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HBx基因对HepG2.2.15细胞MICA-A5.1表达及侵袭、迁移的影响
Effect of hepatitis B x gene on the expression of major histocompatibility complex class I chain-related gene A-A5.1, invasion, and migration of HepG2.2.15 cells
文章发布日期:2020年04月10日  来源:  作者:李沛,刘宇  点击次数:263次  下载次数:48次

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【摘要】:目的 探究HBx基因对HepG2.2.15细胞侵袭、迁移及主要组织相容性复合体(MHC)Ⅰ类链相关基因A (MICA)-A51表达的影响。方法 体外培养HepG2.2.15细胞系(插入HBV全基因组并持续表达的HepG2细胞),随机分为对照组、HBx过表达质粒组、HBx空载质粒组、HBx siRNA组、HBx siRNA阴性对照组,分别转染质粒及siRNA后,以CKK-8法分别检测24 h、48 h后细胞增殖情况,筛选合适药物作用时间;以Transwell侵袭实验和细胞划痕实验检测各组细胞侵袭迁移能力;以免疫印记法检测HBx、MICA-A5.1蛋白和上皮间质转化标志蛋白E-cadherin、Vimentin的表达;以酶联免疫吸附法检测各组细胞培养基中sMICA水平。计量资料多组间比较采用单因素方差分析,进一步两两比较采用SNK q检验。结果 与对照组比较,24 h后,HBx过表达质粒组细胞活力升高,HBx siRNA组细胞活力降低,差异均有统计学意义(q值分别为8.268、4.365,P值分别为<0.001、0.036);48 h后,HBx过表达质粒组细胞活力升高,HBx siRNA组细胞活力降低,差异均有统计学意义(q值分别为12.680、7.523,P值均<0.001)。与对照组比较,HBx过表达质粒组细胞HBx、MICA及Vimentin蛋白表达、细胞培养基中sMICA水平、迁移细胞数目、侵出Transwell小室的细胞数目均升高,E-cadherin表达降低(q值分别为9.427、6.697、10.500、5.042、22.740、15.720、5.258,P值均<0.05); HBx siRNA组细胞HBx、MICA及Vimentin蛋白表达、细胞培养基中sMICA水平、迁移细胞数目、侵出Transwell小室的细胞数目均降低,E-cadherin表达升高(q值分别为8.133、8.828、7.616、7.673、5.391、7.694、6.226,P值均<0.05)。结论 HBx基因调控HepG2.2.15细胞MICA-A5.1表达及侵袭、迁移,上调该基因可促进MICA-A5.1表达,增强HepG2.2.15细胞侵袭转移能力。
【Abstract】:Objective To investigate the effect of hepatitis B x (HBx) gene on the expression of major histocompatibility complex class I chain-related gene A (MICA)-A5.1, invasion, and migration of HepG2.2.15 cells. Methods HepG2.2.15 cells (HepG2 cells with the insertion and continuous expression of whole HBV genome) were cultured in vitro and were randomly divided into control group, HBx overexpression plasmid group, HBx empty plasmid group, HBx siRNA group, and HBx siRNA negative control group. After the transfection of plasmid or siRNA, CCK-8 assay was used to measure cell proliferation after 24 and 48 hours to screen out the appropriate duration of drug action; the Transwell invasion test and wound-healing assay were used to observe the changes in cell invasion and migration abilities; immunoblotting was used to measure the expression of HBx, MICA-A5.1, and the marker proteins for epithelial-mesenchymal transition E-cadherin and vimentin; ELISA was used to measure the level of soluble MICA (sMICA) in cell culture medium. A one-way analysis of variance was used for comparison of continuous data between multiple groups, and the SNK q test was used for further comparison between two groups. Results Compared with the control group after 24 hours, the HBx overexpression plasmid group had a significant increase in cell viability and the HBx siRNA group had a significant reduction in cell viability (q=8.268 and 4.365, P<0.001 and 0.036); compared with the control group after 48 hours, the HBx overexpression plasmid group had a significant increase in cell viability and the HBx siRNA group had a significant reduction in cell viability (q=12.680 and 7.523, both P<0.001). Compared with the control group, the HBx overexpression plasmid group had significant increases in the protein expression of HBx, MICA, and vimentin in cells, the level of sMICA in cell culture medium, the number of migrating cells, and the number of cells out of the Transwell chamber, as well as a significant reduction in the expression of E-cadherin (q=9.427, 6.697, 10.500, 5.042, 22.740, 15.720, and 5.258, all P<0.05); the HBx siRNA group had significant reductions in the protein expression of HBx, MICA, and vimentin in cells, the level of sMICA in cell culture medium, the number of migrating cells, and the number of cells out of the Transwell chamber, as well as a significant increase in the expression of E-cadherin (q=8.133, 8.828, 7.616, 7.673, 5.391, 7.694, and 6.226, all P<0.05). Conclusion The HBx gene regulates the expression of MICA-A5.1 in HepG2.2.15 cells and the invasion and migration of HepG2.2.15 cells, and upregulation of the HBx gene can promote the expression of MICA-A5.1 and enhance the invasion and migration abilities of HepG2.2.15 cells.
【关键字】:肝炎病毒,乙型; Hep G2 细胞; 基因,MHCⅠ类
【Key words】:hepatitis B virus; Hep G2 cells; genes,MHC classⅠ
【引证本文】:LI P, LIU Y. Effect of hepatitis B x gene on the expression of major histocompatibility complex class I chain-related gene A-A5.1, invasion, and migration of HepG2.2.15 cells[J]. J Clin Hepatol, 2020, 36(4): 808-812. (in Chinese)
李沛, 刘宇. HBx基因对HepG2.2.15细胞MICA-A5.1表达及侵袭、迁移的影响[J]. 临床肝胆病杂志, 2020, 36(4): 808-812.

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