乙肝病毒S基因Pre-S区突变的人肝癌细胞HepG2稳定株构建及其生物学行为变化
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摘要
目的构建稳定过表达乙肝病毒(HBV) S基因Pre-S区突变(Pre-S1缺失突变、Pre-S2缺失突变)的HepG2细胞株,并观察该突变对HepG2细胞生物学行为的影响。方法以NCBI中HBV JX661479. 1的Pre-S/S片段序列信息为模板,设计并确定Pre-S1突变、Pre-S2突变的核苷酸序列,采用全基因合成法获得目的片段并定向导入带有FLAG标签的p LVX慢病毒质粒载体(p Lenti-CMV-3FLAG-PGK-Puro),PCR、双酶切、Sanger测序技术检测重组质粒中目的片段的准确性。将HepG2细胞随机分为5组,空白对照组不处理,p LVX-vector组、野生型组、Pre-S1突变组、Pre-S2突变组分别感染p LVX-vector、p LVX-Pre-S/S、p LVX-Pre-S1 mut/S、p LVX-Pre-S2 mut/S慢病毒液;利用嘌呤霉素筛选HepG2稳定株,采用Western blotting法鉴定目的蛋白,分别采用克隆形成、细胞划痕试验观察HepG2细胞稳定株增殖、迁移能力。结果构建的p LVX-Pre-S/S、p LVX-Pre-S1 mut/S、p LVX-Pre-S2 mut/S重组表达质粒PCR电泳产物与理论值相符,经EcoRⅠ和Bam HⅠ双酶切后电泳产物与理论值相符; Sanger测序结果显示,p LVXPre-S1 mut/S、p LVX-Pre-S2 mut/S突变型除突变序列外,其他序列与HBV S基因野生型序列完全相同。空白对照组HepG2细胞全部死亡,其余组HepG2细胞部分存活。在野生型组、Pre-S1突变组、Pre-S2突变组43 k D分子量位置检测到目的条带表达,而p LVX-vector组无法检测到相应条带。与其他组比较,Pre-S2突变组HepG2的第14天克隆形成数增多、细胞相对迁移距离增大(P均<0. 05)。结论成功构建了HBV Pre-S/S基因野生型及Pre-S突变型的HepG2细胞稳定株,HBV的S基因Pre-S2缺失突变导致肝癌HepG2细胞增殖及迁移能力增强。
Objective To construct the HepG2 cell lines stably overexpressing hepatitis B virus( HBV) S gene Pre-S region mutation( Pre-S1 deletion mutation and Pre-S2 deletion mutation),and to observe the effect of this mutation on the biological behavior of HepG2 cells. Methods The sequence of Pre-S/S fragment of HBV virus was determined based on JX661479. 1 sequence in NCBI,the deletion fragment of Pre-S1 and Pre-S2 mutation was designed subsequently. The target fragment was obtained by whole-genome synthesis method and inserted into the p LVX lentiviral plasmid vector( p LentiCMV-3FLAG-PGK-Puro) with FLAG tag. We detected the accuracy of the target fragment in recombinant plasmid by PCR,double digestion and Sanger sequencing. HepG2 cells were randomly divided into 5 groups: the blank control group,the p LVX-vector group,wild type group,Pre-S1 mutation group,and Pre-S2 mutation group,and the cells in the latter four groups were transfected with p LVX-vector,p LVX-Pre-S/S,p LVX-Pre-S1 mut/S,and p LVX-Pre-S2 mut/S lentiviral solution,respectively. The HepG2 stable strain was screened by puromycin,and the expression of the target protein in HepG2 cells was identified by Western blotting. Colony Formation and scratch test were used to detect the impact of hepatitis B virus Pre-S mutation on the proliferation and migration abilities of HepG2 cells,respectively. Results The constructed recombinant expression plasmids of p LVX-Pre-S/S,p LVX-Pre-S1 mut/S,and p LVX-Pre-S2 mut/S were conformed to the theoretical values through PCR electrophoresis analysis,and the electrophoretic products after digestion of EcoR I and Bam H I were in accordance with the theoretical values. Sanger sequencing results showed that the mutations of p LVX-PreS1 mut/S and p LVX-Pre-S2 mut/S were identical to those of wild type of HBV S except mutation sequence. In the blank control group,all HepG2 cells died,and in the other groups,partial HepG2 cells survived. The expression of the target band was detected in the wild type group,Pre-S1 mutant group and Pre-S2 mutant group at 43 k Da molecular weight position,but not in p LVX-vector group. Compared with the other groups,the number of clones and the relative migration distance on the 14 th day of HepG2 cells in the Pre-S2 mutant group increased( all P < 0. 05). Conclusions The stable strain of HepG2 cells with wild and mutant HBV Pre-S/S genes is successfully constructed. The deletion of HBV S protein Pre-S2 results in the enhancement of proliferation and migration of hepatocellular carcinoma HepG2 cells
