The Relationship Between Post-operative HBV Reactivation And Toll Like Receptor 9 In HBV Related Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the ten most commonly occurring solid cancers worldwide and is the second cause of death from malignancy.In our country,60%HCC are developed from hepatitis B virus(HBV)infection.We describe it as HBV related hepatocellular carcinoma(HBV-HCC).HBV-HCC patients had two different states,one is malignant tumor,the other is chronic HBV infection.The commonly utilized modalities for treating HCC include liver transplantation,surgical resection,local ablation therapy,transcatheter arterial chemoembolization(TACE)and so on.Many studies show that HBV reactivation is common in HBV-HCC patients who receive anti-tumor treatments.Postoperative HBV reactivation may aggravate the development of liver inflammation,speed up the process of cirrhosis,and finally influence the prognosis of HBV-HCC patients.Toll-like receptor 9(TLR9)is a kind of conservative transmembrane signaling receptor family which take an important role in immune response.It is also a member of pattern recognition receptors(PPR).TLR9 is highly expressed on plasmacytoid dendritic cells and B lymphocyte,weakly expressed on liver cell,kuffer cells,stromal cells and tumor cells.HBV DNA is known as covalently closed circular DNA(cccDNA).HBVDNA can be recognized by plasma dendritic cells(pDCs)through toll-like receptor 9(TLR9)which activates the NF-κB signal pathway to up-regulate the expression of interferon regulatory 7(IRF-7),resulting in secretion of type I IFN and inflammatory cytokines,and induction of DC maturation and the adaptive immune response.Many studies show that DC are functionally impaired in patients with chronic hepatitis B.CHB patients do not express appropriate levels of the genes in the TLRs pathway which lead to impaired immune response against HBV.Moreover,we can control hepatitis B virus replication by Toll-like receptor signaling pathways.But till now,we had no relevant literature about the mechanism of HBV reactivation in HBV-HCC patients undergoing operation.It is important to analysis the risk factors related to HBV reactivation,more importantly,to analysis the immuse mechanism along with HBV reactivation.And then we can find new targets for antiviral reactivation treatment.This research was a comprehensive study which combined clinical medicine and basic medicine together.Between March 2015 and December 2015,Consecutive patients with HBV-related HCC who underwent hepatic resection were prospectively enrolled in this study.In the first part,we collected the clinical datas of these patients and made afollow-up.Then we clarified the risk factors related to HBV reactivation in HBV-HCC patients.In the second part,preoperative and postoperative peripheral blood of HBV-related HCC patients were collected.Plasmacytoid dendritic cells(pDCs)were enrichmented through positive immunomagnetic sand method using the mini-MACS system.Then we analyzed whether TLR9 signaling molecules(TLR9,MyD88,IRAK1,TRAF6,TAK1,IRF7)were involved in the process of HBV reactivation.Plasma of those patients was also collected.Whether cytokines(IFN-α、IL-6、IL-12、TNF-α)were involved in the process of HBV reactivation was also analyzed.In the third part,we collected the tumor tissue and para-carcinoma tissue,and detected the expression differences of TLR9 signaling molecules in the two groups.Part one: 34 consecutive patients were collected from March 2015 to December 2015.We collected the clinical datas of these patients such as demographic information(age,gender),the background of hepatitis,Blood routine examination(white blood cell count and blood platelet count),liver function(total bilirubin,alanine aminotransferase,aspartate transaminase,albumin and prealbumin),renal function(creatinine and blood urea nitrogen),tumor marker(alpha fetal protein),two pairs of semi-hepatitis(HBsAg,anti-HBs,HBeAg,Anti-HBe,Anti-HBc),HBV DNA load,intraoperative situation(the size and location of tumor,tumor thrombus,inflow blood occlusion time,blood loss).After operation,we made a follow-up.Then patients were divided into two groups(the HBV reactive group and the non-reactive group)according to the standard of HBV reactivation.We analyzed the clinical datas and determined the clinical risk factors related to HBV reactivation.The risk factors were evaluated by uni-and