袁婷,韦四喜,夏英,夏万松,李红羽,杜洪,金泳,黄海
YUAN Ting,WEI Sixi,XIA Ying,XIA Wansong,LI Hongyu,DU Hong,JIN Yong,HUANG Hai

• 1:贵州医科大学附属医院临床检验中心
• 2:贵州医科大学
• 3:贵州中医药大学第一附属医院检验科
• 4:贵州省第二人民医院检验科

摘要(Abstract)：

目的探讨脑型糖原磷酸化酶(PYGB)对胃癌(GC)细胞凋亡的影响及其机制。方法收集29例初诊GC患者的一般临床资料和GC组织及距肿瘤边缘> 5 cm的癌旁组织作为组织标本,同时选取GC细胞系HGC-27、MGC-803、MKN45、AGS及人胃黏膜上皮细胞GES-1作为细胞标本,采用实时荧光定量PCR(RT-qPCR)检测2种组织和上述5种细胞中PYGB mRNA的表达,采用蛋白印迹法(Western blot)检测各种细胞PYGB蛋白的表达,采用χ~2检验分析PYGB mRNA表达与患者临床病理特征的关系;选择PYGB mRNA表达量最高的AGS细胞作后续研究,采用RNA干扰技术根据PYGB基因合成3对siRNA(si-PYGB 1～3)序列,以Lipofectamine~(TM)3000为转染试剂转染胃癌细胞AGS,分为Control组(空白对照)、si-NC组(阴性对照)、si-PYGB 1组(si-PYGB1)、si-PYGB 2组(si-PYGB 2)和si-PYGB 3组(si-PYGB 3),分别采用RT-qPCR和Western blot检测各组AGS细胞PYGB在mRNA和蛋白水平上的表达;保留干扰效果最好的si-PYGB 2组,采用流式细胞术检测si-NC组和si-PYGB 2组细胞周期、凋亡及细胞内活性氧(ROS)水平。结果与癌旁组织比较,GC组织中PYGB mRNA水平表达上调(P <0.05);与GES-1比较,GC细胞AGS和MKN45中PYGB mRNA和蛋白水平上表达上调、GC细胞HGC-27和MGC-803中表达下调(P <0.05);与si-NC组比较,si-PYGB 2组AGS细胞S期比例下降、G2/M期比例上升(P <0.05),同时细胞凋亡率上升、细胞内ROS水平增加(P <0.05)。结论沉默PYGB可诱导AGS细胞凋亡,其机制可能与细胞内ROS水平增加有关。
Objective To investigate the effect and mechanism of brain glycogen phosphorylase(PYGB) on the apoptosis of gastric cancer(GC) cells. Methods The cancer tissues(GC tissues) of 29 newly diagnosed GC patients and the adjacent tissues more than 5 cm from the tumor edge were selected as tissue specimens,and general clinical data of the patients were collected. At the same time,GC cell strains HGC-27,MGC-803,MKN45,AGS,and human gastric mucosal epithelial cells GES-1 were used as cell specimens. Real time fluorescent-quantitative PCR(RT-qPCR) was used to detect the expression of PYGB mRNA in these two tissues and the above mentioned 5 cells; Western blot was used to detect expression of PYGB protein in various cells. The χ~2 test was used to analyze the relationship between the expression of PYGB mRNA and the clinical pathological characteristics of the patients; selecting the AGS cells with the highest expression of PYGB mRNA for follow-up research.Using RNA interference technology to synthesize 3 pairs of siRNA(si-PYGB 1-3) sequences based on the PYGB gene,and transfecting the GC AGS cell with Lipofectamine~(TM)3000 as the transfection reagent [dividing them into control group(blank control),si-NC group(negative control),si-PYGB 1 group(si-PYGB 1),si-PYGB 2 group(si-PYGB 2),and si-PYGB 3 group(si-PYGB 3) ]. Using RT-qPCR and Western blot to detect the expression of PYGB mRNA and protein levels in AGS cell of each group respectively. Keeping 2 groups of si-PYGB with the best interference effect. Flow cytometry was adopted to detect cell cycle,apoptosis and intracellular reactive oxygen species(ROS) levels in si-NC group and si-PYGB 2 group. Results Compared with adjacent tissues,the expression of PYGB mRNA in GC tissues was up-regulated(P < 0. 05). Compared with GES-1,the expression of PYGB mRNA and protein level in GC cells AGS and MKN45 was up-regulated,and the expression in GC cells HGC-27 and MGC-803 was down-regulated(P < 0. 05). Compared with the si-NC group,the ratio of AGS cells in the S phase of the si-PYGB 2 group decreased,and the ratio of G2/M phase increased(P < 0. 05). At the same time,the apoptosis rate increased and the intracellular ROS level increased(P < 0. 05). Conclusion Silencing PYGB can induce apoptosis of AGS cells,and the mechanism may be related to the increase of intracellular ROS levels.

