Nampt及高糖高胰岛素微环境对人顺铂耐药肺癌细胞株增殖和转移的影响

Effects of Nampt and High Glucose and High Insulin Microenvironment on Proliferation and Metastasis of Human Cisplatin-resistant Lung Cancer Cell Lines

目的:探究烟酰胺磷酸核糖转移酶(Nampt)及高糖高胰岛素(HG+HI)微环境对人顺铂耐药肺癌细胞增殖和转移的影响。方法:将人顺铂耐药细胞株A549/DDP分为6组(n=6):对照组(control)、高糖高胰岛素干预组(HH,使用添加30 mmol/L的葡萄糖和500 mU/L的胰岛素的培养基培养72 h)、分别转染sh-NC和sh-Nampt组(sh-NC和sh-Nampt,使用Lipofectamine 2000将sh-NC和sh-Nampt分别转染到细胞中,转染时间为48 h)、HH干预sh-NC和sh-Nampt组(HH+sh-NC和HH+sh-Nampt)。每组6个重复样本。qRT-PCR检测转染效率,MTT法检测细胞增殖,流式细胞仪检测细胞凋亡,Transwell检测细胞迁移和侵袭,qRT-PCR检测Nampt mRNA,Western blot检测Nampt、Bcl-2、Bax、MMP-2、MMP-9、p-PI3K、PI3K、p-AKT和AKT蛋白表达。结果:与对照组和sh-NC组比较,sh-Nampt组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量降低,而细胞凋亡率升高,Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平降低,Bax蛋白表达水平升高(P<0.005)。与对照组和sh-NC组比较,HH组和HH+sh-NC组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量升高,Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平升高,Bax蛋白表达水平降低(P<0.005)。与HH组和HH+sh-NC组比较,HH+sh-Nampt组的Nampt mRNA和蛋白表达水平、相对细胞活力、迁移和侵袭细胞数量降低,细胞凋亡率升高,Bcl-2、MMP-2、MMP-9、Nampt、p-PI3K和p-AKT蛋白表达水平降低,Bax蛋白表达水平升高(P<0.005)。结论:高糖高胰岛素微环境可能通过上调Nampt/PI3K/AKT信号通路诱导人顺铂耐药肺癌细胞的增殖和转移。

Objective:To reveal the effects of nicotinamide phosphoribosyltransferase(Nampt)and high glucose and high insulin(HG+HI)microenvironment on proliferation and metastasis of human cisplatin-resistant lung cancer cell lines.Methods:Human cisplatin resistant cell line A549/DDP was divided into 6 groups(n=6):control group,high glucose and high insulin intervention group(HH,cul-tured with 30 mmol/L glucose and 500 mU/L insulin for 72 h),sh-NC and sh-Nampt transfected groups(Lipofectamine 2000 was used to transfect sh-NC and sh-Nampt into cells respectively,the transfection time was 48 h),HH intervention sh-NC and sh-Nampt groups(HH+sh-NC and HH+sh-Nampt).Each group had 6 replicates.The transfection efficiency was detected by qRT-PCR.Cell proliferation was detected by MTT method,apoptosis was detected by flow cytometry,cell migration and invasion were detected by Transwell,Nampt mRNA was detected by qRT-PCR,and protein expression of Nampt,Bcl-2,Bax,MMP-2,MMP-9,p-PI3K,PI3K,p-AKT and AKT were detected by Western blot.Results:Compared with control group and sh-NC group,the expression level of Nampt mRNA and protein,rel-ative cell viability,the number of migratory and invasive cells decreased,the rate of apoptosis increased,the expression level of Bcl-2,MMP-2,MMP-9,Nampt,p-PI3K and p-AKT protein decreased,and the expression level of Bax protein increased in sh-Nampt group(P<0.05).Compared with control group and sh-NC group,the expression level of Nampt mRNA and protein,relative cell viability,the number of migratory and invasive cells increased,the expression level of Bcl-2,MMP-2,MMP-9,Nampt,p-PI3K and p-AKT protein in-creased,and the expression level of Bax protein decreased in HH group and HH+sh-NC group(P<0.05).Compared with HH group and HH+sh-NC group,the expression level of Nampt mRNA and protein,relative cell viability,the number of migratory and invasive cellsdecreased,the rate of apoptosis increased,the expression level of Bcl-2,MMP-2,MMP-9,Nampt,p-PI3K and p-AKT protein decreased,and the expression level of Bax protein increased in HH+sh-Nampt group(P<0.05).Conclusion:High glucose and high insulin microenvironment may induce proliferation and metastasis of human cisplatin-resistant lung cancer cells by up-regulating Nampt/PI3K/AKT signaling pathway.