Construction of the human hepatocellular carcinoma cell line stably expressed mutated hepatitis B virus X genes and changes of its biological behavior / 中国热带医学

@#Abstract: Objective　To construct HepG2, Huh7 cell lines stably express hepatitis B virus X (HBx) mutant (C1653T, T1753C), and explore their effect on the biological behavior of hepatocellular carcinoma cells. Methods　The lentivirus plasmid of pLVX-HBxC1653T-IRES-tdTomato, pLVX-HBxT1753C-IRES-tdTomato were obtained by PCR site mutagenesis according to wild type ayr HBx. Double enzyme digestion and Sanger sequencing were performed for accuracy of plasmid. Blank HepG2 and Huh7 cells were used as the control group, HepG2, Huh7 cells were infected by pLVX-HBx-IRES-tdTomato, pLVX-HBxC1653T-IRES-tdTomato, and pLVX-HBxT1753C-IRES-tdTomato lentivirus solution, then monoclonal cell was selected by 0.6 μg/mL puromycin. Immunostaining and Western Blot were performed for the verification of stable strains. CCK8 assay was performed for the proliferation capacity of stable strains. Western Blot was performed for expression of EMT-related signal molecules in cells. The independent samples t-test was used for comparison between two groups. Results Double enzyme digestion and Sanger sequencing showed that that the size of the cut fragments of recombinant lentiviral plasmids was correct, and the point mutation location and base substitution were correct, suggesting that the plasmid of pLVX-HBx-IRES-tdTomato, pLVX-HBxC1653T-IRES-tdTomato, pLVX-HBxT1753C-IRES-tdTomato were constructed successfully. Immunostaining and Western blot showed that HBX were expressed in stable strains, while there was no HBX expression in the blank control group, indicating that the HepG2 and Huh7 cell lines stably expressing HBx, HBxC1653T, HBxT1753C were successfully constructed. CCK8 assay showed that the proliferation capacity of HBx and mutant were enhanced compared to the control group (P<0.01), HBx C1653T displayed further additive the effect compared to HBx (P<0.05). Moreover, HBxC1653T mutation also significantly upregulated N-cadherin expression and downregulated E-cadherin expression, thus promoting the occurrence of EMT. Conclusions　HepG2 and Huh7 cell lines stably expressing HBx, HBxC1653T, HBxT1753C were successfully constructed, HBxC1653T mutation significantly enhanced the proliferation of HCC cells and epithelial to mesenchymal transition occurrence.

Two cases of female-restricted X-linked syndromic mental retardation-99 caused by USP9X gene variations / 中华实用儿科临床杂志

Objective:To summarize and analyze the clinical and genotype features of female-restricted X-linked syndromic mental retardation-99(MRXS99F, OMIM: 300968)caused by USP9 X gene mutation, and to improve the clinicians′ understanding of the disease. Methods:Clinical data and genotypes of 2 children with MRXS99F treated in the Children′s Hospital of Nanjing Medical University in March 2020 (case 1) and June 2020 (case 2) were analyzed, and the relevant databases at home and abroad were reviewed to summarize the clinical characteristics and gene variation characteristics of the disease.Results:The 2 cases were 6 months old (case 1) and 5 years old (case 2), both showed psychomotor retardation.Case 1 presented a short stature, pigment abnormality, characteristic facial features, hypotonia, recurrent respiratory tract infections, laryngeal cartilage hypoplasia, atrial septal defect, feeding difficulty, hearing loss and brain hypoplasia.Case 2 had abnormal electroencephalogram.As confirmed by whole-exome sequencing, two children carried c. 6972+ 1G>A, c.6437C>T of USP9 X, respectively.Neither of the 2 variations was previously reported.Twenty-two cases of MRXS99F caused by USP9 X gene mutation were reported in 4 literatures globally, and 24 cases were combined with this study.The clinical manifestations of 20/22 children had special faces.All of them accompanied mental retardation combined with motor and language retardation, and carried neonatal variation. Conclusions:This is the first case report of MRXS99F induced by USP9 X gene variation in China.MRXS99F caused by functional deletion and variation of USP9 X gene is mainly characterized by psychomotor retardation, language disorder, special face and multiple congenital malformations.For children with unexplained growth retardation, special face and multiple congenital malformations, genetic testing like high-throughput sequencing should be carried out as early as possible to determine the etiology.