关键词(KeyWords)： 胃肿瘤;糖原磷酸化酶,脑型;RNA干扰;细胞周期;细胞凋亡;活性氧
stomach neoplasms;glycogen phosphorylase,brain-type;RNA interference;cell cycle;apoptosis;reactive oxygen species(ROS)

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(82060442);; 贵州省创新人才团队项目(2019-5610);; 贵州省卫生计生委科学技术基金项目(gzwjkj 2018-1-072);; 贵阳市科技计划项目[筑科合同(2019)9-2];; 贵阳中医学院科研项目[(2018)101]

作者(Author): 袁婷,韦四喜,夏英,夏万松,李红羽,杜洪,金泳,黄海
YUAN Ting,WEI Sixi,XIA Ying,XIA Wansong,LI Hongyu,DU Hong,JIN Yong,HUANG Hai

参考文献(References)：

• [1]SUNG H,FERLAY J,SIEGEL R L,et al.Global cancer statistics 2020:GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries[J].CA:a Cancer Journal for Clinicians,2021,70(4):313.
• [2]李源,吴钟华,林嗣涵,等.胃癌分子生物学研究现状[J].中国实用外科杂志,2021,4(1):66-71.
• [3]李明,张洪涛.进展期胃癌分子靶向治疗及免疫治疗的研究进展[J].癌症进展,2020,18 (17):1733-1735.
• [4]周东辉,吴璇,钭赉振,等.晚期胃癌分子靶向治疗和免疫治疗的研究进展[J].浙江医学,2020 (3):203-208.
• [5]KHAN T,SULLIVAN M A,GUNTER J H,et al.Revisiting glycogen in cancer:a conspicuous and targetable enabler of malignant transformation[J].Frontiers in Oncology,2020,10:592455.
• [6]MATHIEU C,DUPRET J M,RODRIGUESLIMA F.The structure and the regulation of glycogen phosphorylases in brain[J].Advances in Neurobiology,2019,23:125-145.
• [7]NEWGARD C B,HWANG P K,FLETTERICK R J.The family of glycogen phosphorylases:structure and function[J].Critical Reviews in Biochemistry and Molecular Biology,1989,24(1):69-99.
• [8]NEWGARD C B,LITTMAN D R,VAN G C,et al.Human brain glycogen phosphorylase.cloning,sequence analysis,chromosomal mapping,tissue expression,and comparison with the human liver and muscle isozymes[J].The Journal of Biological Chemistry,1988,263(8):3850-3857.
• [9]SHIMADA S,MAENO M,AKAGI M,et al.Immunohistochemical detection of glycogen phosphorylase isoenzymes in rat and human tissues[J].The Histochemical Journal,1986,18(6):334-338.
• [10]DURAN J,GUINOVART J J.Brain glycogen in health and disease[J].Molecular Aspects of Medicine,2015,46:70-77.
• [11]路春峰,迟晓明,李浩,等.糖原磷酸化酶同工酶脑型在脓毒症心肌损伤早期的诊断价值[J].临床急诊杂志,2019,20(12):931-934,940.
• [12]FAN Z,ZHANG Z,ZHAO S,et al.Dynamic variations in brain glycogen are involved in modulating isoflurane anesthesia in mice[J].Neuroscience Bulletin,2020,36(12):1513-1523.
• [13]XIA B,ZHANG K,LIU C.PYGB promoted tumor progression by regulating Wnt/β-Catenin pathway in gastric cancer[J].Technology in Cancer Research&Treatment,2020,19:1533033820926592.
