PIM1-Induced Cytoplasmic Expression of RBMY Mediates Hepatocellular Carcinoma Metastasis.

BACKGROUND & AIMS: Metastasis indicates a grave prognosis in patients with hepatocellular carcinoma (HCC). Our previous studies showed that RNA binding motif protein Y-linked (RBMY) is potentially a biomarker for poor survival in HCC patients, but its role in metastasis is largely unclear. METHODS: A total of 308 male patients with primary HCC were enrolled. RBMY expression was traced longitudinally by immunostaining from the manifestation of a primary HCC tumor to the formation of a distant metastasis, and its upstream regulators were screened with a protein microarray. A series of metastasis assays in mouse models and HCC cell lines were performed to explore new functional insights into RBMY. RESULTS: Cytoplasmic expression of RBMY was associated with rapid distant metastasis (approximately 1 year after resection) and had a predictive power of 82.4% for HCC metastasis. RBMY conferred high migratory and invasive potential upon phosphorylation by the provirus integration in Moloney 1 (PIM1) kinase. Binding of PIM1 to RBMY caused mutual stabilization and massive translocation of RBMY from nuclei to mitochondria, thereby preventing mitochondrial apoptosis and augmenting mitochondrial generation of adenosine triphosphate/reactive oxygen species to enhance cell motility. Depletion of RBMY suppressed Snail1/zinc finger E-box binding homeobox transcription factor 1-mediated epithelial-mesenchymal transition and dynamin-related protein 1-dependent mitochondrial fission. Inactivation and knockout of PIM1 down-regulated the expression of RBMY. In nude mice, cytoplasmic RBMY promoted liver-to-lung metastasis by increasing epithelial-mesenchymal transition, mitochondrial proliferation, and mitochondrial fission, whereas nuclear-restricted RBMY impeded the mitochondrial switch and failed to induce lung metastasis. CONCLUSIONS: This study showed the regulation of HCC metastasis by PIM1-driven cytoplasmic expression of RBMY and suggested a novel therapeutic target for attenuating metastasis.

Hepatitis B virus X gene impacts on the innate immunity and immune-tolerant phase in chronic hepatitis B virus infection.

BACKGROUND AND AIMS: The immunologic features involved in the immune-tolerant phase of chronic hepatitis B (CHB) virus (HBV) infection are unclear. The hepatitis B virus X (HBx) protein disrupts IFN-ß induction by downregulating MAVS and may destroy subsequent HBV-specific adaptive immunity. We aimed to analyse the impacts of genetic variability of HBx in CHB patients on the immune-tolerant phase during long-term follow-up. METHODS: Children with CHB in the immune-tolerant phase were recruited and followed longitudinally. HBx gene sequencing of infecting HBV strains was performed, and the effects of HBx mutations on the immune-tolerant phase were assessed. Restoration of the host immune response to end the immune-tolerant phase was investigated by immunoblotting, immunostaining, ELISA and reporter assays of MAVS/IFN-ß signalling in liver cell lines, patient liver tissues and the HBV plasmid replication system. RESULTS: A total of 173 children (median age, 6.92 years) were recruited. Patients carrying HBx R87G, I127V and R87G + I127V double mutations exhibited higher cumulative incidences of immune-tolerant phase breakthrough (p = .011, p = .006 and p = .017 respectively). Cells transfected with HBx R87G and I127V mutants and pHBV1.3-B6.3 replicons containing the HBx R87G and I127V mutations exhibited statistically increased levels of IFN-ß, especially under poly(I:C) stimulation or Flag-MAVS cotransfection. HA-HBx wild-type interacted with Flag-MAVS and enhanced its ubiquitination, but this ability was diminished in the R87G and I127V mutants. CONCLUSIONS: HBx suppresses IFN-ß induction. R87G and I127V mutation restored IFN-ß production by preventing MAVS degradation, contributing to curtailing the HBV immune-tolerant phase in CHB patients.

