Role Of Transcription Factor FoxO3a In HBV Replication And The Underlying Mechanisms

【Background】Hepatitis B virus (HBV) infection is a significant public health problem affecting an estimated 350–400 million individuals worldwide.The disease is endemic in China, where a national survey in 2002 showed a 9% rate of HBsAg in the general population. Around 120 million people in China are carriers of HBV. During a 5-year period, 10–20% of patients with chronic hepatitis developed cirrhosis, 6–15% of the people with cirrhosis and chronic hepatitis progressed to hepatocellular carcinoma (HCC), in which the 5-year survival is less than 5%. However, the treatments currently approved for chronic hepatitis B patients, including alpha interferon and four nucleoside analogues that inhibit viral DNA polymerase, are limited by low rates of sustained response, side effects, and the emergence of drug resistance. Therefore, it is necessary and urgent to establish new antiviral strategies to treat this disease. Although there are many factors including HBX, cell cycle, specific hormones and inflammatory cytokines that affect HBV replication, transcriptional regulation of HBV genome is the most critical steps in HBV replication. However, to date, there is still lack of general awareness of HBV genome transcriptional regulation. Studies have shown that some liver-cell-enriched transcription factors such as HNF1, HNF3, HNF4 and C/ EBP play important roles in the transcriptional regulation of HBV. However, these findings did not fully reveal the mechanism of HBV transcriptional regulation. This suggests that there may be other unknown transcription factors that regulate the transcription of HBV genome, and a network in which those known and unknown transcription factors interact to co-regulated the HBV transcription and replication. Therefore, screening for the unknown transcription factors that regulated the HBV replication and clarification of their roles and the underlying mechanisms may be contributive to finding new targets for antiviral therrapy.【Aims】To screen for the transcription factors related to HBV replication and to clarify their roles and the underlying mechanisms in HBV replication, with the aim of better elucidating the mechanisms of HBV replication and laying a foundation for formulating novel anti-HBV therapeutic strategies.【Methods】1. OATFA was performed to detect the difference in activity of transcription factors between HBV stable replication cell line HepG2.2.15 and its parental cell line HepG2. 2. ELISA was carried out for validation of OATFA results. 3. Immunocytochemical method was performed to detect the subcellular localization of FoxO3a transcription factor in cell lines HepG2.2.15 and HepG2. 4.The levels of phospho-FoxO3a in cell lines HepG2.2.15 and HepG2 were detected by Western blot. 5. The clinical data of patients with high-copy and low-copy hepatitis B virus DNA and their fresh live tissues were collected; Levels of phospho-FoxO3a protein in tissues were detected by western blot and the relationship between the activity of FoxO3a and HBV replication were analyzed. 6.The siRNA expression vectors of FoxO3a (FoxO3a-siRNA) were constructed and transfected into HepG2.2.15 cells to down-regulate the expression of FoxO3a. 7. The effect of FoxO3a-siRNA on hepatitis B virus DNA levels and mRNA levels in HepG2.2.15 cells were determined by Real-time fluorescence PCR;The effect of FoxO3a-siRNA on hepatitis B virus protein levels were determined by ELISA. 8. HBV promoter/enhancer truncated sequence were cloned and the luciferase reporter gene vector were constructed; The effect of FoxO3a on transcriptional activity of HBV was evaluated by reporter gene assay. 9. The binding of FoxO3a with HBV enhancer or promoter in cell were verified by chromatin immunoprecipitation (CHIP).10. The binding site of FoxO3a in HBV promoter/enhancer was determined by EMSA. 11. The effect of FoxO3a-siRNA on cell cycle in HepG2.2.15 cells was evaluated using flow cytometry and the effect on level of cell-cycle-related proteins was assessed by Western blot. 12.The effect of FoxO3a-siRNA on cell growth of HepG2.2.15 was determined by MTT assay.