Hepatitis B virus X protein: The X factor in chronic hepatitis B virus disease progression

Introduction: Hepatitis B virus (HBV) is the most common aetiological factor causing hepatocellular carcinoma (HCC). HBx gene plays an enigmatic role in HBV-related HCC. In this study we have analysed amino acid substitutions in HBx from HBV-infected individuals of different clinical stages. Materials and Methods: HBV-infected individuals (n = 93) were recruited in the study. DNA was extracted from plasma, amplified, and DNA sequencing was performed using specific primers targeting HBx gene (540 bp). Results: Among the study participants, 57% had chronic HBV infection, 30% had chronic liver disease (CLD) and 13% had HBV related HCC. Genotypes such as D1, D2, D3, A1, C2 and B2 were identified of which genotype D2 was predominant (78%). HBxC-terminal deletion was observed in four hepatitis B e antigen (HBeAg) negative participants with CLD. The frequency of aminoacid substitution in proapoptotic domain was higher in HBeAg negative participants including I127V (34%), K130M (34%), V131I (40%). The frequency of double mutation (K130M+V131I) and triple mutation (I127V+K130M+V131I) were found to be higher (32% and 36%) in HBeAg negative participants. Also, we identified L5M substitution (4.3%) in HBeAg positive participants with advanced liver disease. Conclusion: In HBx gene, aminoacid substitutions at positions 127, 130, 131 are associated with poor expression of HBeAg. We suggest screening for HBx aminoacid substitutions especially in patients with HBeAg negative chronic HBV infection to predict the clinical outcome and enable early treatment to prevent disease progression.

Influence of hepatitis B virus X gene on apoptosis of hepatic cells mediated by Fas / 中华肝脏病杂志

Objective@#To investigate the influence of hepatitis B virus X gene (HBx) on apoptosis of hepatic cells mediated by Fas in HePG2 cells.@*Methods@#HBx eukaryotic vector pcDNA3.1(+)-X was transfected into HEPG2 cells with lipofectamine, and the null vector pcDNA3.1(+) and untransfected HEPG2 were used as normal controls. The cells were collected 72 h after transfection, and the expression of HBx mRNA and protein was determined using RT-PCR and Western blot, respectively. The mRNA expression of apoptosis-related genes Bcl-2 and Bax mRNA was also determined using RT-PCR. Cytotoxicity and apoptosis were evaluated using CCK-8 and flow cytometry, respectively, after HepG2-HBx and HepG2-3.1 cells were treated with stimulatory monoclonal antibody anti-Fas CH11. The t test was used for pairwise comparison.@*Results@#The cell line HepG2-HBx was successfully established, as confirmed by RT-PCR and Western blot, and RT-PCR results showed that HepG2-HBx cells had significantly higher expression of Bcl-2 mRNA than HepG2-3.1 and HepG2 cells (P < 0.05), but had significantly lower expression of Bax mRNA than HepG2-3.1 and HepG2 cells (P < 0.05); CCK-8 and flow cytometry showed that anti-Fas CH11 had a lower cytotoxicity to HepG2-HBx cells and allowed for a lower apoptosis rate of HepG2-HBx cells compared with HepG2-3.1 and HepG2 cells.@*Conclusions@#HBx can inhibit apoptosis of hepatic cells mediated by the Fas pathway.

Mutaciones K130M y V131I del gen X en aislados venezolanos del virus de hepatitis B / K130M and V131L Mutations on the X gene of hepatitis B virus in Venezuelan isolates

Aproximadamente el 50% de los carcinomas hepatocelulares (CHC) en el mundo están etiológicamente asociados con la infección por el virus de hepatitis B (VHB). Se han descrito 10 genotipos del VHB (A-J). En Venezuela y en varios países latinoamericanos predomina el genotipo F. Las mutaciones K130M y V131I presentes en la proteína HBx del VHB han sido asociadas al desarrollo del CHC. El objetivo de este trabajo fue estudiar la variabilidad genética de la proteína HBx del VHB circulante en pacientes venezolanos, con el fin de correlacionar estas mutaciones con los parámetros clínicos y virológicos de la enfermedad. Se analizó la secuencia del gen X del VHB, mediante amplificación por PCR de un fragmento de ese gen, en 45 pacientes infectados (35 crónicos y 10 agudos). Se observó una mayor frecuencia de las mutaciones K130M y V131I en pacientes de 25 o más años y con infección crónica. La presencia de estas mutaciones fue significativamente menor en el subgenotipo F3, comparado con el genotipo C. Estos resultados refuerzan la hipótesis de que el subgenotipo F3, predominante en Venezuela, podría estar asociado a una progresión menos severa de la enfermedad que la descrita para otros subgenotipos americanos, como F1b o F2.