Genetic variants of NTCP gene and hepatitis B vaccine failure in Taiwanese children of hepatitis B e antigen positive mothers.

BACKGROUND AND AIMS: Hepatitis B virus (HBV) vaccine failure remains a hurdle to the global elimination of HBV infections in the vaccination era. We aimed to elucidate the relationships between HBV entry receptor sodium taurocholate co-transporting polypeptide (NTCP) and vaccine failure in children born to highly infectious mothers. METHODS: The genetic variants rs7154439, rs4646285, rs4646287, and rs2296651 were genotyped in 170 children with chronic HBV infections and 138 control children of mothers positive for hepatitis B e antigen (HBeAg). All children received hepatitis B immunoglobulin and complete HBV vaccination. Total RNAs from 82 adult non-tumor liver tissues were quantified for NTCP, type I interferons and interferon-induced transmembrane protein 3 (IFITM3) levels. RESULTS: A higher rate of the GA/AA genotype (28.3% vs. 15.3%, p = 0.006) of the genetic variant rs4646287 in intron 1 of the NTCP gene was detected in control children compared to the carrier children. The rs4646287 G > A genotype was associated with younger ages at which spontaneous HBeAg seroconversion occurred (10.8 ± 8.4 vs. 14.6 ± 8.7 years, p = 0.003) in chronic HBV-infected children. Unique correlation patterns of NTCP and innate immunity-related genes (type I interferons and IFITM3) were found in HBV-infected liver tissues with the rs4646287 G > A genotype. CONCLUSION: The rs4646287 G > A genotype of the NTCP gene may be associated with lower risk for HBV vaccine failure in children born to highly infectious mothers. The protective effect of rs4646287 G > A was also present in carrier children, evidenced by earlier spontaneous HBeAg seroconversion.

RBMY, a novel inhibitor of glycogen synthase kinase 3ß, increases tumor stemness and predicts poor prognosis of hepatocellular carcinoma.

UNLABELLED: Male predominance of hepatocellular carcinoma (HCC) occurs particularly among young children aged 6-9 years, indicative of a possible role of the Y chromosome-encoded oncogene in addition to an androgenic effect. The discovery of oncogenic activation of RBMY (RNA-binding motif on Y chromosome), which is absent in normal hepatocytes but present in male HCC tissues, sheds light on this issue. Herein, we report on a critical hepatocarcinogenic role of RBMY and its ontogenic origin. During liver development, the Ser/Thr phosphorylated RBMY is expressed in the cytoplasm of human and rodent fetal livers. It is then silenced in mature hepatocytes and restricted to scarce expression in the bile ductular cells. Upon hepatocarcinogenesis, a noteworthy increase of cytoplasmic and nuclear RBMY is observed in HCC tissues; however, only the former is expressed dominantly in hepatic cancer stem cells and correlates significantly to a poor prognosis and decreased survival rate in HCC patients. Cytoplasmic expression of RBMY, which is mediated by binding to nuclear exporter chromosome region maintenance 1 and further enriched upon Wnt-3a stimulation, confers upon tumor cells the traits of cancer stem cell by augmenting self-renewal, chemoresistance, cell-cycle progression, proliferation, and xenograft tumor growth. This is achieved mechanistically through increasing Ser9 phosphorylation-inactivation of glycogen synthase kinase 3ß by RBMY, thereby impeding the glycogen synthase kinase 3ß-dependent degradation of ß-catenin and eventually inducing the nuclear entry of ß-catenin for the transcription of downstream oncogenes. CONCLUSION: RBMY is a novel oncofetal protein that plays a key role in attenuating glycogen synthase kinase 3ß activity, leading to aberrant activation of Wnt/ß-catenin signaling, which facilitates malignant hepatic stemness; because of its absence from normal human tissues except the testis, RBMY represents a feasible therapeutic target for the selective eradication of HCC cells in male patients.

Copy-number variations in hepatoblastoma associate with unique clinical features.

