miR-660-5p通过靶向ZBTB4/HK2轴对肝癌细胞增殖、迁移、侵袭与糖酵解的影响  

miR-660-5p Promotes the Proliferation,Migration,Invasionand Glycolysis of Hepatocellular Carcinoma Cells by Targeting theZBTB4/HK2 Axis

作　　者：金丽娟 连志强 马晓燕 王刚 范海涛 杨俊 张爱芸[2] IN Lijuan;LIAN Zhiqiang;MA Xiaoyan;WANG Gang;FAN Haitao;YANG Jun;ZHANG Aiyun(Ningxia Hui Autonomous Region Combined Hospital of Traditional Chinese and Western Medicine,Yinchuan 750011,China;General Hospital of Ningxia Medical University,The First Clinical Medical College of Ningxia Medical University,Yinchuan 750004,China)

机构地区：[1]宁夏回族自治区中西医结合医院,银川750011 [2]宁夏医科大学总医院,宁夏医科大学第一临床医学院,银川750004

出　　处：《宁夏医科大学学报》2025年第1期14-22,共9页Journal of Ningxia Medical University

基　　金：宁夏卫生健康系统科学研究课题(2021-NW-026)。

摘　　要：目的 探究miR-660-5p/ZBTB4/HK2轴在肝癌细胞增殖、迁移、侵袭和糖酵解中的作用。方法 选择人正常肝上皮THLE-2细胞和肝癌Huh-7、MHCC97H、HepG2细胞,验证ZBTB4表达水平。在ZBTB4表达水平适中的肝癌细胞中转染ZBTB4过表达载体或siRNA,使用CCK-8、Transwell侵袭、划痕、乳酸生成和葡萄糖摄取实验检测细胞增殖、迁移、侵袭能力和糖酵解水平。在转染ZBTB4过表达载体的肝癌细胞同时联合HK2过表达载体共转染,验证ZBTB4调控HK2介导糖酵解。通过双荧光素酶实验检测miR-660-5p与ZBTB4的靶向关系,并在细胞中共转染miR-660-5p inhibitor和ZBTB4 siRNA,验证miR-660-5p对ZBTB4的调控作用。结果 ZBTB4在3种肝癌细胞中的mRNA和蛋白表达均低于THLE-2细胞(P均<0.01),其在MHCC97H细胞中表达适中。与Vector组相比,ZBTB4 OE组ZBTB4C mRNA和蛋白表达均上调、HK2表达均下调(P均<0.05),24 h、48 h和72 h细胞OD值均降低(P均<0.05),迁移率、侵袭细胞数、乳酸含量和葡萄糖摄取量均减少(P均<0.05)。与过表达ZBTB4相比,干扰ZBTB4后MHCC97H细胞的迁移率、侵袭细胞数、乳酸含量和葡萄糖摄取量均升高(P均<0.05)。进一步,在转染ZBTB4过表达载体的同时共转染HK2过表达载体,HK2表达上调,迁移率、侵袭细胞数、细胞OD值、乳酸含量和葡萄糖摄取量均上调(P均<0.05)。双荧光素酶实验证实ZBTB4是miR-660-5p的靶基因(P<0.01),而共转染miR-660-5p inhibitor和ZBTB4 siRNA后,ZBTB4表达下调、HK2表达上调,细胞OD值、侵袭细胞数和乳酸含量均上调(P均<0.05)。结论 miR-660-5p通过调控ZBTB4/HK2轴促进肝癌细胞增殖、迁移、侵袭和糖酵解水平。Objective To investigate the role of miR-660-5p/ZBTB4/HK2 axis in the proliferation,migration,invasion and glycolysis of hepatocellular carcinoma cells.Methods Human normal hepatic epithelial THLE-2 cells and hepatocellular carcinoma Huh-7,MHCC97H and HepG2 cells were selected to verify ZBTB4 expression.Subsequently,ZBTB4 overexpression vectors or siRNAs were transfected in hepatocellular carcinoma cells with moderate ZBTB4 expression.CCK-8,transwell invasion,scratch,lactate production and glucose uptake assays were performed to detect the cell proliferation,invasion,migration and glycolysis.Next,ZBTB4 overexpression vector was co-transfected with HK2 overexpression vector to verify the regulatory relationship between ZBTB4 and HK2 for glycolysis.Finally,dual luciferase assay was performed to detect the targeting relationship between miR-660-5p and ZBTB4,and miR-660-5p inhibitor and ZBTB4 siRNA were co-transfected in cells to verify the regulatory effect of miR-660-5p on ZBTB4.Results The expression of ZBTB4 was significantly lower in all three types of hepatocellular carcinoma cells than that of THLE-2 cells(P all<0.01),and its expression was moderate in MHCC97H cells.Compared with the veotor group,ZBTB4 expression was up-regulated and HK2 expression was down-regulated in the ZBTB4 overexpression group(P all<0.05).The OD values of the cells were reduced at 24 h,48 h,72 h,and the migration rate,the number of invasive cells,the lactate acid content,and the glucose uptake were all reduced(P all<0.05).In contrast to overexpression of ZBTB4,the results of migration rate,number of invasive cells,lactate content and glucose uptake of MHCC97H cells after interference with ZBTB4 were increased(P all<0.05).Further,co-transfection of HK2 overexpression vector along with ZBTB4 overexpression vector resulted in up-regulation of HK2 expression,and up-regulation of migration rate,number of invading cells,OD value,lactate content and glucose uptake(P all<0.05),and dual-luciferase assay confirmed that ZBTB4 was a target gene of

关 键 词：肝癌 miR-660-5p 锌指和BTB结构域4 糖酵解 

分 类 号：R735.7[医药卫生—肿瘤]