Approximately 50% of the hepatocellular carcinomas (HCC) in the world are etiologically associated to hepatitis B virus (HBV) infection. Ten HBV genotypes (A-J) have been described in Venezuela and in other Latin American countries where the F genotype predominates. The K130M and V131I mutations present in the HBx protein of HBV have been associated with the development of HCC. The aim of this work was to study the genetic variability of HBx protein from HBV circulating in Venezuelan patients, in order to correlate these mutations with clinical and virus factors involved in the disease. The X HBV gene sequence was analyzed by PCR amplification of that gene in 45 infected patients (35 with chronic and 10 with acute stages of hepatitis). A higher frequency K130M and V131I mutations was observed in subjects 25 years of age and older with chronic infection. The presence of these mutations was significantly lower in the F3 subgenotype compared with genotype C. These results support the hypothesis that the F3 subgenotype, predominant in Venezuela, could be associated with a less severe progression of the disease than that described for other American subgenotypes, such as F1b or F2.

Mutation analysis of hepatitis B virus X gene in hepatitis B virus-associated glomerulonephritis / 中华肾脏病杂志

Objective To determine whether mutation of Hepatitis B virus (HBV) X gene is associated with hepatitis B virus-associated glomerulonephritis (HBV-GN).Methods The venous blood was collected from 50 patients with HBV-GN and 60 patients with asymptomatic HBV carriers (control group).Serum HBV DNA was extracted to determine the serum titer of HBV-DNA and then polymerase chain reaction (PCR) was used to detect the HBV X gene mutation.Results (1)There were not statistical significance between age and gender in HBV-GN group and control group (P ＞0.05).There were not statistical significance of serum replication level of HBV DNA in HBV-GN with X gene mutation and control group (P ＞ 0.05).Urine protein excretion in HBV-GN group with or without X gene mutation was found with statistical significance (P ＜ 0.05).(2)Nucleotide mutations [84％ (42/50)] resulted in amino acid substitution in HBV-GN.Nucleotide mutations changed in transfunction control region of X gene,including position nt1653,nt1726,nt1727,nt1730,nt1753,nt1762 and nt1764.(3)Nucleotide mutations [8％(5/60)] resulted in amino acid substitution in control group.Nucleotide mutations changed in position nt1632 and nt1635,located in non-functional region.Conclusions HBV X gene mutations and the subsequent amino acid substitutions are found in HBV-GN.The urine protein excretion level increases in patients with X mutation,suggesting that these mutations may play an important role in the pathogenesis of HBV-GN.

Research progress on HBx gene in pathogenesis of hepatitis B virus-associated glomerulonephritis / 中华临床感染病杂志

HBx gene is a multifunctional regulator,which has extensive trans-activating effects,and can activate transcription factors,inhibit DNA repair and regulate cell proliferation,differentiation and apoptosis.In recent years,the role of HBx gene in pathogenesis of hepatitis B virus-associated glomemlonephritis (HBV-GN) has been extensively studied,and the results show that HBx can promote glomerular mesangial cell proliferation,and induce damage or apoptosis of podocytes and renal tubular epithelial cells.This paper reviews the research progress on biological characteristics of HBx and its role in pathogenesis of HBV-GN.

Distribution of Salmonella paratyphi A outer membrane protein X gene and immune-protective effect related to its recombinant expressed products / 中华流行病学杂志