PURPOSE: Hepatoblastoma is a rare childhood liver malignancy with limited relevant cytogenetic data. This study aimed to discover common genomic copy-number variations (CNVs) in subjects with hepatobalstoma and its relevance to the clinical course. METHODS: Gene copy-number was systemically rated by high-resolution comparative genomic hybridization (CGH) DNA oligonucleotide microarray. The study group consisted of 12 children (7 males and 5 females) with hepatoblastoma and another 20 healthy individuals (10 males and 10 females) as controls. The influence of recurrent CNVs on clinical outcomes was analyzed. RESULTS: Four highly recurrent CNVs were identified in these 12 hepatoblastoma children after comparison with controls, including a gain on 1p13.3 (n = 3, 25%) and losses on 5p15.33 (n = 4, 33.3%), 16q12.2 (n = 4, 33.3%), and 19q13.42 (n = 3, 25%). The most prevalent sites of genomic deletion were 5p15.33 and 16q12.2. Zinc finger, DHHC-type containing 11 (ZDHHC11) and DHHC-type containing 11B (ZDHHC11B) were mapped to 5p15.33, which was associated with a lower rate of survival with native liver (p = 0.03). The carboxylesterase 4-like (CES4) gene that mapped to 16q12.2 was associated with smaller tumor size at presentation. CONCLUSIONS: Deletions of 5p15.33 (33.3%) and 16q12.2 (33.3%) are the most frequent hepatoblastoma-related events in our patient group with 5p15.33 microdeletion as a potential biomarker for the fate of survival with native liver.

Male germ cell-specific RNA binding protein RBMY: a new oncogene explaining male predominance in liver cancer.

Male gender is a risk factor for the development of hepatocellular carcinoma (HCC) but the mechanisms are not fully understood. The RNA binding motif gene on the Y chromosome (RBMY), encoding a male germ cell-specific RNA splicing regulator during spermatogenesis, is aberrantly activated in human male liver cancers. This study investigated the in vitro oncogenic effect and the possible mechanism of RBMY in human hepatoma cell line HepG2 and its in vivo effect with regards to the livers of human and transgenic mice. RBMY expression in HepG2 cells was knocked down by RNA interference and the cancer cell phenotype was characterized by soft-agar colony formation and sensitivity to hydrogen-peroxide-induced apoptosis. The results revealed that RBMY knockdown reduced the transformation and anti-apoptotic efficiency of HepG2 cells. The expression of RBMY, androgen receptor (AR) and its inhibitory variant AR45, AR-targeted genes insulin-like growth factor 1 (IGF-1) and insulin-like growth factor binding protein 3 (IGFBP-3) was analyzed by quantitative RT-PCR. Up-regulation of AR45 variant and reduction of IGF-1 and IGFBP-3 expression was only detected in RBMY knockdown cells. Moreover, RBMY positive human male HCC expressed lower level of AR45 as compared to RBMY negative HCC tissues. The oncogenic properties of RBMY were further assessed in a transgenic mouse model. Liver-specific RBMY transgenic mice developed hepatic pre-cancerous lesions, adenoma, and HCC. RBMY also accelerated chemical carcinogen-induced hepatocarcinogenesis in transgenic mice. Collectively, these findings suggest that Y chromosome-specific RBMY is likely involved in the regulation of androgen receptor activity and contributes to male predominance of HCC.

Human interleukin-10 genotypes are associated with different precore/core gene mutation patterns in children with chronic hepatitis B virus infection.

