、CDK2、CDH1、TWIST1、SNAI2)mRNA表达水平,Western blot检测糖酵解相关蛋白(G6PD、PKM2、HK1和LDHA)水平。利用实时无标记细胞分析(real time cellular analysis,RTCA)、流式细胞术分析乳酸对NSCLC细胞A549增殖、迁移、周期等生物学特性的影响。结果两个乳酸处理组相较对照组糖酵解降低,糖酵解最大能力及糖酵解储备均降低且10 mmol/L乳酸处理组比5 mmol/L组降低更显著(P<0.05),糖酵解关键酶在mRNA和蛋白水平下调。10 mmol/L乳酸处理组与对照组相比,对脂肪酸的依赖性增强,对谷氨酰胺的氧化能力减弱(P<0.05);RTCA结果显示两个乳酸处理组相较对照组细胞迁移降低,增殖能力降低,10 mmol/L乳酸处理组较5 mmol/L组增殖和迁移能力下降更为显著(P<0.05)。两个乳酸处理组CCNE1、CCND1、CCNB1、TWIST1基因m RNA水平下调,CDK2、CDH1基因mRNA水平上调。流式结果显示10 mmol/L乳酸处理组G0/G1期细胞增多(P<0.01),S期细胞数量降低(P<0.05),G2/M期细胞数量降低(P<0.05)。结论加入外源乳酸处理后,A549细胞糖酵解能力降低,且与乳酸浓度呈负相关,细胞氧化底物变化,随着乳酸浓度的增加其细胞增殖及迁移能力显著降低,通过细胞周期结果分析后推测加入外源乳酸可能使A549细胞抑制在G1期。

Background One of the important characteristics of tumor is abnormal metabolism.As a metabolite,lactic acid also participates in metabolism.Objective To investigate the effects of lactic acid on glycolysis pressure,proliferation,migration and cycle of A549 non-small cell lung cancer cells(NSCLCs).Methods Human non-small cell lung cancer cell A549 was cultured and divided into control group,5 mmol/L and 10 mmol/L lactic acid treatment groups.The control group did not receive any treatment.After 24 hours of lactic acid treatment in the other two groups,the glycolytic stress and mitochondrial substrate selection in A549 cells after lactate treatment were analyzed by Seahorse Cell Metabolism Analyzer.The mRNA level of glycolysis related genes(HK3,LDHA,LDHB,PKM)and cell cycle and migration related genes(CCNE1,CCND1,CCNB1,CDK2,CDH1,TWIST1,SNAI2)were detected by qPCR,Western blotting was used to detect the level of glycolytic related proteins(G6PD,PKM2,HK1 and LDHA).Real time cellular analysis(RTCA)and flow cytometry were used to analyze the effects of lactic acid on proliferation,migration,cycle and other biological characteristics of NSCLC A549 cells.Results Compared with the control group,the glycolysis of the two lactic acid treatment groups decreased,the maximum capacity of glycolysis and glycolysis reserve decreased,and the reduction of the 10 mmol/L lactic acid treatment group was more significant than that of the 5 mmol/L group(P<0.05).The key enzymes of glycolysis were down-regulated at the level of mRNA and protein.Compared with the control group,the 10 mmol/L lactic acid treatment group was more dependent on fatty acids,and its oxidation ability to glutamine was decreased(P<0.05);RTCA results showed that the cell migration and proliferation ability of the two lactic acid treatment groups were lower than those of the control group,and the proliferation and migration ability of the 10 mmol/L lactic acid treatment group was significantly lower than that of the 5 mmol/L group(P<0.05).The mRNA levels of CCNE1,CCND1,CCNB1 and TWIST1 genes were down-regulated and the mRNA levels of CDK2 and CDH1 genes were up-regulated in the two lactic acid treatment groups.Flow cytometry showed that the number of G0/G1 phase cells increased(P<0.01),while the number of S phase cells and G2/M phase cells decreased(P<0.05,respectively).Conclusion After adding exogenous lactic acid treatment,the glycolysis ability of A549 cells decreases,and it is negatively correlated with the concentration of lactic acid.The change of the oxidation substrate of the cells decreases significantly with the increase of the concentration of lactic acid,which suggests that adding exogenous lactic acid can inhibit A549 cells in G1 phase through the analysis of cell cycle results.