Objective To determine the distribution and sequence conservation of outer membrane protein X (ompX) gene in Salmonella paratyphi A isolates as well as the immunogenicity and irnmono-protection of ompX gene products.Methods OmpX gene in Salmonella paratyphi A isolates was detected by PCR and the amplification products were sequenced after the T-A cloning process.OmpX gene product was expressed with E.coli expression system and the expressed rOmpX was extracted by Ni-NTA affinity chromatography.SDS-PAGE and Bio-Rad Gel Image Analyzer were applied to examine the expression and yield of rOmpX.Both antigenicity and immune-reactivity of rOmpX were detected by immune-diffusion test,ELISA and Western blot assay.The immuneprotective effect of rOmpX against infection of Salmonella paratyphi in mice was determined and the agglutinative titers of sera from rOmpX-immunized mice was measured by micro-Widal' s test.Results All the tested Salmonella paratyphi A isolates had ompX gene with high nucleotide or amino acid sequence identity (99.2％-100.0％ or 98.4％-100.0％).When rOmpX was induced to rabbits to produce high level antibody and combined with antiserum against whole cell of Salmonella paratyphi A,the results displayed a positive Western hybridization signal.Results from ELISA demonstrated that 95.6％ (65/68) of the serum samples from paratyphoid-A patients were positive on rOmpX antibody.Mice that were immunized with 100 μg or 200 μg rOmpX displayed an immune-protective rate of 93.3％ (14/15) or 100.0％ (15/15).Sera from those rOmpX-immunized mice provided 1 ∶ 10-1 ∶ 40 agglutination titers in both H antigens of Salmonella paratyphi A and Salmonella typhi.Conclusion The recombinant expression product of ompX gene could be used as a candidate antigen for developing genetic engineering vaccines against Salmonella paratyphi A infection.

Mechanism on hepatitis B virus X gene-induced hepatic steatosis / 第二军医大学学报

Objective To investigate the effect of liver X receptor α (LXRα) gene silencing on lipid metabolism-related genes in HepG2. 2. 15 cells. Methods HepG2. 2. 15 cells were divided into blank control group (without transfection), negative control group (transfected with HK plasmid), and shLXRα group(transfected with shLXRα plasmid). The shLXRα plasmids carrying LXRα gene were constructed and were used to transfect HepG2. 2. 15 cells using Polyjet™ reagent. Green fluorescent protein and LXRα protein expression were examined by fluorescence microscope and Western blotting analysis 24-96 h after transfection, so as to identify the best interference time. Then cells were treated with agonis T090131 fo 2 o 4 h, and the content of triglyceride (TG) was observed to detect the degree of steatosis by biochemical assay. The expression of sterol regulatory element binding protein-lc (SREBP-lc) mRNA was detected by RT-PCR and the expression of hepatitis B virus X (HBx) protein and fatty acid synthase (FAS) protein was tested by Western blotting analysis. Results The shLXRa plasmid was constructed and transfected into HepG2. 2. 15 cells successfully. Compared with blank and negative control groups, LXRα protein was markedly decreased in the shLXRα group, with the lowest level found at 48-72 h after transfection (P<0. 01). Afte cell wer stimulate wit T090131, HBx and FAS protein expression, the content of TG, and SREBP-lc mRNA expression gradually increased with the prolongation of stimulation period, and there was no significant difference in HBx expression at the same time point between different groups. FAS protein, TG contents, and SREBP-lc mRNA in shLXRα group were significantly lower than those in the other two groups (P<0. 01). Conclusion HBx can regulate lipid metabolism through LXRa/SREBP-lc/FAS pathway.

HBV X Gene Transfection Upregulates IL-1β and IL-6 Gene Expression and Induces Rat Glomerular Mesangial Cell Proliferation / 华中科技大学学报（医学）（英德文版）

The X gene of HBV encodes a 17-KD protein, termed HBx, which has been shown to function as a transcriptional trans-activator of a variety of viral and cellular promoter/enhancer elements. The aim of this study was to investigate the effect of HBx on gene expression of interleukin (IL)-1β and IL-6, and proliferation of rat mesangial cells in vitro. The X gene of HBV was amplified by PCR assay, and inserted into the eukaryotic expression vector pCI-neo. The structure of recombinant pCI-neo-X plasmid was proved by restrict endonuclease digestion and sequencing analysis. pCI-neo-X was transfected into cultured rat mesangial cell line in vitro via liposome. HBx expression in transfected mesangial cells was detected by Western blot. The IL-1β and IL-6 mRNA expression in those cells was assayed by semiquantitative RT-PCR. Mesangial cell proliferation was tested by MTT. The results showed that HBx was obviously expressed in cultured mesangial cell line at 36th and 48th h after transfection. The expression of IL-1β and IL-6 mRNA was simultaneously increased. The cell proliferation was also obvious at the same time. It was concluded that HBx gene transfection could induce IL-1β and IL-6 gene expression and mesangial cell proliferation. HBx may play a critical role in mesangial cell proliferation through upregulation of the IL-1β and IL-6 gene expression.