OBJECTIVE: To elucidate the association between human interleukin-10 (IL-10) genotypes and hepatitis B virus (HBV) precore/core gene mutation in children with chronic HBV infection. STUDY DESIGN: The study group comprised of 21 children with chronic HBV infection with spontaneous hepatitis B e antigen (HBeAg) seroconversion who were followed for more than 10 years. Another nine children without HBeAg seroconversion served as the control subjects. Sera at the immune tolerance and inflammatory phase (alanine aminotransferase, >80 IU/L) were subjected to HBV precore/core sequence analysis. IL-10 -1082 polymorphism was also determined. RESULTS: HBV precore/core gene mutation increased significantly more in the inflammatory phase than in the tolerance phase (G1896A, 76.2% versus 4.8%; C1913A, 33.3% versus 0%; C2189A, 28.6% versus 4.8%; G2304A, 52.4% versus 14.3%) in study group (n = 21) but not the control group (n = 9). Subjects with the G/G genotype at the IL-10-1082 polymorphism site had higher C2189A mutation rate than the A allele carriers (P = .02). C2189A mutation carriers are associated with more viral load decrement from tolerance to inflammatory phase (P = .01) and earlier spontaneous HBeAg seroconversion (P = .01). CONCLUSIONS: The G/G genotype at the IL-10 -1082 polymorphism is associated with higher C2189A mutations, lower HBV viral load at immune inflammatory phase, and earlier spontaneous HBeAg seroconversion than A allele carriers.

Effect of puberty onset on spontaneous hepatitis B virus e antigen seroconversion in men.

BACKGROUND: Male predominance is a remarkable phenomenon in hepatitis B virus (HBV)-related liver disease. This study elucidated the effects of puberty on spontaneous hepatitis B virus e antigen (HBeAg) seroconversion in boys. METHODS: One-hundred HBeAg-positive chronic HBV-infected males recruited at younger than 10 years of age who had been followed for >10 years were selected randomly from our long-term followed cohort into this study. Serum testosterone levels, androgen receptor exon-1 CAG repeat number and steroid 5alpha reductase type II (SRD5A2, valine vs leucine alleles) polymorphism were determined. Serial clinical data, HBV genotype, and spontaneous HBeAg seroconversion age were also analyzed. RESULTS: Seventy-two subjects had spontaneous HBeAg seroconversion during the follow-up period. Subjects with serum testosterone levels > or =2.5 ng/mL at 15 years old (earlier-onset puberty, n = 87) had earlier HBeAg seroconversion (median age, 13.2 vs 22.5 years; hazard ratio = 2.95; P = .005), higher peak alanine aminotransferase levels when HBeAg positive (305.7 +/- 372.7 vs 154.8 +/- 126.0 IU/L; P = .006), and a greater HBV viral load reduction from 10 to 20 years of age (1.6 +/- 2.4 vs 0.2 +/- 1.4 log10 copies/mL; P = .009) than those with serum testosterone levels <2.5 ng/mL (later-onset puberty, n = 13). Valine allele carrier at the SRD5A2 V89L polymorphism was also associated with earlier spontaneous HBeAg seroconversion (median age, 11.7 vs 18.7 years; hazard ratio = 1.88; P = .028). CONCLUSION: Earlier-onset puberty and increased SRD5A2 enzyme activity are associated with earlier HBeAg seroconversion, higher serum alanine aminotransferase levels, and a greater HBV viral load decrement in chronic HBV infected males.

Pre-S2 deletions of hepatitis B virus and hepatocellular carcinoma in children.

UNLABELLED: The cause of early oncogenesis in hepatitis B virus (HBV)-related childhood hepatocellular carcinoma (HCC) remains unclear. This study investigated whether pre-S deletion of HBV is related to childhood HCC. By using nested polymerase chain reaction, we compared the pre-S sequence of HBV from sera of children with HCC against control children with similar chronic HBV infection. The HBV in sera of children with HCC had a significantly higher rate of pre-S deletion than that of children with chronic HBV infection (p = 0.008). All except one of the pre-S deletions from the HCC group involved the pre-S2 region, whereas no pre-S2 deletion was found in the chronic HBV group (p = 0.003). There was a trend whereby genotype-C sera had a higher rate of pre-S2 deletion than genotype-B sera (p = 0.11). A multivariate logistic regression model revealed that pre-S deletion was an independent risk factor for HCC in children (odds ratio: 36.69, p = 0.015). In conclusion, pre-S2 deletion does not need to take decades to occur; its presence in nearly half of children with HCC, in contrast to its absence in children with chronic HBV infection, suggests a link between pre-S2 deletion and HCC development in children. ABBREVIATIONS: :

Mutations of T-cell epitopes in the hepatitis B virus surface gene in children with chronic infection and hepatocellular carcinoma.