• [14]赵娟娟,韦四喜,严芝强,等.胃癌长链非编码RNASNHG16异常表达及其临床病理意义[J].东南大学学报(医学版),2017(36):551-554.
• [15]石学淑,张永杰,祁向争.胃癌免疫治疗相关生物标志物的研究进展[J].癌症进展,2021,19(4):342-344.
• [16]王倩,商建,王晓月,等.晚期胃癌分子靶向药物治疗及免疫治疗新进展[J].武汉大学学报(医学版),2021,42(3):407-412.
• [17]WANG Z,HAN G,LIU Q,et al.Silencing of PYGBsuppresses growth and promotes the apoptosis of prostate cancer cells via the NF-κB/Nrf2 signaling pathway[J].Molecular Medicine Reports,2018,18 (4):3800-3808.
• [18]ZHANG S,ZHOU Y,ZHA Y,et al.PYGB siRNA inhibits the cell proliferation of human osteosarcoma cell lines[J].Molecular Medicine Reports,2018,18 (1):715-722.
• [19]郭培玲,马铭,闫兰芳,等.HIF-2α表达对低氧环境下胃癌细胞增殖、凋亡和细胞周期的影响[J].国际检验医学杂志,2021,42(8):975-978.
• [20]齐丽莎,王靖怡,王雅蕾,等.内复制及其在肿瘤发生发展中的作用[J].中国肿瘤临床,2018,45 (20):1071-1074.
• [21]WU X,HUANG W,QUAN M,et al.Inhibition of brain-type glycogen phosphorylase ameliorates high glucose-induced cardiomyocyte apoptosis via Akt-HIF-1αactivation[J].Biochemistry and Cell Biology,2020,98(4):458-465.
• [22]ZHOU Y,JIN Z,WANG C.Glycogen phosphorylase Bpromotes ovarian cancer progression via Wnt/β-catenin signaling and is regulated by miR-133a-3p[J].Biomedicine&Pharmacotherapy,2019,120:109449.
• [23]XIAO L,WANG W,HUANGFU Q,et al.PYGB facilitates cell proliferation and invasiveness in non-small cell lung cancer by activating the Wnt-β-catenin signaling pathway[J].Biochemistry and Cell Biology,2020,98(5):565-574.
• [24]ZHAN Y,CHEN R,WANG T,et al.Glycogen phosphorylase B promotes cell proliferation and migration through PI3K/AKT pathway in non-small cell lung cancer[J].Experimental Lung Research,2021,47(3):111-120.
• [25]CIRCU M L,AW T Y.Reactive oxygen species,cellular redox systems,and apoptosis[J].Free Radical Biology&Medicine,2010,48(6):749-762.
• [26]LEE W,LEE D G.Reactive oxygen species modulate itraconazole-induced apoptosis via mitochondrial disruption in candida albicans[J].Free Radical Research,2018,52(1):39-50.
• [27]CUI L,BU W,SONG J,et al.Apoptosis induction by alantolactone in breast cancer MDA-MB-231 cells through reactive oxygen species-mediated mitochondrion-dependent pathway[J].Archives of Pharmacal Research,2018,41(3):299-313.
• [28]GALADARI S,RAHMAN A,PALLICHANKANDY S,et al.Reactive oxygen species and cancer paradox:to promote or to suppress[J].Free Radical Biology and Medicine,2017,104:144-164.

文章评论(Comment)：