miRNA-3677-3p介导HBx促肝癌肿瘤干性和化疗抵抗的机制研究  

Study on the mechanism of miRNA-3677-3p mediating HBx to promote hepatocellular carcinoma tumor dryness and chemotherapy resistance

作　　者：何恒正 全养雅[2] 程发辉 黎慧娟 周坚 HE Hengzheng;QUAN Yangya;CHENG Fahui;LI Huijuan;ZHOU Jian(Department of Minimally Invasive Surgery,Hunan Provincial Brain Hospital,Changsha,410007,China;Department of Gastroenterology,Hunan Provincial Brain Hospital;Department of Quality Control,Hunan Provincial Brain Hospital)

机构地区：[1]湖南省脑科医院微创外科,长沙410007 [2]湖南省脑科医院消化内科 [3]湖南省脑科医院质控科

出　　处：《中国中西医结合消化杂志》2022年第8期565-572,共8页Chinese Journal of Integrated Traditional and Western Medicine on Digestion

基　　金：长沙市自然科学基金项目(No:kq2014189)。

摘　　要：目的:探讨miRNA-3677-3p/FOXA3/Wnt/β-catenin通路介导HBx促进肝癌肿瘤干性和化疗抵抗的分子机制。方法:TargetScan预测miRNA-3677-3p的靶基因,培养HEK293T细胞后双荧光素酶实验分析miRNA-3677-3p与FOXA3的靶向关系。培养人肝癌细胞株Huh7和正常肝细胞株HL-7702,RT-qPCR检测细胞内miRNA-3677-3p及FOXA3的表达情况,分别过表达或干扰miRNA-3677-3p、FOXA3在Huh7细胞中的表达后,通过CCK-8、Transwell和流式细胞仪分别检测Huh7细胞增殖活力、侵袭数目和CD133、EpCAM阳性表达率。培养耐药的Huh7/ADM细胞,分析miRNA-3677-3p与FOXA3对细胞化疗抵抗的影响,Westernblot检测Wnt/β-catenin通路相关蛋白表达。上调HBx在Huh7细胞中的表达,分析Huh7细胞肿瘤干性和化疗抵抗,检测miRNA-3677-3p基因表达情况。最后分析下调miRNA-3677-3p表达对裸鼠成瘤的影响。结果:Huh7细胞中miRNA-3677-3p表达高于HL-7702细胞,FOXA3表达低于HL-7702细胞。下调miRNA-3677-3p表达降低了Huh7细胞增殖活力、侵袭数目和CD133、EpCAM的阳性表达,提高了细胞的化疗抵抗。miRNA-3677-3p靶向FOXA3,FOXA3表达上调降低了Huh7细胞增殖、侵袭数目和CD133、EpCAM的阳性表达,上调了细胞的化疗抵抗。上调miRNA-3677-3p靶向FOXA3激活了Wnt/β-catenin通路,并促进了Huh7细胞发展。过表达HBx后能够促进miRNA-3677-3p表达和Huh7细胞增殖与侵袭。下调miRNA-3677-3p表达在体内能抑制肿瘤发生。结论:miRNA-3677-3p/FOXA3/Wnt/β-catenin通路通过介导HBx调控肝癌肿瘤干性和化疗抵抗。Objective:To explores the molecular mechanism of miRNA-3677-3p/FOXA3/Wnt/β-catenin pathway mediating HBx to promote tumor stemness and chemotherapy resistance of liver cancer.Methods:TargetScan predicted the target genes of miRNA-3677-3p,and the target relationship between miRNA-3677-3p and FOXA3 was analyzed by dual luciferase experiments after culturing HEK293T cells.Human hepatoma cell line Huh7 and normal liver celline HL-7702 were cultured,and the expressions of miRNA-3677-3p and FOXA3 in the cells were detected by RT-qPCR.Overexpression or interference of miRNA-3677-3p and FOXA3 expression in Huh7 cells was performed,respectively.Afterwards,the proliferation activity,invasion number and positive expression rate of CD133 and EpCAM of Huh7 cells were detected by CCK-8,Transwel and flow cytometry,respectively.The drug-resistant Huh7/ADM cells were cultured,and the effects of miRNA-3677-3p and FOXA3 on the chemoresistance of cells were analyzed.Western blot was used to detect the expression of Wnt/β-catenin pathway-related proteins.The expression of HBx in Huh7 cells was up-regulated,the tumor stemness and chemotherapy resistance of Huh7 cells were analyzed,and the expression of miRNA-3677-3p gene was detected.Finally,the effect of down-regulated miRNA-3677-3p expression on tumorigenesis in nude mice was analyzed.Results:The expression of miRNA-3677-3p in Huh7 cells was higher than that in HL-7702 cells,and the expression of FOXA3 was lower than that in HL-7702 cells.Down-regulation of miRNA-3677-3p expression reduced the proliferation activity,invasion number and positive expression of CD133 and EpCAM in Huh7 cells,and improved the chemoresistance of cells.miRNA-3677-3p targeted FOXA3,and up-regulation of FOXA3 expression decreased the proliferation,invasion number and positive expression of CD133 and EpCAM in Huh7 cells,and improved the chemoresistance of cells.Up-regulation of miRNA-3677-3p targeting FOXA3 activated the Wnt/β-catenin pathway and promoted the development of Huh7 cells.Overexpression of HBx

关 键 词：miRNA-3677-3p FOXA3 WNT/Β-CATENIN 肝癌 

分 类 号：R735.7[医药卫生—肿瘤]