multi-unconditional logistic model used SPSS 20.0.On univariate analysis,1)We found that those patients with a higher pre-ALT or pre-AST(>100U/L)have a higher incidence of HBV reactivation(100.0% vs 20.0%,P=0.001).2)Patients who had a long history of antiviral treatment had a lower incidence of HBV reactivation(56.3% vs 5.6%,P=0.001);3)Patients with a higher postoperative AST had a higher incidence of HBV reactivation(66.7% vs 21.4%,P=0.027).The result of maternal exposed factors multi-unconditional logistic model showed that antiviral therapy(without)was significantly related with HBV reactivation(HR=13.908;95%CI,1.137-170.067,P=0.039).Part two: To further understand the mechanism of HBV reacativation in HBV-HCC patients,We collected the pre-and post-operative peripheral blood of these patients.Plasmacytoid dendritic cells(pDCs)of peripheral blood were concentrated throughpositive immunomagnetic selection using the mini-MACS system.And the relative expression levels of TLR9,MyD88,IRAK1,TRAF6,TAK1,IRF7 were evaluated by Real-Time PCR techniques.Meanwhile the cytokine(TNF-α,IL-6,IL-12,IFN-α)were tested by Elisa test.Then all datas were analyzed with SPSS 20.0.The results showed that1)HBV reactivation do not correlated to the number of DC of peripheral blood.2)TLR9,MyD88,IRAK1,TRAF6,TAK1,IRF7 mRNA of pre and post-operation in the reactivate group were lower than the non-reactivate group.The statistical analysis showed: The relative expression levels MyD88 mRNA of pre-operation in reactivate group is significantly lower than the non-reactivate group(0.39,0.26-2.59 vs 1.03,0.64-2.62,P=0.042).The relative expression level of Pre-IRF7 mRNA in reactivate group is(0.67,0.28-1.00),which in reactivate group is(2.42,1.19-5.46)(P=0.008);The relative expression levels of post-operative TLR9 mRNA in reactivate group is significantly lower than which in non-reactivate group(0.27,0.12-0.32 vs 0.63,0.28-1.09,P=0.009);The expression levels of pro-TRAF6 mRNA in reactivate group is(0.69,0.49-0.98),which is significantly lower than TRAF6 mRNA in non-reactivate group(1.02,0.80-1.79)(P=0.025);Pro-TAK1 mRNA in reactivate group is(0.22,0.14-0.62),which is significantly lower than non-reactivate group(0.54,0.29-1.04)(P=0.038);The relative expression levels of pro-IRF7 mRNA in reactivate group is significantly lower than which in non-reactivate group(0.26,0.18-0.52 vs 0.66,0.32-1.02,P=0.020).And post-operative TNF-α in HBV reactivate group is lower than the non-reactivate group.It is correlated with postoperative viral reactivation(11.44±2.04 pg/ml vs 14.78±3.55 pg/ml,P=0.002).Part three: Tumor tissue and para-carcinoma tissue of these patients were also collected.We evaluated TLR9 and MyD88 signaling pathway molecular(MyD88,IRAK1,TRAF6,TAK1,IRF7)expression levels and compared the differences between the two groups in tumor tissue and para-carcinoma tissue by Real-time PCR,immunohistochemistry,Western Blotting technique.And we found TLR9 of paracarcinoma tissue in reactivate group was higher than the non-reactivate group(6.35,4.43-14.17 vs 0.66,0.28-1.64,P=0.006).Moreover,we found that TLR9 protein is also higher in reactivate group than non-reactivate group.But the expression levels of MyD88,IRAK1,TRAF6,TAK1,IRF7 had no difference between the two groups.Conclusions: 1)Antiviral therapy(without)is the independent risk factor of pro-operative HBV reactivation.It showed that antiviral therapy can significantly decrease the perioperative reactivation in patients with hepatitis B virus-related hepatocellularcarcinoma undergoing liver resection.2)Postoperative HBV reactivation has nothing to do with the number of DC,which maybe is mainly related to DC dysfunction.3)TLR9 is a double-edged sword in HBV reactivation.The lower expression of TLR9 in DC is related to the HBV reactivation.TLR9 of para-carcinoma tissue in reactivate group is higher than the non-reactivate group.So we speculated that TLR9 is a double-edged sword in HBV reactivation.TLR9 of DC play an essential role in the recognition of invading pathogens via microbial molecular motifs,comprising a bridge between the innate and adaptive immune responses,and promote the release of cytokines.But TLR9 of para-carcinoma tissue can help HBV invading from the immune surveillance,which lead to the replication of HBV in the liver cells.3)Expression of postoperative TNF-α may be a cause of HBV reactivation.