BACKGROUND AND OBJECTIVES: Hepatitis B virus (HBV) infection induces an interaction of host immune responses against virus antigen-presenting hepatocytes. The emergence of mutants is a strategy through which the virus can escape host attacks and produce chronic infection. In this study, we aimed to investigate mutations of the human leukocyte antigen-A2-restricted T-cell epitope (TCE) in chronic HBV-infected children. METHODS: In total, 441 chronic HBV-infected children were longitudinally followed-up every 6 months. They were divided into hepatitis B e antigen (HBeAg) (-) (N = 60) and HBeAg (+) (N = 381) groups according to this seromarker at their enrollment. The HBeAg (+) group was further divided into HBeAg (+/-) (N = 229) and HBeAg (+/+) (N = 152) groups, depending on the occurrence of spontaneous HBeAg seroconversion. Twenty-five children with HBV-related hepatocellular carcinoma (HCC) were also recruited. TCE mutations were examined using the latest serum samples, and serum samples at enrollment were used if TCE mutations were present in the latest serum samples in the seroconverters. HBV genotypes and liver enzymes were also analyzed. RESULTS: The HBeAg (+/+) group had a lower TCE mutation rate (7.9%) than that of the HBeAg (+/-) (29.2%), HBeAg (-) (26.7%), and HCC (28.0%) groups. In the HBeAg (+/-) group, TCE mutations were present before HBeAg seroconversion in 11.9% of the subjects. Those with TCE mutations showed HBeAg seroconversion at an older age (16.1 +/- 5.3 yr vs 12.7 +/- 5.7 yr, P < 0.001) and with higher peak alanine aminotransferase (ALT) levels (median 175 U/L vs 119 U/L, P = 0.03) than those without TCE mutations. CONCLUSIONS: TCE mutations tended to be positively associated with HBeAg seroconversion and higher ALT levels in chronic HBV-infected children.

Viremia profiles in children with chronic hepatitis B virus infection and spontaneous e antigen seroconversion.

BACKGROUND & AIMS: This study investigated the viremia profiles in children with chronic hepatitis B virus (HBV) infection and spontaneous hepatitis B e antigen (HBeAg) seroconversion. METHODS: Fifty-eight children with chronic HBV infection met the following criteria: normal alanine aminotransferase (ALT) level at enrollment, followed up for more than 10 years, no antiviral treatment, and having undergone spontaneous HBeAg seroconversion during follow-up evaluation. They were grouped according to the post-HBeAg seroconversion HBV-DNA levels: (1) low viremia: transient or never 10(4) copies/mL or greater (n=35) (2) fluctuating high viremia: 10(4) copies/mL or greater at least twice at intervals more than 1 year apart (n=23). Abdominal sonography, ALT, and HBV-DNA levels were assessed annually. Another 14 nonseroconverted children served as controls. The precore mutant (nt1896) and genotypes were examined. RESULTS: The initial HBV-DNA level of the 58 seroconverters was 10(8.4+/-1.0) copies/mL and decreased to 10(2.9+/-2.0) copies/mL at the end of follow-up period. Their mean ages at enrollment, at peak HBV-DNA, at peak ALT, at HBeAg seroconversion, and at final follow-up were 7.0 +/- 3.7, 13.4 +/- 5.8, 16.3 +/- 6.0, 17.2 +/- 5.8, and 23.7 +/- 4.1 years, respectively. The precore mutant appeared more often in the fluctuating-high-viremia group than in the low-viremia group (60.9% vs 22.9%, P=.004). HBV genotypes had no effect on the viremia profiles. After HBeAg seroconversion, none had persistent abnormal ALT levels. CONCLUSIONS: Generally, these young seroconverters had decreased viral loads, normal ALT levels, and uneventful courses after HBeAg seroconversion. A longer follow-up period is necessary to elucidate the significance of HBeAg seroconversion occurring in childhood and young adulthood.

