Studies On The Roles And Mechanisms Of HBC/HCV Contributes To HCC Proliferation And Metastasis

Part I: Studies on the Roles and mechanisms of HBV/HCV contributes to HCC proliferation and metastasisHepatocellular carcinoma(HCC)is currently one of the most commonmalignant tumors around the world,the incidence of HCC ranks fifth,and the mortality rate ranks third in all malignancies.Due to the concealed onset of the disease,symptoms are not obvious at early stage,disease progress is rapid,so the majority of patients diagnosed at late stage or when distant metastasis has occurred and unqualified for best treatment,hence leading to poor prognosis and highmortality.The etiology and exact molecular mechanism of HCC is not yet clear,which is currently considered as a multi-causative factors and multi-step complex process,hepatitis B virus(HBV)and hepatitis C virus(HCV)infection are the most common cause of HCC,accounting for about 70-85%.Previous studies have shown that HCC patients with hepatitis B virus infection had poorprognosis than the virus-free patients.Our previous study showed virus re-activation after partial hepatectomy was common;hepatitis virus reactivation played an important role in the development of HCC and could affect tumor recurrence and postoperative survival.Previous studies had shown that postoperative continuous antiviral therapy was expected to reduce the viral load,liver inflammation and thus eased the liver load,delayed recurrence and improved patient outcomes.Our previous study showed that antiviral therapy could not play a direct role in tumor,but can reduce chronic liver inflammation by inhibiting the hepatitis virus,thereby reducing the chance of recurrence.There is still controversy over whether antiviral therapy was possible to anti-tumor.Therefore,it was necessary to study the mechanism of viral replication in HCC recurrence.Theaims of our study were to identify the genes and signaling pathways related to HCCcaused by hepatitis virus infection by RNA sequencing and further clarify the specific mechanism of changes in related genes and pathways caused by hepatitis virus infection.Next,we would focus on these genes and signaling pathways and figure out how those genes and pathways enhanced HCC cell proliferation and metastasis.This study could not only clarify the specific mechanism of the development of HCC caused by hepatitis virus,but also provide more personalized treatment for future antiviral therapy.First of all,in this study,RNA sequencing was used to detect normal liver tissue,HCC tumor tissues without hepatitis virus infection,and HCC tumor tissuesinfected with hepatitis C virus.The results obtained in our study are as follows: using RNA sequencing and bioinformatics analysis,we found that P54 nrb was up-regulated in HCC tumor tissues infected with HBV or HCV,indicatingthat P54 nrb played an important role in HCC tumor tissues infected with HBV or HCV.By IHC,we found that high expression of P54 nrb was related to severalclinicpathological features of multiple malignancies,high expression of P54 nrbhad poor prognostic,and multivariate analysis showed that high expression of P54 nrbwas an independent factor in HCC.We found that P54 nrb promoted HCC cell proliferation?invasion and migration in vitro.Using animal experiments in nude mice,we found that P54 nrb could promote HCC cells in vivo,indicating that P54 nrb played an important rolein the development of HCC.In order to explore the molecular mechanism of P54nrbup-regulatedin virus-infected tumor tissues,we found that the transcription factor FoxD3 had a potential binding site in the promoter region of P54 nrb by bioinformatics analysis and previous published literature.We used the technique of chromatin immunoprecipitation,quantitative PCR,western blot and double luciferase reporter to show that FoxD3 could directly target the promoter region of P54 nrb,which inhibited the transcription of P54 nrb.Further analysis of clinical tissue samples,we found that the expression of FoxD3 and P54 nrbwere negatively correlated in the tumor tissues with virus infection.Thus we speculate that viral infection leaded to a decrease in the expression of FoxD3 in the tumor and thus promoted an abnormally high expression of P54 nrb.In recent years,it had been reported that FoxD3 was very low in tumor tissue closely related to methylation regulation.The methylation level of FoxD3 promoter region was tested by methylation-specific PCR(qMSP).It was found that FoxD3 was down regulated in HBV/HCV-HCC tumors and its low expression closely related to