外泌体传递血管生成抑制蛋白-2参与乙肝后发生肝癌的机制  

The Mechanism of Exosomal Delivery of the Vasohibin 2 in Post-hepatitis B Liver Cancer Development

作　　者：宋欢欢[1] 韦朝阳 赵蕾[1] 聂尚燕[1] 郭香娟[1] 王敏[1] 张肖[1] 卢力飞 SONG Huan-huan;WEI Zhao-yang;ZHAO Lei;NIE Shang-yan;GUO Xiang-juan;WANG Min;ZHANG Xiao;LU Li-fei(Department of Infectious Diseases,Handan City Central Hospital;Department of Internal MedicineⅡ,Lingbei Hospital of General Universal China Coal Group,Handan 056000,China)

机构地区：[1]邯郸市中心医院感染性疾病科 [2]通用环球中煤岭北医院内二科,河北邯郸056000

出　　处：《南昌大学学报（医学版）》2024年第6期85-91,共7页Journal of Nanchang University：Medical Sciences

基　　金：河北省卫生健康委员会科研项目(20220689);邯郸市科学技术研究与发展计划项目(19422083020ZC)。

摘　　要：目的分析肝细胞肝癌(HCC)患者血清中感染乙型肝炎病毒X蛋白(HBx)的HepG2(HepG2.2.15)细胞外泌体(Exo)微小RNA-200(miRNA-200)的表达情况,及HepG2.2.15细胞中血管生成抑制蛋白-2(VASH2)表达水平,探讨miRNA-200、VASH2在HepG2细胞侵袭、迁移中的作用。方法选取HCC患者15例(研究组)和健康受试者15例(对照组),比较2组血清Exo miRNA-200表达水平。从HepG2.2.15细胞中分离出Exo,随后将其分为过表达组(转染miRNA-200 mimic)和阴性对照组(转染miRNA-NC),采用透射电镜、Western blot和纳米颗粒跟踪进行Exo鉴定,采用qRT-PCR、Western blot检测各组HepG2.2.15细胞中VASH2表达情况。在补充Exo的HepG2细胞中分析过表达Exo组(miRNA-200过表达组的Exo)和对照组(未转染组的Exo)细胞的增殖、迁移、侵袭及细胞周期情况。在转染后12、24、48、72 h采用CCK-8法评估HepG2细胞增殖率;在转染后24h采用Transwell法评估HepG2细胞迁移和侵袭数量;在转染后48 h采用流式细胞术分析细胞周期,采用Western blot检测VASH2蛋白表达水平。采用荧光素酶报告基因检测评估miRNA-200是否靶向VASH2。结果与对照组相比,研究组血清Exo miRNA-200表达水平显著降低(P<0.01)。与阴性对照组相比,过表达组HepG2.2.15细胞的VASH2 mRNA和蛋白表达水平均显著降低(P<0.01)。过表达Exo组与对照组细胞增殖率比较差异无统计学意义(P>0.05),过表达Exo组迁移细胞和侵袭细胞数量较对照组显著减少(P<0.01),HepG2细胞在Exo过表达前后G1期、S期、G2期分布差异无统计学意义(P>0.05),过表达Exo组VASH2蛋白的相对表达水平显著低于对照组(P<0.01)。与miR-NC+VASH2 WT组相比,miRNA-200 mimic+VASH2 WT组的荧光素酶活性明显降低(P<0.01),而miRNA-200 mimic+VASH2 MUT组与miRNC+VASH2 MUT组的荧光素酶活性比较差异无统计学意义(P>0.05),表明miRNA-200靶向VASH2。结论miRNA-200在HCC患者血清Exo中低表达,上调miRNA-200可抑制HepG2.2.15细胞VASH2表达,�Objective To analyze miRNA-200 levels in serum and exosomes from HepG2 cells infected with the HBx protein of the hepatitis B virus,to investigate the expression of Vasohibin 2(VASH2)in HepG2 cells,and to elucidate the impact of miRNA-200 and VASH2 on the invasion and migration capabilities of HepG2.2.15 cells.Methods 15 patients with hepatocellular carcinoma(HCC)(study group)and 15 healthy subjects(control group)were selected for the study;the serum Exo miRNA-200 expression levels were compared between the 2 groups.Exo was isolated from HepG2.2.15 cells,which were subsequently divided into an overexpression group(transfected with miRNA-200 mimic)and a negative control group(transfected with miRNA-NC);TEM,Western blot,and tracking of nanoparticles were used to characterize exosomes;Western blot and qRT-PCR were used to detect VASH2 expression in each group of HepG2.2.15 cells.The proliferation,migration and invasion of cells as well as cell cycle status in the overexpressed Exo group(Exo from the miRNA-200 overexpressed group)and the control group(Exo from the untransfected group)were analyzed in HepG2 cells supplemented with Exo.The proliferation rate of HepG2 cells was evaluated using the CCK-8 method at 12,24,48,and 72 hours after transfection.The number of migrating and invading HepG2 cells was assessed using the Transwell method at 24 hours after transfection.Cell cycle analysis was performed using flow cytometry at 48 hours after transfection,and the expression level of VASH2 protein was detected by Western blot.Luciferase reporter gene assay was used to evaluate whether miRNA-200 targeted VASH2.Results Serum Exo miRNA-200 expression level was significantly reduced in the study group compared to the control group(P<0.01).Both VASH2 mRNA and protein expression levels were significantly reduced in HepG2.2.15 cells in the overexpression group compared with the negative control group(P<0.01).There was no statistically significant difference in cell proliferation rate between the overexpression Exo group and the

关 键 词：肝癌 乙肝病毒X蛋白 外泌体 微小RNA-200 血管生成抑制蛋白-2 

分 类 号：R735.7[医药卫生—肿瘤]