Comparison of X gene Sequences of Hepatitis B Virus Isolates in Hepatitis B Induced Liver Cirrhosis Patients and HBV Carriers / 中华医院感染学杂志

OBJECTIVE To discover diversity of X gene sequences of hepatitis B virus isolates in hepatitis B induced liver cirrhosis patients and HBV carriers.METHODS DNA fragment including X gene sequences of hepatitis B virus was amplified,sequencing PCR products was preformed.The PCR products of three liver cirrhosis patients and three HBV symptomless carriers were cloned into pGEMT Easy vectors.Positive clones with target sequences were selected out for sequencing.Sequence comparison was made to find the identity.RESULTS A comparison of T1762/A1764,G1719T,T1727G/A,G1730C and T1753C mutations in a core promoter between the liver cirrhosis patients and the HBV carriers showed that the HBV isolates from the former had higher frequencies of mutation than the latter.The X promoter region of the HBV isolates from the liver cirrhosis patients showed higher frequencies of mutation than the isolates from the HBV carriers.Additionally,the homology between clones of X gene from one individual with liver cirrhosis averages 91.3-99.7%,that of HBV carriers averages 96-100%.CONCLUSIONS The core and X promoter region of the HBV isolates from the liver cirrhosis patients show the higher frequencies of mutation than the isolates from the HBV carriers.There are HBV quasispecies which possess great variation in the liver cirrhosis patients.

Hepatitis B Virus X Protein Upregulates Tumor Necrosis Factor-? Expression of Rat Mesangial Cell Line Via Extracellular Regulated Protein Kinases Pathway / 实用儿科临床杂志

Objective To investigate the extracellular regulated protein kinases (ERKs) pathway of hepatitis B virus(HBV) X protein(HBx) on glomerular mesangial cell(GMC) proliferation of rat and tumor necrosis factor(TNF)-? expression.Methods The HBV X gene was amplified by polymerase chain reaction(PCR),inserted into the eukaryotic expression vector PCI-neo and confirmed by restrict endonuclease digestion and sequence analysis.PCI-neo contained HBV X gene (PCI-neo-X) was transfected into cultured GMC via liposome.GMC proliferation,TNF-? and its mRNA expression were investigated in the condition of with or without U0126 in the culture media.HBx,ERK1/2 and phosphorynated-ERK1/2 (p-ERK1/2) expression in GMC were assessed by Western blot.TNF-? mRNA expression was assessed by semi-quantitative reverse transcriptase-PCR (RT-PCR).TNF-? level in supernatants was measured by enzymelinked immunosorbent assay(ELISA).GMC proliferation was assessed by methyl thiazolyl tetrazolium(MTT).Results HBx expression was found in transfected GMC,and became prominent at 36 and 48 hours after transfection whether with or without U0126 in culture media.TNF-? mRNA expression was significantly decreased in U0126 group compared with U0126-free group.TNF-? levels in supernatants in PCI-neo-X transfection without U0126 group were (189.0?18.1) ng/L at 36 hours and (172.3?24.3) ng/L at 48 hours after transfection,respectively.In contrast,TNF-? levels in supernatants with U0126 were (65.6?11.6) ng/L and (84.0?24.6) ng/L,respectively.The TNF-? le-vels in the latter groups were significantly lower than the formers (Pa

Effect of hepatitis B virus X gene and hepatitis C virus C gene coexpression protein on telomerase activity of HepG2 cells / 第三军医大学学报

Objective To establish an experimental model of HCV C-HBV X coexpression protein and explore its effect on tolemerase activity. Methods The HBV X gene was recovered by enzyme digestion, cloned into PBK-CMV and PBK-HCV C, and the recombinant plasmids PBK-X and PBK-X-C were obstained. The plasmids PBK-CMV, PBK-X, PBK-HCV C and PBK-X-C were transfected into HepG2 cells with liposome. After selected with G418, positive colonies were obtained. The reverse transcription PCR and Western blotting were used to detect HBV X and HCV core protein expression and PCR-ELISA for tolemerase activity. Results The recombinant plasmid PBK-X-C could express HBV X and HCV core protein efficiently. The telomerase activity of the cells coexpressed HCV C-HBV X protein was higher than that of cells expessed HBV X, HCV C and vector only. Conclusion HBV X-HCV C coexpression protein can increase the telomerase activity of HepG2 cells, which suggests that HBV and HCV can cooperate with carcinogenesis.