Objective To construct the HepG2 cell lines stably overexpressing hepatitis B virus( HBV) S gene Pre-S region mutation( Pre-S1 deletion mutation and Pre-S2 deletion mutation),and to observe the effect of this mutation on the biological behavior of HepG2 cells. Methods The sequence of Pre-S/S fragment of HBV virus was determined based on JX661479. 1 sequence in NCBI,the deletion fragment of Pre-S1 and Pre-S2 mutation was designed subsequently. The target fragment was obtained by whole-genome synthesis method and inserted into the p LVX lentiviral plasmid vector( p LentiCMV-3FLAG-PGK-Puro) with FLAG tag. We detected the accuracy of the target fragment in recombinant plasmid by PCR,double digestion and Sanger sequencing. HepG2 cells were randomly divided into 5 groups: the blank control group,the p LVX-vector group,wild type group,Pre-S1 mutation group,and Pre-S2 mutation group,and the cells in the latter four groups were transfected with p LVX-vector,p LVX-Pre-S/S,p LVX-Pre-S1 mut/S,and p LVX-Pre-S2 mut/S lentiviral solution,respectively. The HepG2 stable strain was screened by puromycin,and the expression of the target protein in HepG2 cells was identified by Western blotting. Colony Formation and scratch test were used to detect the impact of hepatitis B virus Pre-S mutation on the proliferation and migration abilities of HepG2 cells,respectively. Results The constructed recombinant expression plasmids of p LVX-Pre-S/S,p LVX-Pre-S1 mut/S,and p LVX-Pre-S2 mut/S were conformed to the theoretical values through PCR electrophoresis analysis,and the electrophoretic products after digestion of EcoR I and Bam H I were in accordance with the theoretical values. Sanger sequencing results showed that the mutations of p LVX-PreS1 mut/S and p LVX-Pre-S2 mut/S were identical to those of wild type of HBV S except mutation sequence. In the blank control group,all HepG2 cells died,and in the other groups,partial HepG2 cells survived. The expression of the target band was detected in the wild type group,Pre-S1 mutant group and Pre-S2 mutant group at 43 k Da molecular weight position,but not in p LVX-vector group. Compared with the other groups,the number of clones and the relative migration distance on the 14 th day of HepG2 cells in the Pre-S2 mutant group increased( all P < 0. 05). Conclusions The stable strain of HepG2 cells with wild and mutant HBV Pre-S/S genes is successfully constructed. The deletion of HBV S protein Pre-S2 results in the enhancement of proliferation and migration of hepatocellular carcinoma HepG2 cells
引文
[1] EASL Clinical Practice Guidelines. Management of hepatocellular carcinoma[J]. J Hepatol,2018,69(1):182-236.
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[7]European Association for the Study of the Liver. EASL 2017 clinical practice guidelines on the management of hepatitis B virus infection[J]. J Hepatol,2017,67(2):370-398.
[8]Lin CM,Wang GM,Jow GM,et al. Functional analysis of hepatitis B virus pre-S deletion variants associated with hepatocellular carcinoma[J]. J Biomed Sci,2012,19:17.
[9]Wang HC,Chang WT,Chang WW,et al. Hepatitis B virus preS2 mutant upregulates cyclin A expression and induces nodular proliferation of hepatocytes[J]. Hepatology,2005,41(4):761-770.
[10] Wang HC,Wu HC,Chen CF,et al. Different types of ground glass hepatocytes in chronic hepatitis B virus infection contain specific pre-S mutants that may induce endoplasmic reticulum stress[J]. Am J Pathol,2003,163(6):2441-2449.
