MCM3AP, a novel HBV integration site in hepatocellular carcinoma and its implication in hepatocarcinogenesis

Abstract

A novel HBV integration site involved in hepatocarcinogenesis was investigated. The HBV DNA integration sites were detected by Alu-PCR in hepatocellular carcinoma tissues, matched surrounding liver tissues in 30 patients with hepatitis B-related hepatocellular carcinoma (HCC) and 3 cases of normal liver tissues. The integration sites and flanking sequences in human genome were sequenced and blasted, and the expression of integrated HBV genes was determined by reverse transcriptase-polymerase chain reaction (RT-PCR). The influence of the up-regulated expression of integrated genes on hepatocarcinogenesis was analyzed. Nineteen integration sites of HBV DNA into HCC tissues were obtained by RT-PCR and sequencing. These genes encoding proteins were: LOC51030, LOC157777, minichromosome maintenance complex component 3 associated protein (MCM3AP), MCTP1, SH3 and multiple ankyrin repeat domains 2 isoform 2, CCDC40, similar to HCG2033532, mitochondrial ribosomal S5 pseudogene 4. One of them was integrated into the intron of MCM3AP. RT-PCR demonstrated that the expression levels of MCM3AP mRNA in HCC tissues, matched surrounding liver tissues and normal liver tissues were in a descendent order. The ratio of MCM3AP mRNA to the GAPDH mRNA in these three tissues was 1.07375, 0.21573, 0.06747 respectively, with the difference being statistically significant among them (P<0.05). Meanwhile, the expression levels of MCM3AP mRNA from HCC tissues in which HBV DNA integrated into MCM3AP were still significantly higher than those without HBV DNA integrated into MCM3AP. It was concluded that the HBV DNA integration sites into human genome were random, and MCM3AP was a new site. The up-regulated MCM3AP mRNA may affect flanking sequences which promote the hepatocarcinogenesis.

Keywords

HBV integration / Alu-PCR / hepatocellular carcinoma / minichromosome maintenance complex component 3 associated protein

References

[1] MatsudaY., IchidaT.. Impact of hepatitis B virus X protein on the DNA damage response during hepatocarcinogenesis. Med Mol Morphol, 2009, 42(3): 138-142

[2] ParkN.H., ChungY.H.. Molecular mechanisms of hepatitis B virus-associated hepatocellular carcinoma. Korean J Hepatol, 2007, 13(3): 320-340

[3] Bonilla GuerreroR., RobertsL.R.. The role of hepatitis B virus integrations in the pathogenesis of human hepatocellular carcinoma. J Hepatol, 2005, 42(5): 760-777

[4] BlumbergB.S.. Primary and secondary prevention of liver cancer caused by HBV. Front Biosci (Schol Ed), 2010, 2: 756-763

[5] CheminI., ZoulimF.. Hepatitis B virus induced hepatocellular carcinoma. Cancer Lett, 2009, 286(1): 52-59

[6] TamoriA., YamanishiY., KawashimaS., et al.. Alteration of gene expression in human hepatocellular carcinoma with integrated hepatitis B virus DNA. Clin Cancer Res, 2005, 11(16): 5821-5826

[7] TaborE.. Pathogenesis of hepatitis B virus-associated hepatocellular carcinoma. Hepatol Res, 2007, 37(Suppl2): S110-S114

[8] LeeA.T., LeeC.G.. Oncogenesis and transforming viruses: the hepatitis B virus and hepatocellularcarcinoma—the etiopathogenic link. Front Biosci, 2007, 12: 234-245

[9] LuoK.X.. . Hepatitis B Basic Biology and Clinical Science, 20012nd ed.Beijing, People’s Medical Publishing House, 572

[10] TsaiC.C., HuangK.W., ChenH.F., et al.. Gene expression analysis of human hepatocellular carcinoma by using full-length cDNA library. J Biomed Sci, 2006, 13(2): 241-249

[11] MinamiM., PoussinK., BrechotC., et al.. A novel PCR technique using Alu-specific primers to identify unknown flanking sequences from the human genome. Genomics, 1995, 29(2): 403-408

[12] RemusD., BeuronF., TolunG., et al.. Concerted loading of Mcm2-7 double hexamers around DNA replication origin licensing. Cell, 2009, 139(4): 719-730

[13] GambichlerT., ShternM., RotterdamS., et al.. Minichromosome maintenance proteins are useful adjuncts to differentiate between benign and malignant melanocytic skin lesions. J Am Acad Dermatol, 2009, 60(5): 808-813

[14] BraunK.A., BreedenL.L.. Nascent transcription of MCM2-7 is important for nuclear localization of the minichromosome maintenance complex in G1. Mol Biol Cell, 2007, 18(4): 1447-1456

[15] YangZ.Y., WenJ.G.. The expression and significance of MCM protein family in carcinoma. J Clin Res, 2004, 21(9): 1032-1035

[16] KageshitaT., KuwaharaK., OkaM., et al.. Increased expression of germinal center-associated nuclear protein (GANP) is associated with malignant transformation of melanocytes. J Dermatol Sci, 2006, 42(1): 55-63

[17] GiaginisC., VgenopoulouS., VielhP., et al.. MCM proteins as diagnosis and prognostic tumor markers in the clinical setting. Histol Histopathol, 2010, 25(3): 351-370

[18] LinD.I., AggarwalP., DiehlJ.A.. Phosphorylation of MCM3 on Ser-112 regulates its incorporation into the MCM2-7 complex. Proc Natl Acad Sci USA, 2008, 105(23): 8079-8084

[19] LeeY.S., HaS.A., KimH.J., et al.. Minichromosome maintenance protein 3 is a candidate proliferation marker in papillary thyroid carcinoma. Exp Mol Pathol, 2010, 88(1): 138-142

[20] MusahlC., HolthoffH.P., LeschR., et al.. Stability of the replicative MCM3 protein in proliferating and differentiating human cells. Exp Cell Res, 1998, 241(1): 260-264

[21] HaS.A., ShinS.M., NamkoongH., et al.. Cancer-associated expression of minichromosome maintenance 3 gene in several human cancers and its involvement in tumorigenesis. Clin Cancer Res, 2004, 10(24): 8386-8395

[22] OsmanW., LaineS., ZilliacusJ.. Functional interaction between the glucocorticoid receptor and GANP/MCM3AP. Biochem Biophys Res Commum, 2006, 348(4): 1239-1244

[23] TakeiY., SwietlikM., TanoueA., et al.. MCM3P, a novel acetyltransferase that acetylates replicatin protein MCM3. EMBO Rep, 2001, 2(2): 119-123

[24] GaoH.F., TuH.. Identification of hepatitis B virus integration sites in hepatocellular carcinomas. Chin J Hepatol, 2004, 12(9): 567-568

[25] FujimuraS., XingY., TakeyaM., et al.. Increased expression of germinal center-associated nuclear protein RNA-prim ase is associated with lymphomagenesis. Cancer Res, 2005, 65(13): 5925-5934