【Results】1. OATFA revealed that the activities of 12 transcription factors were significantly different (>1.5 fold) in HBV stable replication cell line HepG2.2.15 and its parental cell line HepG2.The activities of HNF(1,3,4), C/EBP,FoxO (1,3a,4),FOXI1, SF-1, GR, SRY, COUP-TF1 were up-regulated in HepG2.2.15 cells, while those of AP1,AP2,SP(1,2,3,4),Egr(1,2,3)were down-regulated. Among them, 5 transcription factors family members have not yet been reported to be associateed with HBV replication, which are transcription factors FoxO (1,3a,4),Egr(1,2,3),FOXI1, SRY and AP2;The activity of FoxO(1,3a,4)and AP2 were the most dramatically changed. 2. ELISA assay demonstrated that the activities of FoxO(1,3a,4)were increased and the activity of AP2 were decreased in HepG2.2.15 cells compared with its counterpart;These results were in consistence with those of OATFA. 3 Immunofluorescence cytochemistry showed that FoxO3a localized mainly in cytoplasm in HepG2 cells, and in nuclear and cytoplasm in HepG2.2.15 cells. 4. Western blot results showed that the expression level of phospho-FoxO3a protein in HepG2 cells was higher than that of HepG2.2.15 cells. 5. Western blot results showed that the expression level of phospho-FoxO3a protein in live cirrhotic tissues with high-copy hepatitis B virus DNA were higher than that of live cirrhotic tissues with low-copy hepatitis B virus DNA. 6. PSilencer3.1/FoxO3a-siRNA expression vector were transfected into HepG2.2.15 cells and western blot results showed that the protein expression of FoxO3a was significantly reduced by siRNA. 7. Real-time quantitative PCR results showed that down-regulated expression of FoxO3a by siRNA could decreased the HBV DNA level of the cell supernatants and intracell of HepG2.2.15. 8. Real-time quantitative PCR results showed a significant inhibition of the level of HBV mRNA in HepG2.2.15 cell supernatant by knockdown of FoxO3a with siRNA. 9.ELISA results showed that decreased expression of FoxO3a by siRNA significantly reduced the HBeAg and HBcAg protein levels, which was time-dependent. 10.Through bioinformatics analysis, we found that HBV promoter/enhancer regions have four potential FREs (FoxO3a DNA- binding sequence: (G/C) (T/A) AA (C/T)AA; Dual luciferase reporter gene assay showed that: 1) FoxO3a protein increased the transcriptional activity of HBV; 2) The potential FRE of FoxO3a transcriptional regulation of the activity of HBV is located in FRE1 (HBV genomic regions 955-1184 bp). 11.CHIP experiments further confirmed that FoxO3a could bind to the sequence of HBV enhancerⅠin HepG2.2.15. 12. EMSA results further confirmed that the binding sites of FoxO3a with HBV enhancerⅠsequence is in the region 1127-1134bp (5'-GTAAACAA-'3). 13. Flow cytometry assay showed that down-regulated expression of FoxO3a by siRNA lead to the cell cycle progression in HepG2.2.15 cell. Western blot results showed that knockdown of FoxO3a by siRNA increased the expression of cyclinD1 and cyclinD2, but reduced the expression of P27KIP1. 14.MTT assay demonstrated that down-regulated expression of FoxO3a by siRNA promoted HepG2.2.15 cells growth in vitro.【Conclusions】1. The activity of host cell transcription factors in HBV stable replication cell line is different from its parental cell line. Many known and unknown transcription factors may be involved in the replication of hepatitis B virus. 2. The activity of FoxO3a transcription factor was increased in cells and liver tissues with hepatitis B virus infection. The activity of FoxO3a was correlated with HBV replication status. 3. FoxO3a transcription factor could enhance HBV hepatitis B virus replication in vitro. 4.There are two different pathways by which FoxO3a induced HBV replication in HepG2.2.15. Firstly, FoxO3a transcription factor could bind with HBV enhancerⅠsequence region 1127-1134bp (5'-GTAAACAA-3') and directly activate the HBV transcription; Secondly, FoxO3a transcription factor resulted in cell cycle G1/S phase arrest through up-regulating expression of P27KIP1 and down-regulating expression of cyclinD1 and cyclinD2, thus modulating HBV replication in HepG2.2.15 cells.