摘要
[目的]检测RNA干扰自噬相关基因16L1 (autophagy-related gene 16L1,ATG16L1)表达后胃癌细胞的自噬活性,观察在ATG16L1敲减下顺铂(DDP)对胃癌细胞凋亡的影响。[方法]用ATG16L1 siRNA技术构建ATG16L1低表达的人胃癌BGC823细胞株作为干扰组,转染阴性干扰的BGC823细胞作为阴性转染组,未转染的BGC823细胞作为对照组;Western blot检测各组细胞中ATG16L1的表达和自噬标志物微管相关轻链蛋白3(microtubule-associated protein 1A/1B-light chain 3,LC3)及死骨片蛋白1 (sequestosome 1,P62)的表达;免疫荧光观察LC3II情况。CCK-8实验检测DDP处理后各组细胞增殖能力,计算IC50值。经DDP刺激后,流式细胞术检测细胞凋亡,再用Western blot检测凋亡相关蛋白Bcl-2、Cleaved caspase-3、Cleaved caspase-9蛋白表达。最后应用公共数据库Kaplan-Meier对多组胃癌患者mRNA表达谱进行分析,预测ATG16L1的表达水平对患者预后的影响。[结果] ATG16L1 siRNA干扰组较对照组蛋白表达水平明显降低(P<0.001);LC3II水平下降(P<0.01),P62水平上升(P<0.01),并且LC3荧光强度下降(P<0.05)。经DDP诱导后,干扰组细胞增殖明显受抑(P<0.001),细胞凋亡率明显增加(P<0.01);ATG16L1干扰组中促凋亡分子cleaved caspase-3、cleaved caspase-9表达比对照组明显增加(P<0.001,P<0.01),抗凋亡蛋白Bcl-2表达减少(P<0.001)。公共数据库Kaplan-Meier表达谱芯片分析结果显示高表达ATG16L1组较低表达组预后差,差异有统计学意义(P=0.001)。[结论] RNA干扰ATG16L1抑制胃癌细胞自噬,并能够明显增强顺铂诱导人胃癌细胞的凋亡,提示靶向干扰ATG16L1抑制细胞自噬后可能通过调控细胞凋亡提高胃癌细胞对化疗药物的敏感性。
[Purpose]To investigate the effect of RNA knockdown of the ATG16 L1 gene on autophagy and cisplatin-induced apoptosis in gastric cancer cells.[Methods]Gastric cancer BGC-823 cells were transfected with ATG16 L1 siRNA or siRNA negtive control,thus the cells were divided into 3 groups:siRNA ATG16 L1,siRNA NC and control.Expression of autophagy-related proteins was detected by Western blot and immunofluorescence;CCK-8 assay was used to detect cell proliferation and IC50 value was calculated.The cells were treated with cisplatin(DDP),cell apoptosis was detected by flow cytometry,and the expressions of apoptosis-related proteins Bcl-2,cleaved caspase-3 and cleaved caspase-9 were detected by Western blot.Kaplan-Meier plotter was used to analyze the on-line database of gastric cancer patients.The value of ATG16 L1 mRNA expression in prognosis of gastric cancer patients was analyzed.[Results]The expression of ATG16 L1 protein significantly decreased(P<0.001),LC3 II levels were decreased(P<0.01),P62 expresstion was elevated(P<0.01)and the fluorescence intensity of LC3 II was decreased(P<0.01)in siRNA interference group,compared with the control group.With the downregulation of ATG16 L1 gene,cell proliferation was inhibited(P<0.001),the rate of apoptosis was increased(P<0.01),the expression of cleaved caspase-3,cleaved caspase-9 was over-expressed(P<0.001,P<0.01),but the expression of antiapoptotic protein Bcl-2 was down-regulated(P<0.001).The result of Kaplan-Meier plotter showed that the prognosis in the high expression of ATG16 L1 group was poorer(P=0.001).[Conclusion]Downregulation of ATG16 L1 gene inhibits autophagy and promotes cisplatin-induced apoptosis in human gastric cancer cell line BGC823.It suggests that ATG16 L1 may be associated with chemoresistance in human gastric cancer by regulating apoptosis.
作者
张玉梅
林方方
冯凡
陈琦
刘月琴
许文林
沈慧玲
ZHANG Yu-mei;LIN Fang-fang;FENG Fan;CHEN Qi;LIU Yue-qin;XU Wen-lin;SHEN Hui-ling(The Fourth Hospital,Jiangsu University,Zhenjiang212001,China;Medical College ofJiangsu University,Zhenjiang212013,China;People’s Hospital,Jiangsu University,Zhenjiang212002,China)
出处
《中国肿瘤》
CAS
CSCD
北大核心
2019年第8期636-642,共7页
China Cancer
基金
国家自然科学基金(81672913)
江苏省高层次卫生人才“六个一工程”科研项目(LGY2017021)
