The Expression Of Nfbd1gene In Cervical Cancer Samples And Construction Of Retroviral Vector For Expressing NFBD1 SHRNA

NFBD1 (a nuclear factor with BRCT domain 1), also known as DNA damage checkpoints protein (mediator of DNA damage checkpoint protein1 MDC1),is a number of BRCT protein superfamily. Many researchers speculate that NFBD1 may be a potential tumor suppressor gene, because it plays an important role in DNA damage response and DNA repair. However, some studies observe that the NFBD1 is not a typical tumor suppressor gene;it can interact with the p53 protein, inhibite the expression of p53 , and then promote cell apoptosis, which suggesting that NFBD1 may be closely related to cell growth and reproduction, not a tumor suppressor gene. Some other studies show that blocking NFBD1 drugs can increase the effect of chemotherapy or radiotherapy, enhancing NFBD1 drugs can reduce the side effects of chemotherapy or radiotherapy and prevent people to be affected by the harmful effects of high doses of radiation. So,we speculate, NFBD1 may be a very effective drug target for cancer treatment research,and such research will be very significant. Cervical cancer is a serious disease, which is very dangerous for women's health and has a long precancerous stage, if we can discovery and diagnosis this disease eralier , the therapeutic efficacy of cervical cancer will be greatly improved and the fatality rate can be reduced. Because of NFBD1 gene has greate effect in the DNA damage response ,cell growth, cycle, apoptosis, the main purpose of this subject is to investigate the role of NFBD1 in cervical cancer and mechanism of action.We collecte a large number of clinical cases of cervical cancer specimens, detecte the expressing of NFBD1 in cervical cancer tissue and normal tissue, and then build NFBD1 shRNA retroviral vector and detecte its interfering effect on the expressiong of NFBD1 at mRNA level and protein level . Part one The expression of NFBD1 in cervical cancer ABSTRACT Objective : collecting many clinical specimens, comparing the expressing of NFBD1 gene in cervical cancer tissue and normal tissue at mRNA level and protien level. Methods: extracting samples' mRNA and protien, amplifing NFBD1 cDNA by RT-PCR. Then at mRNA level ,we use Real Time PCR to compare the expressiong of NFBD1 in cervical cancer tissue and normal tissue; at the protein level, we detecte the expression of NFBD1 protein by immunohistochemistry experiment. Results: At mRNA level , detecting 39 specimens,about 24 specimens have higher expression of NFBD1 gene in cervical cancer than in the corresponding normal tissues , accounting for 61.5%; at the protein level, testing 60 specimens , about 42 specimens have higher expression of NFBD1 gene in cervical cancer than in the corresponding normal tissues , accounting for 70%. Conclusion: NFBD1 gene has the higher expression in cervical cancer than normal tissue either at the level of mRNA or protein. So we speculate that, NFBD1 gene may have an important effect in promoting cell growth and apoptosis in cancer. Part two Construction retroviral vector of expressing NFBD1 shRNA ABSTRACT Objective: construct NFBD1 shRNA retroviral vector , then multiply the virus in PT 67 cells , infecte SiHa cells with theses virus, detecte the interference effects by extracting RNA and protein . Methods: connecte linearized Psilencer5.1-H1-Retro vector and NFBD1 shRNA fragment, transforming the combinants and then multipling recombinant plasmid in PT 67 packaging cells. Transfecte the SiHa cells with the combinantion virus ,and then extracte the cells' mRNA and protein , identifiy the expression of NFBD1 gene at mRNA level and protien level by Real Time PCR and Western-blotting. Results: The Psilencer-shRNA -NFBD1-1 and Psilencer-shRNA-NFBD1-2's DNA sequences were confirmed correct . The virus amplified in PT 67 packaging cells can well transfect SiHa cells. The results by Real Time PCR and Western blot showed that, after transfecting with the combinantion plasmid Psilencer- shRNA-NFBD1-1 and Psilencer-shRNA-NFBD1-2 , the expression of NFBD1 was lower than before both at mRNA level and protein level, much lower with transfecting Psilencer-shRNA-NFBD1-2. Conclusion: NFBD1 shRNA retroviral vector was successfully constructed, has obviously interference effect, and we can choose Psilencer-shRNA-NFBD1-2 retrov -iral vector for future researches.