Construction and expression of eukaryotic expression vector for hepatitis B virus X gene containing EGFP / 重庆医科大学学报

Objective:To construct eukaryotic expression vector for hepatitis B virus X(HBV X) gene with enhanced green fluorescence protein(EGFP),and confirm its expression in hepatocellular carcinoma(HCC)cell line.Method: HBV X gene was cloned from pcDNA3.1(+)-X by enzyme digestion and inserted to pEGFP-N1.The rector was confirmed by enzyme digestion,PCR assay and sequencing.Then pEGFP-X was transfected to HCC cell line Bel-7402.After transient transfection,expressions of HBV X and EGFP gene were detected by RT-PCR and fluorescence microscope respectively.Results: The results showed that the recombinant plasmid could express HBV X gene efficiently in Bel-7402,which showed green fluorescence.Conclution: The pEGFP-X was constructed and the fused HBV X-EGFP gene was expressed in Bel-7402 successfully.These facilitate the study of the effect of HBV X protein on the development of HCC.

Gene Expression Profile in Response to Hepatitis B Virus X Gene by Using an Adenoviral Vector / 대한간학회지

BACKGROUND/AIMS: Hepatitis B virus (HBV) is the etiological factor for hepatocellular carcinoma (HCC). Numerous evidence has indicated a link between chronic infection with HBV and the development of HCC. Among the four proteins encoded by HBV, Hepatitis B virus X gene(HBx), best characterized as a transcriptional transactivator, gained attention owing to its presumptive role in oncogenesis. Further, HBx has been shown to stimulate signal transduction pathways such as Ras-MAPK pathway, NF-kappaB, and Src kinase. The pleiotropic events caused by HBx may be the key to understanding the HBV-mediated oncogenicity. However, the specific roles of HBx in oncogenesis remain largely elusive. To explore the role of HBx in hepatocarcinogenesis, we examined the deregulation of host genes induced by HBx expression. METHODS: HBx was ectopically expressed in HepG2 cells using a recombinant adenovirus to transiently express HBx. Gene expression profiling of HBx was conducted on cDNA microarrays that contained 1,028 cDNAs. RESULTS: A number of oncogenes and genes that are involved in cell growth, DNA repair, cell cycle regulation, and cell motility were deregulated by HBx. CONCLUSIONS: Theses results suggest that HBx regulates transcription in a way that contributes to the proliferation of hepatocytes, a probable early event of HCC.

The inhibitory effects on hepatitis B virus gene expression in stable expression of DN mutants of hepatitis B virus X gene / 中华传染病杂志

Objective The newly developing gene therapy method and dominant negative mutants were bein g used as new promising HBV therapy method, and a dominant negative mutant of HB V X g ene we have reported in our previous report has some effects both on HBV replica tion and expression in transient expression, but the effects were interfered by persistent secretion of HBV in HepG 2 2.2.15 cell line in the experiment. To mak e sure the effects of dominant negative mutant of HBx gene, we established a HBx DN stably expressing cell clone, and evaluated the effects of HBx dominant negat ive mutant on HBV gene expression. Methods The prev HBx-GFP dominant mutant and the plasmids pRev Xwt, pRev GFP which contain the wild type X gene or GFP gene then transfected into HepG 2 2.2.15 cells by liposome. The HBsAg, HBeAg by in media were as sayed by RIA and HBV-related RNA were assayed by Northern blot. Results The pRev HBx-GFP, GFP and wild type X constructs can be effectively expressed in HepG 2 2.2.15 cells. The stable expressed HBx -GFP can significantly reduce HBeAg, HBeAg in media and the HBV-related RNA in HepG 2 2.2.15 cells, but not for pRev Xwt and pRev GFP. Conclusions The dominant negative mutant pRev HBx-GFP can significantly inhibit the HBV gen e expression. It also suggested that X gene may be one promising target for HBV gene therapy.

