微囊藻毒素-LR诱导的恶性转化肝细胞miRNA表达谱的变化  

Differentially expressed mi RNA profile of hepatocyte malignant transformation induced by microcystin-LR

作　　者：朱雪凤[1,2] 农清清 范誉[1] 李江恒 贾雪姣 费梦雪[1] 马智星[1] 黄秋月 廖娟 胡新梅 ZHU Xue-feng;NONG Qing-qing;FAN Yu;LI Jiang-heng;JIA Xue-jiao;FEI Meng-xue;MA Zhi-xing;HUANG Qiu-yue;LIAO Juan;HU Xin-mei(Department of Environmental Health ,School of Public Health, Guongxi Medical University,Nanning, Guangxi 530021, China)

机构地区：[1]广西医科大学公共卫生学院环境卫生学教研室,广西南宁530021 [2]广西高校高发疾病预防与控制研究重点实验室

出　　处：《环境与健康杂志》2018年第1期10-13,F0003,共5页Journal of Environment and Health

基　　金：国家自然科学基金(81660529;81360420);广西自然科学基金(2015GXNSFAA139120)

摘　　要：目的构建微囊藻毒素-LR(microcystin-LR,MC-LR)诱导的人永生化肝细胞株(WRL68细胞)恶性转化模型,检测mi RNA表达谱,寻找差异表达的mi RNA。方法用10 nmol/L MC-LR对WRL68细胞进行连续染毒至第25代,进行软琼脂集落形成实验及裸鼠成瘤实验。通过mi RNA芯片技术检测和分析对照WRL68细胞和恶性转化细胞的mi RNA表达谱;采用实时荧光定量PCR对芯片结果加以验证;应用Target Scan在线软件预测mi RNA可能调控的靶基因。结果第25代MC-LR染毒组细胞在软琼脂中可以形成集落,能在裸鼠皮下形成肿瘤。芯片检测分析显示,表达差异显著的mi RNA有54个,表达上调有35个,表达下调有19个。实时荧光定量PCR结果显示,hsa-mi R-140-3p、hsa-mi R-19b-1-5p、hsami R-203a和hsa-mi R-494均下调(P<0.05),与芯片检测结果趋势一致。以生物信息学技术分析mi R-494可能调控的靶基因,结果预测到3个与肿瘤相关的潜在靶基因,分别是肿瘤坏死因子受体相关因子3(TNF receptor-associated factor 3,TRAF3)、微管相关肿瘤抑制基因(microtubule associated tumor suppressor 1,MTUS1)和结肠癌转移相关基因(metastasis associated in colon cancer 1,MACC1)。结论 MC-LR诱导肝细胞恶性转化过程中可引起mi RNA表达谱发生显著改变,差异表达的mi RNA可能在肝细胞恶性转化过程中起着重要作用。Objective To construct the malignant transformation of the WRL68 cell line induced by microcystin-LR （MC-LR）, and to identify the differentially expressed miRNAs. Methods Cultured WRL68 cells were continuously exposed to a lower concentration （10 nmol/L） of MC-LR for 25 passages, then the soft agar assay and nude mice tumor formation were carried out. A pilot miRNA expression array analysis was conducted on the malignant transformation cells, followed by validation of the selected miRNAs by RT-PCR. The target genes that may be regulated by miRNA were predicted by TargetScan online software. Results The soft agar assay showed that clonies formation were observed in the MC-LR-treated cells of passage 25. Tumor could be seen in head and neck of each nude mice in MC-LR-treated group in passage 25. The chip detection analysis showed that there were 54 differentially expressed miRNAs, of which 35 were up-regulated and 19 were down-regulation. Quantitative real-time PCR results showed that hsa-miR-140-3p ,hsa-miR-19b-l-5p, hsa-miR-203a and hsa-miR-494 were all down-regulated （P〈0.05）,consistent with the trend of chip results. Moreover, bioinformatics was used to analyze the target genes that might be regulated by miR-494 and three potential target genes associated with cancer were found,such as TNF receptor-associated factor 3 （TRAF3）, microtubule associated tumor suppressor 1 （MTUS1） and metastasis associated in colon cancer 1 （MACC1）. Conclusion MC-LR induces a significant change in miRNA expression profile during the malignant transformation of hepatocytes. And these miRNAs may play an important role in the malignant transformation of hepatocytes.

关 键 词：微囊藻毒素-LR 细胞恶性转化 MIRNA WRL68细胞 

分 类 号：R994.6[医药卫生—毒理学]