摘要
目的:探讨钙网蛋白(CALR)在胃癌组织中的表达水平及其调节AGS、HGC-27细胞转移的作用机制。方法:收集65例胃癌患者肿瘤组织样本和16例大网膜转移组织样本,采用免疫组织化学法检测胃癌及大网膜转移组织中CALR的表达水平;通过小干扰RNA(siRNA)下调AGS和HGC-27细胞中CALR的表达,通过慢病毒转染技术过表达AGS和HGC-27细胞中的CALR,采用Transwell实验和细胞划痕实验检测CALR表达对AGS、HGC-27细胞侵袭和迁移的影响;采用活性氧(ROS)和线粒体三磷酸腺苷(ATP)荧光探针检测AGS、HGC-27细胞ROS水平和ATP荧光强度;采用线粒体膜电位检测试剂盒(JC-1)检测AGS、HGC-27细胞线粒体膜电位水平;Western blot法检测CALR、肌浆/内质网Ca^(2+)转运ATP酶A2(ATP2A2)、ATP酶Na+/K+转运亚基Beta 3(ATP1B3)和ATP合酶-偶联因子6(ATP5J)的蛋白表达水平;建立胃癌裸鼠腹腔移植瘤模型,探究CALR在体内对HGC-27细胞增殖的影响。结果:与癌旁组织比较,CALR在胃癌组织和大网膜转移组织中显著过表达(P<0.01);CALR过表达显著促进AGS、HGC-27细胞侵袭和迁移,而siRNA CALR则抑制胃癌细胞的侵袭和迁移(P<0.01);pcDNA3.1-CALR组AGS、HGC-27细胞中ATP荧光强度、线粒体膜电位水平均显著升高,ROS水平则降低;而siRNA-CALR组ATP荧光强度、线粒体膜电位水平均显著降低,ROS水平则升高(P<0.01);此外,与阴性对照组比较,pcDNA3.1-CALR组AGS、HGC-27细胞中CALR、ATP2A2、ATP1B3和ATP5J蛋白表达水平均显著升高,而siRNA-CALR组CALR、ATP2A2、ATP1B3和ATP5J蛋白表达水平均显著降低(P<0.01);在裸鼠模型中,敲减CALR可显著抑制肿瘤组织的生长,降低肿瘤组织中ATP2A2、ATP1B3和ATP5J蛋白的表达水平。结论:CALR在人胃癌组织及大网膜转移组织中过表达,CALR可通过调节线粒体膜电位和ATP合酶介导线粒体功能,影响胃癌AGS和HGC-27细胞的转移。
AIM:To investigate the expression level of calreticulin(CALR)in gastric cancer tissues and its mechanism of regulation of metastasis in AGS and HGC-27 cells.METHODS:Tumor samples from 65 patients with gastric cancer and greater omental metastatic tissues samples from 16 patients were collected,and the expression level of CALR in gastric cancer and greater omental metastatic tissues was examined by immunohistochemistry.The expression of CALR in AGS and HGC-27 cells was downregulated by siRNA,and overexpression of CALR in AGS and HGC-27 cells was achieved by lentivirus transfection.The effects of CALR expression on the invasion and migration of AGS and HGC-27 cells were determined by Transwell assays and cell Scratch assays.The fluorescence intensities of reactive oxygen species(ROS)and mitochondrial ATP in AGS and HGC-27 cells were detected using ROS and mitochondrial ATP fluorescent probes.The mitochondrial membrane potential was measured using a mitochondrial membrane potential assay kit with JC-1.Protein expression levels of CALR,ATPase sarcoplasmic/endoplasmic reticulum Ca^(2+)transporting 2(ATP2A2),ATPase Na^(+)/K^(+)transporting subunit beta 3(ATP1B3)and ATP synthase-coupling factor 6(ATP5J)in AGS and HGC-27 cells were detected by Western blot.The effect of CALR on the proliferation of HGC-27 cells in vivo was investigated by es-tablishing a xenograft model of gastric cancer in nude mice.RESULTS:Compared to paracancerous tissues,the relative expression of CALR protein was significantly increased in gastric cancer and greater omental metastatic tissues(P<0.01).CALR overexpression significantly promoted the invasion and migration of AGS and HGC-27 cells,while CALR siRNA in-hibited the invasion and migration of gastric cancer cells(P<0.01).The ATP fluorescence intensity and mitochondrial membrane potential of AGS and HGC-27 cells in the pcDNA3.1-CALR group were significantly increased,while ROS lev-els were decreased.In the siRNA-CALR group,ATP fluorescence intensity and mitochondrial membrane potential were significantly decreased,while ROS levels were increased(P<0.01).Compared with those in the negative control group,the protein expression levels of CALR,ATP2A2,ATP1B3 and ATP5J in AGS and HGC-27 cells in the pcDNA3.1-CALR group were significantly increased,while those in the siRNA-CALR group were significantly decreased(P<0.01).In the nude mouse model,CALR knockdown significantly inhibited tumor growth and reduced the protein expression levels of ATP2A2,ATP1B3 and ATP5J in tumor tissues.CONCLUSION:CALR can affect the metastasis of gastric cancer AGS and HGC-27 cells by regulating mitochondrial membrane potential and ATP synthase-mediated mitochondrial function.
作者
李宁
张美航
孟爱国
王沂
刘海颖
王彤
LI Ning;ZHANG Meihang;MENG Aiguo;WANG Yi;LIU Haiying;WANG Tong(Department of Laboratory,North China University of Science and Technology Affiliated Hospital,Tangshan 063000,China;Department of Personnel,North China University of Science and Technology,Tangshan 063000,China;Graduate School,North China University of Science and Technology,Tangshan 063000,China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2023年第11期1973-1983,共11页
Chinese Journal of Pathophysiology
基金
河北省2020年度医学科学研究课题计划(No.20200132)
河北省省属高等学校基本科研业务费研究项目(No.JQN2022021)。
关键词
胃癌
侵袭
迁移
钙网蛋白
线粒体功能障碍
gastric cancer
invasion
migration
calreticulin
mitochondrial dysfunction