Lamivudine treatment in maternally transmitted chronic hepatitis B virus infection patients.

BACKGROUND: Lamivudine treatment in chronic carriers who acquired hepatitis B virus through maternal transmission were investigated. METHODS: A total of 29 subjects (Male:Female, 24:5; mean age, 14.7 +/- 5.6 years) who were hepatitis B e antigen (HBeAg) seropositive for >6 months, alanine aminotransferase (ALT) was >1.3 times of upper limit of normal value, and receiving a 52 week-long treatment, received open-label lamivudine (3 mg/kg per day, maximum 100 mg/day). Another 29 subjects matched for gender, age, liver function, and HBeAg status followed up before the introduction of lamivudine served as the control group. The control group did not receive any treatment and were evaluated at week 52 after the onset of abnormal ALT. Mothers of all study subjects were hepatitis B surface antigen (HBsAg) carriers. A successful treatment response at week 52 was defined as: (i) undetectable hepatitis B virus DNA by real time polymerase chain reaction; (ii) normal ALT; and (iii) HBeAg/anti-HBe seroconversion. Lamivudine-resistant YMDD mutants were checked at week 52. RESULTS: The lamivudine group did not reach a better successful treatment response rate than the control group (17 vs 10%, P = 0.44), except in patients with a baseline ALT >5 times of the upper limit of normal value. YMDD mutants developed in 34% of patients in the lamivudine group. CONCLUSION: Lamivudine treatment is effective for maternally transmitted subjects with high ALT.

Differential integration rates of hepatitis B virus DNA in the liver of children with chronic hepatitis B virus infection and hepatocellular carcinoma.

BACKGROUND AND AIM: Integration of hepatitis B virus-DNA (HBV-DNA) into the host genome, a phenomenon found frequently in hepatocellular carcinomas (HCC) and causally linked to oncogenesis, has not been well characterized in children. The aim of the present study was to determine the prevalence of HBV integration more accurately and to decide whether the integration rate varies at different stages of chronic HBV infection in children. METHODS: Of 13 children with chronic hepatitis, 14 liver biopsy tissues were analyzed. One liver tissue with pure liver cirrhosis, nine non-tumor, and nine tumor liver tissues from children with HCC were analyzed by a very sensitive method, inverse polymerase chain reaction (IPCR). RESULTS: Thirteen genuine viral-host junctional sequences from 23 patients were successfully isolated and proved that IPCR is a useful method in this context. The results also indicated that the detection rate of HBV-DNA integration increased in parallel with the progress of liver histology towards the neoplastic transformation, with 0% in the liver of chronic hepatitis, 22.2% in non-tumor livers of HCC patients, and 66.7% in tumor liver tissues of HCC patients. CONCLUSION: The present results indicate that integration of HBV-DNA into the host genome was rarely confirmed at the early stage of chronic hepatitis in children until the stage of HCC formation.

Longitudinal study on mutation profiles of core promoter and precore regions of the hepatitis B virus genome in children.

