Abstract
Chronic hepatitis B virus (HBV) is the leading cause of hepatocellular carcinoma (HCC). Covalently closed circular DNA (cccDNA) is an intermediate in the life cycle of HBV. HBV-encoded X protein (HBx), a key viral oncoprotein, can be specifically ubiquitylated by male specific lethal 2 (MSL2), which causes upregulation of HBx activity and promotes transcription, cell proliferation and tumor growth. The present study compared the levels of cccDNA, MSL2 mRNA and HBx mRNA in tumor and peri-tumor tissues, and clarified the effect of antiviral therapy on these indicators. Levels of intrahepatic cccDNA, MSL2 mRNA and HBx mRNA were determined using quantitative PCR in patients with HBV-associated HCC who had undergone liver surgery. A total of 50 patients were included in the present study. Prior to surgery, 31 patients had undergone antiviral treatment. Intrahepatic cccDNA levels were significantly higher in the tumor tissues compared with the peri-tumor tissues (P=0.001), particularly in the hepatitis B e antigen-positive (P=0.008), tumor recurrence (P=0.002) and <3 cm tumor size (P=0.003) groups. Furthermore, in patients with preoperative cirrhosis, levels of cccDNA and MSL2 mRNA were significantly higher in tumor tissues compared with that in peri-tumor tissues (P<0.001 and P=0.023, respectively). The expression levels of HBx mRNA in antiviral-treated tumors and peri-tumor tissues were significantly lower compared with those in untreated tissues (P=0.026 and P=0.035). The levels of cccDNA and MSL2 mRNA in the HBx-positive group were significantly higher in tumor tissues compared with those in peri-tumor tissues (P=0.026 and P=0.013). In conclusion, cccDNA participated in the tumorigenesis of HBV-associated HCC, and antiviral therapy was found to modulate hepatocarcinogenesis by decreasing the levels of HBx to inhibit the tumorigenic effect of MSL2 and cccDNA.
Highlights
Hepatitis B virus (HBV) infection is a global health concern that causes >1,000,000 deaths worldwide per year [1]
The extraction procedures for closed circular hepatitis B virus total DNA (DNA) (cccDNA), male specific lethal 2 (MSL2) mRNA and HBV‐encoded X protein (HBx) mRNA were based on the sample to be assayed. cccDNA, MSL2 mRNA, HBx mRNA were stored at ‐20 ̊C from a single HBV‐negative sample, based on a fixed control patient, and were added to each PCR analysis; fold‐changes were determined based on the relative levels of cccDNA, MSL2 mRNA and HBx mRNA
Following infection, which is hepatocyte‐specific, the capsid is transported to the nucleus and the relaxed circular DNA is released and converted into the persistent form, cccDNA
Summary
Hepatitis B virus (HBV) infection is a global health concern that causes >1,000,000 deaths worldwide per year [1]. HCC is the fifth most common type of cancer and the third highest cause of cancer‐associated mortality worldwide, next to lung and stomach cancer [3]. Closed circular DNA (cccDNA) is an important intermediate in the life cycle of HBV. It does not directly participate in HBV replication, but maintains a stable pool within the hepatocyte nucleus [5]. A previous study demonstrated that HCC often develops as HBV replication intensifies during the late stage of hepatitis B [6], a period in which cccDNA becomes predominant in quantity [7]. It may be possible to predict the involvement of cccDNA in HBV‐associated HCC by measuring the levels of intrahepatic cccDNA in paired tumor and peri‐tumor tissues
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