摘要
目的:探讨不同剂量灵芝酸A(GAA)对非小细胞肺癌(NSCLC) PC9细胞的生物活性、糖酵解及其限速酶表达的影响,并阐明其作用机制。方法:体外培养NSCLC PC9细胞,将PC9细胞分为空白对照组、低剂量(25μmol·L^(-1)) GAA组、中剂量(50μmol·L^(-1)) GAA组和高剂量(100μmol·L^(-1)) GAA组。采用噻唑蓝(MTT)法检测各组PC9细胞存活率,Transwell小室实验检测各组PC9细胞迁移细胞数,葡萄糖检测试剂盒检测各组PC9细胞葡萄糖摄取量,三磷酸腺苷(ATP)检测试剂盒检测各组PC9细胞中ATP水平,乳酸检测试剂盒检测各组PC9细胞中乳酸水平,实时荧光定量PCR(RT-qPCR)法检测各组PC9细胞中己糖激酶2(HK2)和M2型丙酮酸激酶(PKM2) mRNA表达水平,Westernblotting法检测各组PC9细胞中HK2和PKM2蛋白表达水平。结果:MTT法,培养24、48和72 h时,与空白对照组比较,中和高剂量GAA组PC9细胞存活率明显降低(P<0.05);培养72 h时,与低剂量GAA组比较,高剂量GAA组细胞存活率明显降低(P<0.05)。Transwell小室实验,与空白对照组比较,中和高剂量GAA组细胞迁移数明显减少(P<0.05);与低剂量GAA组比较,高剂量GAA组细胞迁移数明显减少(P<0.05)。与空白对照组比较,中和高剂量GAA组PC9细胞中葡萄糖摄取量和乳酸水平均明显降低(P<0.05),高剂量GAA组PC9细胞中ATP水平明显降低(P<0.05)。RT-qPCR法,与空白对照组比较,中和高剂量GAA组PC9细胞中HK2和PKM2 mRNA表达水平均明显降低(P<0.05)。Western blotting法,与空白对照组比较,中和高剂量GAA组PC9细胞中HK2和PKM2蛋白表达水平明显降低(P<0.05)。结论:中和高剂量GAA可抑制PC9细胞增殖及迁移的生物活性,降低糖酵解作用,其作用机制可能与抑制关键限速酶HK2和PKM2的表达有关。
Objective:To discuss the effects of different doses of ganoderic acid A(GAA) on the biological activities,glycolysis,and the expression of rate-limiting enzymes of non-small cell lung cancer(NSCLC) PC9 cells,and to clarify the mechanism.Methods:The NSCLC PC9 cells were cultured in vitro and divided into blank control group,low dose(25 μmol·L~(-1)) of GAA group,medium dose(50 μmol·L~(-1)) of GAA group,and high dose(100 μmol·L~(-1)) of GAA group.The methylthiazolydiphenyltetrazolium(MTT) assay was used to detect the survival rates of the PC9 cells in various groups;Transwell chamber assay was used to detect the number of migration cells of the PC9 cells in various groups;the glucose uptakes of the PC9 cells in various groups were detected by glucose assay kit;the levels of ATP in the PC9cells in various groups were detected by ATP assay kit;the levels of lactic acid in the PC9 cells in various groups were detected by lactate assay kit;the expression levels of hexokinase 2(HK2) and pyruvate kinase M2(PKM2) mRNA in the PC9 cells in various groups were detected by real-time fluorescence quantitative PCR(RT-qPCR) method;the expression levels of HK2 and PKM2 proteins in the PC9 cells in various groups were detected by Western blotting method.Results:The MTT assay results showed that,at 24,48,and 72 h of culture,compared with blank control group,the survival rates of the cells in medium and high doses of GAA groups were significantly decreased(P<0.05).At 72 h of culture,compared with low dose of GAA group,the survival rate of the cells in high dose of GAA group was significantly decreased(P<0.05).The Transwell chamber assay results showed that compared with blank control group,the numbers of migration cells in medium and high doses of GAA groups were significantly decreased(P<0.05);compared with low doses of GAA group,the number of migration cells in high dose of GAA group was significantly decreased(P<0.05).Compared with blank control group,the glucose uptakes and levels of lactic acid in the cells in medium and high dose of GAA groups were significantly decreased(P<0.05),and the level of ATP in the cells in high dose of GAA group was significantly decreased(P<0.05).The RT-qPCR results showed that compared with blank control group,the expression levels of HK2 and PKM2 mRNA in the cells in medium and high doses of GAA groups were significantly decreased(P<0.05).The Western blotting results showed that compared with blank control group,the expression levels of HK2 and PKM2 proteins in the cells in medium and high doses of GAA groups were significantly decreased(P<0.05).Conclusion:Medium and high doses of GAA can inhibit the biological activities of proliferation and migration of the PC9 cells,reduce the glycolysis,and its mechanism may be related to the inhibition of the expressions of the key rate-limiting enzymes HK2 and PKM2.
作者
任爱华
董彦伯
苗润芝
吕俞娇
刘岩峰
REN Aihua;DONG Yanbo;MIAO Runzhi;LYU Yujiao;LIU Yanfeng(Department of Anatomy,School of Basic Medicial Sciences,Beihua University,Jilin 132013,China;Department of Surgery,School of Clinical Medical Sciences,Beihua University,Jilin 132013,China)
出处
《吉林大学学报（医学版）》
CAS
CSCD
北大核心
2024年第3期682-688,共7页
Journal of Jilin University：Medicine Edition
基金
吉林省教育厅科学技术研究项目(JJKH20220069KJ,JJKH20180355KJ)
北华大学省级大学生创新创业训练计划项目(202310201135)。
