摘要
目的探讨过氧化物氧化还原酶蛋白1(PRDX1)对人胃癌细胞株的影响及分子机制.方法采用实时定量聚合酶链反应(Real-time PCR)和蛋白质印迹法(Western blot)检测PRDX1在人胃癌细胞株SGC7901、AGS及正常胃黏膜细胞GES-1的表达.合成针对PRDX1的小干扰RNA(siRNA)并制备慢病毒载体LV-PRDX1-siRNA,转染AGS细胞.噻唑蓝(MTT)法检测细胞的活性;流式细胞术(FCM)检测各组细胞凋亡率及线粒体膜电位;Real-time PCR及Westem blot检测凋亡、自噬相关基因的mRNA和蛋白表达水平.结果 SGC7901、AGS细胞中PRDX1的mRNA(1.049±0.124、1.597±0.103)和蛋白(0.873 ±0.135、1.083±0.204)的相对表达水平明显高于GES-1细胞(mRNA:0.451 ±0.060、蛋白:0.305±0.022),差异有统计学意义(F=200.162、48.432,P<0.01).LV-PRDX1-siRNA转染AGS细胞后,细胞的凋亡率[48 h:(19.383 ±2.010)%,96 h:(26.875±4.337)%]明显高于空白对照组[48 h:(4.648±1.146)%、96 h:(8.849 ±1.361)%]、阴性病毒对照组[48 h:(5.539 ±0.778)%、96 h:(9.414±1.183)%,F=206.260、85.681,P<0.01],线粒体膜电位明显降低(F=29.508,P<0.01).LV-PRDX1-siRNA转染后AGS细胞的凋亡相关蛋白B细胞淋巴瘤/白血病-2(bcl-2)、生存素(Survivin)表达降低,bcl-2相关X蛋白(bax)表达增高(F =44.219、32.780、74.801,P<0.01),黑色素瘤凋亡抑制蛋白(Livin)变化不明显(F =0.250,P>0.05);转染后自噬相关蛋白Beclin-1、LC-3Ⅱ/LC-3 Ⅰ均明显增高(F=30.332、24.260,P<0.01).结论 PRDX1在胃癌细胞中表达增高,抑制PRDX1表达能够通过调控凋亡和自噬相关基因而促进细胞凋亡及自噬.
Objective To investigate the effect and molecular mechanism of peroxiredoxin 1 (PRDX1) in human gastric cancer cell lines. Methods The expression of PRDX1 was detected in gastric cancer cell lines SGC7901, AGS and the normal gastric mucosa cell line GES-1 with real-time quantitative polymerase chain reaction (Real-time PCR). Small interfering RNA to PRDX1 was synthetized for the transfection with lentivirus vector of LV-PRDX1-small interfering RNA (siRNA), which was transfected into AGS. Methyl thiazol tetrazolium (MTT) assay was used to test activity of cells. Flow cytometry (FCM) was used to detect the apoptosis rate and mitochondrial trans-membrane potential. Real-time PCR and Western blotting were used to detect the expression of mRNAs and proteins associated with apoptosis and autophagy. Results The expression of PRDX1 mRNA (1.049±0.124, 1.597±0.103) and proteins (0.873±0.135, 1.083±0.204) in SGC7901, AGS cells was significantly higher than that in GES-1 cells (mRNA: 0.451±0.060, and protein: 0.305±0.022), and the difference was significant (F=200.162, 48.432, P<0.01). Results showed that apoptosis rate of AGS cells in LV-PRDX1-siRNA group [48 h:(19.383±2.010)%;96 h:(26.875±4.337)%] increased significantly as compared with blank control group [48 h:(4.648±1.146)%;96 h:(8.849±1.361)%] and negative control group [48 h:(5.539±0.778)%;96 h:(9.414±1.183)%, F=206.260, 85.681, P<0.01], and the mitochondrial trans-membrane potential decreased significantly (F=29.508, P<0.01). After transfection, the expression of apoptosis associated genes bcl-2 and Survivin decreased significantly, and that of bax increased significantly (F=44.219, 32.780, 74.801;all P<0.01). For autophagy associated proteins, Livin expression had no significant change (F=0.250, P>0.05), and the expression of Beclin-1 and LC-3Ⅱ/LC-3Ⅰ increased significantly (F=30.332, 24.260, P<0.01). Conclusion PRDX1 was up-regulated in gastric cancer cells, and inhibition of PRDX1 can regulate apoptosis and autophagy via regulating apoptosis and a
作者
檀碧波
李勇
赵群
范立侨
刘庆伟
Tan Bibo;Li Yong;Zhao Qun;Fan Liqiao;Liu Qingwei(Third Department of Surgery, the Fourth Hospital of Hebei Medical University, Shijiazhuang 050011, China)
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2019年第8期1418-1421,共4页
Chinese Journal of Experimental Surgery
基金
河北省医学科学研究重点课题计划项目(20180484).
