Reasearch On The Nucleotide Mutations Of Hepatitis B Virus Covalently Closed Circular In Hepatitis B Virus-infected Hepatocellular Carcinoma

Obejective: To study expression levels and nucleotide mutations in each region of ccc DNA(covalently closed circular DNA,ccc DNA) in HBV(hepatitis B virus, HBV)related HCC(hepatocellular carcinoma,HCC),investigate the association between nucleotide mutations in different regions and development of HCC. To find key gene factors for HBV-infected HCC based on gene expression data, and provide candidate disease genes for HCC prevention and treatment.Methods: We collected 49 paired matched cancerous tissues(CT)and contiguous noncancerous tissues(CNCT) liver from patients in the First and second Affiliated Hospital of Chongqing Medical University. We firstly measured clinical virology information and levels of intrahepatic ccc DNA and HBV total DNA in CT and CNCT through Tag Man probe q PCR. Then we specifically amplified ccc DNA by rolling circle amplification(RCA). We analyzed ccc DNA and DNA expression levels inCT and CNCT tissues, as well as association between single and combination nucleotide mutations in HBV ccc DNA X and pre C/C gene regions in development of HCC. We downloaded gene expression data of5 paired HBV-infected HCC patients from NCBI, identified HCC related candidate genes through bioinformatics analysis.Results:(1)All of 35 pairs patients’ levels of ccc DNA were detected in matched CT and CNCT samples. Overall intrahepatic ccc DNA levels in CT was significantly lower than that in CNCT(5.19 VS. 9.35 copies/cell,p=0.0033). We got 18 and 16 pairs of ccc DNA sequences in X and pre C/C gene regions through RCA amplification. Mutation ratio of X gene region in CT was significantly higher than that in CNCT(28.63% VS. 20.94%,p=0.0313); T1721 G in X gene region and A1979 Gin pre C/C gene region had a higher mutation numbers in CT than in CNCT(p = 0.04075,OR =10.1598;p = 0.0155,OR = 13.7529), and their combination mutations A1703 G / T1727G、A1762T / G1764 A / T1727G、A1703G / T1727 G /A1762 T / G1764 A also showed a higher mutation numbers in CT.(2) Up regulated genes EGR1, FOS, DUSP1 in HCC high risk modules participated in MAPK signal pathway and the downstream cell cycle pathway.Conclusion: Typical mutations A1762 T / G1764 A, G1896 A /G1899 A, etc. mutate both in CT and CNCT samples, have no significant difference in X and pre C / C gene region. T1721 G and A1979 G singlenucleotide mutations and their combination mutations are more characteristic in our patient samples, they may be associate with development of HCC. Screend genes EGR1, FOS, DUSP1 may be not only biomarkers for early recognition of HCC,but also provide candidate gene for targeted gene therapy of HCC.