The Mechanisms Of 53BP1 And Minichromosome Maintenance Complex Regulating DNA Damage Repair Of Human Hepatocellular Carcinoma Cells

Background and ObjectivePrimary liver cancer is one of the most common malignant tumors in digestive system,and the second leading cause of cancer mortality worldwide.According to statistics,the number of people who died of primary liver cancer each year accounted for more than 50%of the world's primary liver cancer deaths,it threatens seriously people life health.The occurrence and development of hepatocellular carcinoma is a complex process involving multiple causes and multiple factors involved in the formation of multiple stages.Anti cancer gene mutation or deletion,a link activation of proto oncogenes and aberrant cell cycle regulation and DNA repair related gene expression,critical repair may be abnormal liver cell carcinogenesis,there may be a sign of liver cancer cells.Thus,DNA damage repair in cancer cells is a promising approach for the treatment of cancers.P53 binding protein 1(53BP1)plays an important role in the maintenance of genomic stability and the prevention of cancer,which is an important sensor involved in the repair of DNA damage.53BP1 gene is located on human chromosome 15q15-215 with two transcripts of ll.Okb and 6.6kb.The protein encoded by 53BP1 is composed of 1972 amino acid residues,which is combined with the DNA binding region of p53,which promotes the transcription initiation of p53 mediated gene.53BP1 has a wide range of physiological functions,which can quickly accumulate in the DNA double strand breaks and form a nuclear polymer.Therefore,53BP1 is considered to be one of the markers of endogenous DNA double strand damage.minichromosome maintenance protein(MCM)is an important component of eukaryotic DNA replication complex,which can initiate and participate in the process of replication,which is necessary for the replication of all eukaryotic cells.The study found that many aspects of MCM protein mainly involved in higher-order chromosome structure dynamic change,such as the sex chromosome dosage compensation,sister chromatid pairing,chromosome condensation and separation,DNA recombination and DNA damage repair process.Many studies showed that MCM7 protein were highly expressed in breast cancer,lung cancer,Hodgkin's lymphoma and other malignant tumor cells,can be used as one of the indexes to reflect the activity of cancer cell proliferation and determine its biological behavior.In this study,the expression of 53BP1 in hepatocellular carcinoma cell line HepG2 was reduced by transfection,and a stable cell line was constructed to verify the expression level.The effects of 53BP1 on the proliferation of hepatocellular carcinoma cells were analyzed by cell cloning and MTS cell proliferation assay.The relationship between 53BP1 and MCM complex was studied by immunoprecipitation and luciferase reporter gene,respectively.The aim of this study was to elucidate the role of 53BP1 in the regulation of DNA replication by inhibiting the proliferation of DNA and the involvement of MCM in cell injury and cell repair.Finally,the expression of 53BP1 was detected by quantitative PCR and Western blot,and the expression of 53BP1 was detected by immunohistochemical method.The expression level of 53BP1 was compared with the clinical data of patients,and the relationship between 53BP1 expression and survival prognosis of patients with hepatocellular carcinoma was analyzed by multivariate analysis.Materials and methodsChapter 1:the biological role of 53BP1 in human hepatocellular carcinoma cell line HepG2 DNA damageIn this study,we selected the human hepatocellular carcinoma cell line HepG2,by constructing shRNA lentiviral vector,transfected into human hepatocellular carcinoma cell line HepG2 by transfection technology,down regulated the expression of 53BP1 in hepatocellular carcinoma cell line HepG2.And then through the fluorescent quantitative PCR,Western blot and other experimental techniques to verify whether the stable expression of the cell line is successful.Secondly,the effect of 53BP1 on the proliferation of human hepatocellular carcinoma cell line was confirmed by MTS and cell clone formation assay.Finally,the changes of DNA in human hepatocellular carcinoma cell line HepG2 were studied by using bleomycin induced injury.Chapter 2:The combination of 53BP1 and the maintenance of the protein complex of the chromosome promotes chromatin separation and colony formationThe recombinant vectors of 53BP1 and MCMs with tags were constructed and transfected into HepG2 cells.Immunoprecipitation(IP)and mass spectrometry(MS)were performed to identify the possible interactions between 53BP1 and MCMs,and glutathione S-transferase(GST)pull-down assay was carried out to detect the direct interaction.Moreover,the expression of MCM2 and MCM6 was suppressed by specific short hairpin RNAs(shRNAs),and then the chromatin fraction and foci formation of 53BP1 were examined under the condition of DNA damage.Chapter 3:The expression of 53BP1 in human liver cancer tissue and its clinical significanceSurgical specimens of liver cancer and paracancerous tissue in our hospital were collected.The expression of 53BP1 was detected by quantitative PCR and Western blot,and the expression of 53BP1 was detected by immunohistochemical method.The expression level of 53BP1 was compared with the clinical data of patients,and the relationship between 53BP1 expression and survival prognosis of patients with hepatocellular carcinoma was analyzed by multivariate analysis.Results1.We successfully constructed the human hepatoma cell line HepG2 stably transfected with down-regulation of 53BP1 expression.The proliferation ability of shRNA-53BP1 group was stronger than that of shRNA-control group,53BP1 could inhibit the proliferation of human hepatoma cell line HepG2.The DNA damage induced by bleomycin showed that the expression of 53BP1 was up-regulated in bleomycin induced DNA damage model,which indicated that 53BP1 was activated in the process of DNA damage.2.The results showed that MCM2/3/5/6 was immunoprecipitated against the hemaglutinin(HA)-tagged 53BP1 in HepG cell nuclei.GST results revealed that there was a direct interaction between 53BP1 and MCMs complex.Moreover,the non-chromatin level of 53BP1 was significantly increased by downregulation of MCM2 or MCM6,but was statistically decreased the chromatin level.Furthermore,we observed that knockdown of MCM2 or MCM6 could statistically inhibit the foci formation of 53BP1 in HepG cell nuclei upon bleomycin-induced DNA damage(P<0.01).3.The expression of mRNA and protein in 53BP1 was significantly lower in hepatocellular carcinoma than in paracancerous tissues.The results showed that the expression of 53BP1 was lower in hepatocellular carcinoma tissues by immunohistochemistry.Lower expression of 53BP1 is closely related to tumor size and TNM staging.Moreover,53BP1 is considered as a protective factor for overall survival of patients with hepatocellular carcinoma.ConclusionThe results suggest that down-regulation of 53BP1 expression can promote the proliferation of human hepatocellular carcinoma cell line HepG2,and 53BP1 is activated in the process of DNA damage.Furthermore,there is a direct interaction between 53BP1 and MCMs,which is essential for 53BP1 chromatin fraction and foci formation in hepatoma HepG2 cells.53BP1 may be a potential diagnostic marker and therapeutic target for hepatocellular carcinoma.