MolecΜlar Mechanism Of Cisplatin Induced HBV Reactivation And Its Experiments In Vivo

According to the World Health Organization, more than two billion people worldwide have infected with HBV ever and 400 million have became HBV chronic carriers(three-quarters of them are Chinese), which leads to more than one million deaths annually and half of them in China. In China, chronic hepatitis B virus infection-induced hepatitis is an important risk factor for cirrhosis and liver cancer. According to statistics, HBV reactivation phenomenon rarely occurs in asymptomatic carriers and patients with chronic, or if receiving immunosuppressive therapy or chemotherapy, there will be a chance to activate in a resting state or low HBV replication status virus. Transient activation of a large number of hepatitis B virus will induce patients with acute hepatitis, acute liver failure and even fulminant hepatic failure, and interrupt normal chemotherapy,delay the normal treatment and prognosis of cancer patients, severe cases may endanger the lives of patients. In 2011, cancer is ranked third causes of death, incidence and mortality increased year by year. As well, the incidence rate of HBV reactivation from clinical statistics is 14%-66%, HBV reactivation has become an important public health problem and a serious threat to human health in China.Chemotherapeutic drug cisplatin can induce HBV reactivation phenomenon has been confirmed by a number of domestic and foreign clinical trials, but the molecular mechanism of it induces reactivation of hepatitis B virus replication is still unclear. Currently there are two mechanisms to explain this phenomenon: first, chemotherapy drugs such as cisplatin has a strong immune suppression, making the hepatitis B virus immune escape by inhibiting the body’s immune system, a large number of hepatitis B virus replication induced HBV reactivation. Majority of scholars agree with this mechanism. Second, cisplatin can directly contribute to the hepatitis B virus replication. Current domestic and foreign on cisplatin directly contribute to HBV reactivation and molecular mechanisms related research reports is blank. The subject is designed for this research gap, cisplatin can directly promote HBV replication and its associated molecular pathway is the key of research. We first constructed successfully in vitro cyclization from even the HBV DNA(circμlarized HBV DNA) and confirmed: whether in vivo or in vitro, the infection efficiency cyclization and replication of HBV DNA levels were significantly better than the linear HBV DNA(P < 0.05). Hydrodynamic HBV mouse model built with a ring of HBV DNA, and using the backcross method passaged improve existing C57 HBV transgenic mice genetic stability, provide reliable animal model for the experimental study next. Second, choose C57 HBV stable expression mice model and pre-constructed hydrodynamic model of HBV transgenic mice to continue validation experiments of HBV reactivation in vivo. By ELISA, REAL-TIME PCR, Western-blot, Southern-blot, IHC and CHIP experiments confirm whether cisplatin induced HBV reactivation in vivo. Validation experiments of DDP-induced HBV reactivation in vitro have been completed. We explore the molecular mechanisms of cisplatin-induced HBV reactivation: First, screening HBV promoters and liver-enriched transcription factor about HBV reactivation; second, select two representative transcription factor PGC-1α and HNF-4α performed in vivo verification. Discussion association between PGC-1α,HNF-4α and HBV replication by CHIP; building AdsiPGC-1α and AdsiHNF-4α, reverse verify in vitro and in vivo wether PGC-1α and HNF-4α are the key factors of cisplatin-induced HBV reactivation; Through experiments such as ELISA, REAL-TIME PCR, Western-blot screening endoplasmic reticulum stressrelated factors, and explore the correlation and key factor between endoplasmic reticulum stress and HBV reactivation. Experiments suggested that, on the one hand, cisplatin direct regulate expression of HBV replication related transcription factor PGC-1α and HNF-4α enhance activity of HBV core promoter, in turn increases HBV replication at the transcriptional level; On the other hand, cisplatin can cause endoplasmic reticulum stress, UPR induced upregulation of PGC-1α, indirect regulated HBV replication.Conclusion: The chemotherapy drug cisplatin can induce reactivation of HBV in vivo and in vitro. Its molecular pathway may have two: 1. Pathway of PGC-1αand HNF-4α, liver-enriched transcription factor PGC-1α and HNF-4α as a co-activator, can combine together in HBV DNA enhancer Ⅰ and enhanced Ⅱ, cisplatin can regulated the expression of liver-enriched transcription factor PGC-1α and HNF-4α, directly enhance the replication of hepatitis B virus, cause HBV reactivation; 2. Cisplatin can cause endoplasmic reticulum stress(ERS), endoplasmic reticulum stress can induce a slight upregulation of PGC-1α, indirect regulated HBV replication. By studying the molecular mechanisms of cisplatin-induced HBV reactivation, we can find drug targetsto block the phenomenon, clinical preventive medication to reduce the incidence of HBV reactivation, improve the prognosis and quality of life of cancer patients.