[11]Su IJ,Wang HC,Wu HC,et al. Ground glass hepatocytes contain pre-S mutants and represent preneoplastic lesions in chronic hepatitis B virus infection[J]. J Gastroenterol Hepatol,2008,23(8 Pt1):1169-1174.
[12]Pollicino T,Cacciola I,Saffioti F,et al. Hepatitis B virus Pre S/S gene variants:pathobiology and clinical implications[J]. J Hepatol,2014,61(2):408-417.
[13]Milich DR,Jones JE,Mc Lachlan A,et al. Importance of subtype in the immune response to the pre-S(2)region of the hepatitis B surface antigen.Ⅱ. Synthetic Pre-S(2)immunogen[J]. J Immunol,1990,144(9):3544-3551.
[14]Abe K,Thung SN,Wu HC,et al. Pre-S2 deletion mutants of hepatitis B virus could have an important role in hepatocarcinogenesis in Asian children[J]. Cancer Sci,2009,100(12):2249-2254.
[15] Hung JH,Su IJ,Lei HY,et al. Endoplasmic reticulum stress stimulates the expression of cyclooxygenase-2 through activation of NF-kappaB and pp38 mitogen-activated protein kinase[J]. J Biol Chem,2004,279(45):46384-46392.
[2]Trepo C,Chan HL,Lok A. Hepatitis B virus infection[J]. Lancet,2014,384(9959):2053-2063.
[3]Levrero M,Zucman-Rossi J. Mechanisms of HBV-induced hepatocellular carcinoma[J]. J Hepatol,2016,64(Suppl 1):84-101.
[4]Lin CL,Kao JH. Natural history of acute and chronic hepatitis B:The role of HBV genotypes and mutants[J]. Best Pract Res Clin Gastroenterol,2017,31(3):249-255.
[5]Yeung P,Wong DK,Lai CL,et al. Association of hepatitis B virus pre-S deletions with the development of hepatocellular carcinoma in chronic hepatitis B[J]. J Infect Dis,2011,203(5):646-654.
[6]Zhang AY,Lai CL,Huang FY,et al. Evolutionary changes of hepatitis B virus pre-S mutations prior to development of hepatocellular carcinoma[J]. PLo S One,2015,10(9):e139478.
[7]European Association for the Study of the Liver. EASL 2017 clinical practice guidelines on the management of hepatitis B virus infection[J]. J Hepatol,2017,67(2):370-398.
[8]Lin CM,Wang GM,Jow GM,et al. Functional analysis of hepatitis B virus pre-S deletion variants associated with hepatocellular carcinoma[J]. J Biomed Sci,2012,19:17.
[9]Wang HC,Chang WT,Chang WW,et al. Hepatitis B virus preS2 mutant upregulates cyclin A expression and induces nodular proliferation of hepatocytes[J]. Hepatology,2005,41(4):761-770.
[10] Wang HC,Wu HC,Chen CF,et al. Different types of ground glass hepatocytes in chronic hepatitis B virus infection contain specific pre-S mutants that may induce endoplasmic reticulum stress[J]. Am J Pathol,2003,163(6):2441-2449.
[11]Su IJ,Wang HC,Wu HC,et al. Ground glass hepatocytes contain pre-S mutants and represent preneoplastic lesions in chronic hepatitis B virus infection[J]. J Gastroenterol Hepatol,2008,23(8 Pt1):1169-1174.
[12]Pollicino T,Cacciola I,Saffioti F,et al. Hepatitis B virus Pre S/S gene variants:pathobiology and clinical implications[J]. J Hepatol,2014,61(2):408-417.
[13]Milich DR,Jones JE,Mc Lachlan A,et al. Importance of subtype in the immune response to the pre-S(2)region of the hepatitis B surface antigen.Ⅱ. Synthetic Pre-S(2)immunogen[J]. J Immunol,1990,144(9):3544-3551.
[14]Abe K,Thung SN,Wu HC,et al. Pre-S2 deletion mutants of hepatitis B virus could have an important role in hepatocarcinogenesis in Asian children[J]. Cancer Sci,2009,100(12):2249-2254.
[15] Hung JH,Su IJ,Lei HY,et al. Endoplasmic reticulum stress stimulates the expression of cyclooxygenase-2 through activation of NF-kappaB and pp38 mitogen-activated protein kinase[J]. J Biol Chem,2004,279(45):46384-46392.