lncRNA LINC02859介导Wnt通路在甲基硝基亚硝基胍诱发胃癌中的机制研究  

lncRNA LINC02859 mediates Wnt pathway in N-methyl-N'-nitro-N-nitrosoguanidine-induced gastric cancer

作　　者：张雪玲 曾勇 王婷 王建丁 肖登 王俊玲[1] 李成云[1] ZHANG Xueling;ZENG Yong;WANG Ting;WANG Jianding;XIAO Deng;WANG Junling;LI Chengyun(Institute of Health Toxicology,School of Public Health,Lanzhou University,Lanzhou,Gansu 730000,China;Department of Infectious Diseases,Second Hospital of Lanzhou University,Lanzhou,Gansu 730000,China)

机构地区：[1]兰州大学公共卫生学院卫生毒理学研究所,甘肃兰州730000 [2]兰州大学第二医院院感科,甘肃兰州730030

出　　处：《环境与职业医学》2024年第10期1136-1143,共8页Journal of Environmental and Occupational Medicine

基　　金：兰州大学中央高校基本科研业务费专项资金项目(lzujbky-2022-25);甘肃省自然科学基金项目(23JRRA1077)

摘　　要：[背景]胃癌发生是多因素共同作用的复杂过程,长链非编码RNA(lncRNA)在胃癌进程中可作为致癌或抑癌基因发挥重要作用。研究发现,lncRNA LINC02859在胃癌组织中表达下调且与肿瘤的分化程度及病理分期相关,同时在环境致癌物甲基硝基亚硝基胍(MNNG)诱发细胞恶性转化的发生发展中发挥重要作用,但其作用机制尚不明确。[目的]探索胃癌相关lncRNA LINC02859基因在MNNG诱导人正常胃黏膜细胞(GES-1)恶性转化中的作用和潜在机制。[方法]选取甘肃省胃癌高发区110例胃癌患者作为生物标本来源,由医院专业病理医师采集患者癌组织及距肿瘤边缘5 cm以上癌旁正常胃黏膜组织,采用实时荧光定量PCR(RT-qPCR)检测癌及癌旁组织中LINC02859的表达水平。通过高通量测序及生物信息学分析发现与LINC02859共表达关系的基因潜在调控的信号通路。取细胞代数较低且形态正常的GES-1,待细胞融合达到70%~80%时,分别加入0、0.25和0.5μmol·L^(–1)的MNNG染毒液培养48 h后检测LINC02859基因表达水平,采用Cell Counting Kit-8(CCK-8)检测细胞增殖活性,平板细胞克隆形成试验检测细胞克隆形成,划痕实验检测细胞迁移能力,评估MNNG染毒对细胞形态及功能的影响;采用RT-qPCR及免疫印记实验(Western blotting)检测Wnt信号通路关键蛋白的表达水平。[结果]RT-qPCR结果显示,与癌旁组织比较,LINC02859在胃癌组织中呈低表达,差异有统计学意义(P<0.05);Pathway富集分析显示,LINC02859潜在调控Wnt通路。体外恶性转化实验提示,MNNG暴露后,恶性转化第五代细胞(MC-5)与对照组细胞相比,其形态发生改变、集落形成数量增加、增殖和迁移能力高于GES-1细胞;与正常GES-1细胞比较,0.25和0.5μmol·L^(–1)MNNG暴露的GES-1细胞,LINC02859基因表达量均降低,0.5μmol·L^(–1)MNNG暴露后,Wnt通路关键蛋白转录因子7(TCF7)、Axis抑制剂(Axin1)、磷酸化糖原合成酶激酶-3-Beta(p-GSK-[Background]Gastric carcinogenesis is a multifactorial and complex process,in which long noncoding RNAs(lncRNAs)play important roles as oncogenes or antioncogenes.Research has found that the expression of lncRNA LINC02859 is down-regulated in gastric cancer tissues and correlated with the degree of tumor differentiation and TNM stage,and also plays an important role in the development of malignant transformation of cells induced by environmental carcinogen NDOI methyl-N'-nitro-N-nitrosoguanidine(MNNG),but its mechanism of action is still unclear.[Objective]To explore the role and potential regulatory mechanism of gastric cancer-associated lncRNA LINC02859 in MNNG-induced malignant transformation of human normal gastric mucosal cells(GES-1).[Methods]A total of 110 gastric cancer patients from a high incidence area of gastric cancer in Gansu Province were selected,and their cancer tissues and normal gastric mucosa tissues adjacent to the cancer were collected to detect the expression level of LINC02859 by real-time quantitative PCR(RT-qPCR).High-throughput sequencing and bioinformatics analysis of the tissues were used to identify the potential signaling pathways regulated by the genes co-expressed with LINC02859.GES-1 cells at 70%-80%cell fusion with low cell passage number and normal morphology were incubated with 0,0.25 and 0.5μmol·L^(–1)MNNG solution for 48 h and the LINC02859 expression level was detected.Cell proliferation activity was detected by Cell Counting Kit-8(CCK-8),clone formation was detected by plate clone formation assay,and cell migration ability was detected by scratch assay to evaluate the effects of MNNG on cell morphology and function.The expression levels of key proteins of Wnt signaling pathway were detected by RT-qPCR and Western blotting.[Results]The RT-qPCR results showed that LINC02859 was lowly expressed in the gastric cancer tissues compared with the paracancerous tissues,and the difference was statistically significant(P<0.05).The pathway enrichment analysis showed that LINC02859

关 键 词：胃癌 lncRNA LINC02859 甲基硝基亚硝基胍 恶性转化 WNT通路 

分 类 号：R11[医药卫生—公共卫生与预防医学]