methylation.DNA methyltransferase 1(DNMT1)was a key enzyme for DNA replication and maintenance normal methylation.DNMT1 overexpression played an important role in tumorigenesis,but whether DNMT1 induced FoxD3 promoter methylation in viral-infected tumor tissues was still unknown.We found that the expression level of DNMT1 in clinic HCC samples by RT-PCR and that DNMT1 could indeed regulate the degree of methylation in the FoxD3 promoter region.HBx protein and HCV core protein play an important biological role in the development of hepatocellular carcinoma,and can regulate a number of important transcriptional expressions.In our study,it was identified that HBx protein and HCV core protein could activate the transcription of DNMT1 and upregulate the expression of DNMT1.We obtained the HBx protein and HCV analysis,classical molecular biology techniques,etc.At molecular level as well as cell level,we validated that P54 nrb binds to the promoter region of the N-Ras gene and promotes the transcription of the N-Ras gene,thereby activating the RAF / ERK / MEK signaling pathway and play a role in promoting the proliferation and metastasis of hepatocellular carcinoma.Finally,we detected the expression of DNMT1/FoxD3/P54nrb/N-Ras in 2 groups of clinical patients.The antiviral therapy was able to reverse the expression change of the signal pathway.Hence,we infered that antiviral therapy was possible to inhibit the occurrence and development of the tumor by inhibiting the activation of the signaling pathway so as to achieve its antitumor effect.In summary,we hadstudied the role of viral infection in the development of HCC,especially the epigenetic regulation of the involvement of specific genes,in order to further understand the complex signal pathways frombrand new perspective and provided a new way of thinking for antiviral therapy.Part II: TMED3 promotes hepatocellular carcinoma progression via IL-11/STAT3 signalingHepatocellular carcinoma(HCC)is currently the world's most common malignant tumors,ranks the sixth of incidence of malignant tumors,while the mortality rate isthe third highest of tumor-related death.With the matureof surgical resection?liver transplantation and some adjuvant therapy,five-year survival rate of HCC has been improved.However,the risk of postoperative recurrence,intrahepatic and extrahepatic metastasis of HCC is still large.As the concealed onset of liver cancer,symptoms are not obvious at the early stage.While the course of disease progress rapidly,patients often diagnosed lately with the occurrence of vascular cancer thrombosis,sub-foci,incomplete and multi-center when effective treatment is limited.Hence the postoperative recurrence and metastasis rate are high,resulting in the prognosis of HCC.Therefore,a comprehensive understanding of the molecular mechanism of recurrence and metastasis of HCC is required and more effective therapies are in need.Hence,it is important to find molecular markers that are important for prognosis and treatment.It has been reported that TMED3 can inhibit tumor invasion and metastasis in colon cancer.However,the expression and biology role of TMED3 in HCC is still unknown.Therefore,our study aims to understanding the role of TMED3 in the progression of HCC and to provide new potential therapeutic targets for the treatment of HCC.The expression of TMED3 in HCC tissues was significantly higher than that in adjacent tissues by Real-Time and immunohistochemical experiment,and we found that high expression of TMED3 was closely related to invasive clinical and pathological features.High TMED3 expression in the HCC tissues had poor Recurrence-free survival(RFS,P = 0.0128)and overall survival(OS,P =0.0155).Multivariate analysis showed that high TMED3 expression was an important prognosis factor in HCC patients after surgery and was anindependent risk factor.We also confirmed that TMED3 could significantly promote HCC cells invasion and metastasis,but could not affect HCC cell proliferationin vitro and vivo.By microarray analysis and molecular biology experiments,we found that TMED3 increased IL11 expression?induced IL11 autocrine?and activated IL11 / STAT3 signaling pathway.In conclusion,our study found that the expression of TMED3 was significantly associated with prognosis and tumor metastasis by analyzing the clinical HCC samples.In the mechanism,we found that TMED3 influence the invasion and metastasis of HCC cells by affecting IL-11 / STAT3 signaling pathway.In summary,our study found a potential target for HCC treatment,providing new insights into the treatment of HCC.