摘要
目的通过研究miRNA-145a-5p在肝前体细胞和小鼠感染乙肝病毒X(hepatitis B virus X,HBx)不同时间的变化情况,初步探讨HBx导致肝癌发生的机制。方法通过慢病毒将HBx基因转染到14-19肝前体细胞中,构建HBx-EGFP-14-19细胞系,并通过肝门静脉将HBx-EGFP-14-19细胞悬液注入KM小鼠体内,建立具有完整免疫系统的HBx小鼠模型,并分别于30、90、180、360 d收集肝脏组织。通过qRT-PCR和Western blot检测HBx在HBx-EGFP-14-19细胞和HBx小鼠中的表达。将miR-145a-5p mimics和inhibitor转染到HBx-EGFP-14-19细胞和饲养180 d HBx小鼠中,qRT-PCR检测miR-145a-5p、c-Myc、Kras、细胞凋亡和细胞周期因子的RNA表达,Western blot检测c-Myc、Kras、细胞凋亡和细胞周期因子的蛋白质表达,CCK-8检测细胞增殖。结果成功构建能够稳定表达HBx的细胞模型和动物模型。HBx过表达后,miR-145a-5p在HBx-EGFP-14-19细胞和HBx小鼠中的表达下调,而c-Myc和Kras的表达升高。使用miR-145a-5p模拟物(mimic/agomir)可以抑制细胞增殖并下调c-Myc、Kras、Bcl-2、CDK4、Cyclin E、CyclinD1的表达,并上调Bad和Bax的表达,而miR-145a-5p抑制剂(inhibitor/antagomir)与miR-145a-5p模拟物组的结果相反。结论 HBx过表达导致小鼠肝癌发生,其机制可能与HBx调节miR-145a-5p及其靶基因c-Myc和Kras的表达和抑制细胞凋亡有关。
Objective To investigate the changes of miRNA-145 a-5 p in liver progenitor cells and mice infected with hepatitis B virus X protein(HBx) at different time points to preliminarily explore the mechanism of HBx causing liver tumor. Methods HBx gene was transfected into 14-19 liver precursor cells by lentivirus vector to construct HBx-EGFP-14-19 cell line, and then the suspension of obtained cells was injected into KM mice through hepatic portal vein to establish an HBx model with a complete immune system. The liver tissues were collected on days 30, 90, 180, and 360. The expression of HBx in HBx-EGFP-14-19 cells and HBx mice was detected by qRT-PCR and Western blotting. After miR-145 a-5 p mimic and inhibitor was transfected into the HBx-EGFP-14-19 cells and HBx mice having been fed for 180 d, respectively, the RNA expression of miR-145 a-5 p, c-Myc, Kras, apoptosis-related and cell cycle-related factors was detected by qRT-PCR, and the protein expression of them were detected by Western blotting. The cell proliferation was detected by CCK8 assay. Results The cell model capable of stably expressing HBx and HBx mouse model were established successfully. Overexpression of HBx induced the down-regulation of miR-145 a-5 p in HBx-EGFP-14-19 cells and HBx mice, while enhanced the expression of c-Myc and Kras. MiR-145 a-5 p mimic/agomir inhibited cell proliferation and down-regulated the expression of c-Myc, Kras, Bcl-2, CDK4, CyclinE, CyclinD1, and up-regulated the expression of Bad and Bax, while miR-145 a-5 p inhibitor/antagomir led to the opposite results from the mimic. Conclusion Overexpression of HBx leads to the occurrence of liver tumor in mice, and its mechanism may be related to its regulation of miR-145 a-5 p and its target genes c-Myc and Kras and inhibition for cell apoptosis.
作者
杜彬
周梦瑶
毋楠
张思遥
黄欣
况钦
吴勇
冯涛
DU Bin;ZHOU Mengyao;WU Nan;ZHANG Siyao;HUANG Xin;KUANG Qin;WU Yong;FENG Tao(Molecular Medicine and Cancer Research Center,Biochemistry and Molecular Biology Research Center,College of Pharmacy,Chongqing Medical University,Chongqing,400016,China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2021年第18期1751-1761,共11页
Journal of Third Military Medical University