Precore nucleotide 1896 and core promoter mutations may account for hepatitis B e antigen (HBeAg) seroconversion in chronic hepatitis B virus (HBV) infection, yet the mutational profiles of the core promoter are largely unknown in children. An age-matched, case-control study enrolled 110 chronic HBV-infected children, including 55 HBeAg seroconverters and 55 nonseroconverters. Precore and core promoter genes of HBV were sequenced and the serum viral genomes were genotyped from three serial serum samples of the seroconverters and from one serum sample of the nonseroconverters. Higher frequency of A1775G and G1799C mutation rates and lower frequency of A1752G mutation rate were found in the seroconverters. Precore 1896 mutation appeared more in seroconverters than in nonseroconverters (45.5% versus 10.9%; p < 0.001). 1762 + 1764 mutation rates were not different between the seroconverters (9.1%) and the nonseroconverters (5.5%). Genotype B was the major type. Genotype C was associated with core promoter 1762 + 1764 mutations in the seroconverter group (p = 0.023). The conclusions of this study include the following: 1) mutations of core promoter at nucleotide position 1752, 1775, and 1799 have significant correlations with HBeAg seroconversion; 2) core promoter 1762 + 1764 mutations play a minimal role in HBeAg seroconversion; 3) precore 1896 mutant accounted for half of childhood HBeAg seroconversion; 4) genotype C is associated with 1762 + 1764 mutations during the process of HBeAg seroconversion.

RBMY, a male germ cell-specific RNA-binding protein, activated in human liver cancers and transforms rodent fibroblasts.

The RNA-binding motif (RRM) gene on Y chromosome (RBMY), encoding a male germ cell-specific RNA-binding protein associated with spermatogenesis, was found inserted by hepatitis B virus (HBV) DNA in one childhood hepatocellular carcinoma (HCC). This study is aimed to explore the oncogenic potential of the RBMY protein. The RBMY transcripts, expressed exclusively in the testis of normal people, were detected by reverse transcription-polymerase chain reaction in 36% of HCCs from 90 males and in 67% of hepatoblastoma from six boys. The nontumor liver counter parts, cirrhotic liver tissues from children with biliary atresia, and other types of cancers, such as bile duct, colon, stomach, lung, prostate, and kidney, were all negative for RBMY expression. One to four types of RBMY transcripts, including wild type and variants with N-terminal RRM deletion, C-terminal SRGY (serine-arginine-glycine-tyrosine) boxes deletion, or deletion of both domains, were found in the testis and liver cancer tissues. The wild-type RBMY protein was expressed in the nucleus and demonstrated its tumorigenicity by transformation of mouse fibroblast NIH3T3 cells and in vivo tumor formation. The RBMY variant protein with deletion of C-terminal exons 9-12 was trapped in the cytoplasm and showed decreased tumorigenicity. Our results suggest that RBMY is a new candidate oncogene specific for male liver cancer.

Characterization of integration patterns and flanking cellular sequences of hepatitis B virus in childhood hepatocellular carcinomas.

Hepatitis B virus (HBV) DNA integration into host chromosomes is detected in more than 80% of HBV-related hepatocellular carcinomas (HCC), yet its significance in tumor development remains obscure. In this study, we re-examined the integration pattern of HBV in childhood HCC tissues, which has less environmental confounding factors than adult HCC. The HBV junctions and flanking cellular sequences were amplified from five childhood HCC patients by the inverse polymerase chain reaction (IPCR) method using primers located near HBV direct repeats (DR) 1 and 2. The viral junctions in nine of the ten obtained IPCR clones were demonstrated to be located near HBV DR1, and their patterns were classified to type I integrants. Southern blot analyses demonstrate that the cellular junctions derived from two of the five HCC tissues were male specific and contained sequences homologous to human long interspersed DNA elements (LINE-1). HBV integrant of one HCC tissue (1217T) was integrated into a RNA binding motif Y chromosome (RBMY) gene. The expression of RBMY, which is normally found only in male germ cells, was detected in HCC tissue 1217T by RT-PCR but not in the corresponding non-tumor liver tissue. The prevalence of RBMY expression in liver tissues from the tumor and non-tumor parts of ten other HCC children and seven biliary atresia (BA) children was studied by RT-PCR. No RBMY transcripts were detected in the non-tumor parts of HCC patients or the cirrhotic livers of BA children, whereas 30% (three of ten) of HCC tissues specifically expressed RBMY. The results indicate that HBV integration and activation of RBMY gene expression in liver cells may be associated with the development of childhood HCC.