ANALYSIS OF X GENE MUTATIONS IN HEPATITIS B VIRUS GENOME POSITIVE FOR ANTI-HBE BETWEEN ASYMPTOMATIC CARRIER AND PATIENTS WITH CHRONIC ACTIVE HEPATITIS / 解放军医学杂志

To elucidate whether the mutations in X region of hepatitis B virus (HBV) might be responsible for the different clinical profiles in cases positive for antibody to hepatitis B e antigen. The nucleotide sequences of X gene regions in serum HBV were examined in 14 asymptomatic carriers (AsC) and 14 chronic active hepatitis (CAH) patients with antibody to hepatitis e antigen. The results showed that 12 of 14 AsCs (85.7%) had insertions, deletions or point mutations in nucleotide sequence of X region resulting in truncation of the X protein by creating frame shift mutation or a new stop codon, whereas no patient with CAH had those X gene mutations( P

Construction of double-copy and x gene deleted hepatitis B virus DNA expression plasmid and cells transfection study / 第二军医大学学报

Objective:To construct double copy and x gene deleted HBV expression plasmid and study its expression in Hep3B cell line. Methods:The double copy HBV DNA ( adr Ⅰ) was used to inactivate HBV x gene by inserting mutation and gene recombination. The inserted 55 bp DNA sequence was synthesized artificially; the insertion point was ApaL Ⅰ of x gene area. After recombination, an x gene defected HBV plasmid containing single P, S and C gene was constructed,which can express in mammalian cell line. Another plasmid carrying double copy HBV DNA with normal x gene was constructed as contrast. Both were used to transfect Hep3B cells. Then the cells were screened by G418 and HBV virus in culture medium were isolated and detected by fluorescence quantitative PCR. Results: Plasmids pcDNA3 KN F1F2 and pcDNA3 ES HBV2 were constructed successfully. After cell transfection, the HBV DNA was highly expressed with both plasmids on the 3 rd, 6 th,14th day. Conclusion: The plasmids constructed can express in Hep3B cell line and cause HBV replication; x gene defected HBV gene has no effect on HBV replication in Hep3B cell line.

Mutations of DNA repair associated gene, APEX in human colorectal cancer

Structural and functional changes in the major apurinic/apyrimidinic DNA endonuclease (APEX) gene in human colorectal cancers were investigated. DNAs were prepared from surgically removed 25 human colorectal tissues and direct sequencing of PCR-amplified APEX gene covering the entire protein coding region was performed. Point mutations in 3 and silent mutations in 3 out of 25 colorectal cancer patients were found. Base substitutions in intron II were also found in 2 patients. T C or some A G transitions were the most typical pattern of the mutations. AP DNA endonuclease (APE) activities in normal and tumor tissues were 65.7 EU/mg and 21.7 EU/mg, respectively. APEX protein was detected in both normal and tumor tissues and no remarkable difference in the amount of APEX protein between colorectal cancer tissues and their normal counterparts was observed. The incidence of APEX gene mutation in colorectal cancer was 12% which is relatively lower than that of other genes associated with colorectal tumor, but a significant reduction of APE enzyme activities in tumor tissues, especially in those with APEX mutations, was observed. These results indicate that the decreased APE enzyme activity might be closely related to the colorectal tumorigenesis, although no quantitative correlation between APE enzyme activity and APEX content exists.

EFFECT OF HEPATITIS B VIRUS X-GENE HETEROGENEITY ON ITS TRANSACTIVATION / 解放军医学杂志

Polymerase chain reaction(PCR) was employed to amplify the whole HBV X region from the serum of patients with chronic hepatitis B virus(HBV) infection, and then the PCR products were subcloned into pGEM Teasy vectors. Clones were randomly selected to be sequenced. Comparison of the cloned sequence was made to look for the differene. The sequencing results showed that each sequence of selected clones was differenct and there were HBV quasispecies groups in patients. There was hot deletion region near the 3' end of X gene. To address whether the mutations were responsible for the transactivating effect,the wild type(wt) and the mutants of HBV X genes were subcloned into the EcoRI site of pcDNA3 1( ) vectors, and the recombinant plasmids were cotransfected into HepG2 cells with reporter plasmid pSV lacZ,respectively. The activity of ? galactosidase controlled by SV40 early promoter/enhancer was detected, which reflected the transactivating function of HBx protein. The cotransfection results indicated that the wt HBV X gene acted as a transactivator on the SV40 early promoter/enhancer, and the mutations which caused premature termination of the X open reading frame reduced their transactivating effects to certain extent.