Study On The Expression And Function Of NFBD1/MDC1 In Cervical Cancer

NFBD1(nuclear factor with BRCT domains protein 1),also named as MDC1(mediator of DNA damage checkpoint protein 1), is an important molecular that participates in DNA damage response. Recent research also hints that it may participate in tumorigenesis and development of tumor. In the present study, we have mainly investigated the expression of NFBD1/MDC1 in cervical cancer and its role in cell growth, cell cycle and apoptosis, and explored its molecular mechanism. The results are summarized as following 5 parts.(1) Expression of NFBD1/MDC1 in cervical cancer.Using real time PCR, we found substantial increases in NFBD1 mRNA levels in 24 of 39 cases (61.5%) of cervical cancer tissue specimens compared with the corresponding adjacent cervical normal tissue specimens. Using immunohistochemical staining method, we found substantial increases in NFBD1 protein levels in 42 of 60 cases (70%) of cervical cancer tissue specimens compared with the corresponding adjacent cervical normal tissue specimens from the same patient. The expression differences of NFBD1 between cervical cancer and the normal cervical tissue suggest that NFBD1 may have some roles in the genesis and development of cervical cancer.(2) Construction of retroviral vector containing NFBD1 shRNA and production of retrovirus.We designed and synthesized some siRNAs according to NFBD1 gene sequence, then we cloned them to the retroviral vector pSilencer5.1 H1-Retro, and confirmed the reconstructed plasmids by sequencing. The correct plasmids were transfected into HeLa, SiHa and CaSki cells using Lipofectamine2000 respectively, 72 h after transfection, the expression of NFBD1 mRNA was detected by RT-PCR. The better plasmid which had the obvious NFBD1 knockdown effect was selected and transfected into PT67 cell line which could package retrovirus using Lipofectamine2000. We collected the retrovirus and infected HeLa, SiHa and CaSki cells, 72 h after infection, the expression of NFBD1 mRNA and protein was detected by RT-PCR, real time PCR and western blot respectively. In the results, we found the expression of NFBD1 was downregulated obviously in the NFBD1 shRNA group compared with the negative control and without treatment group. This indicated that NFBD1 shRNA carried by retrovirus could inhibit the expression of NFBD1 effectively. At the same time, we constructed the pBABE retroviral vector expressing EGFP and transfected into PT67 cells. We collected the retrovirus expressing EGFP and infected HeLa, SiHa and CaSki cells. We could observe the EGFP expression under fluorescence microscope, this also indicated that retrovirus could infect HeLa, SiHa, CaSki cells and express in these cells.(3) Influences of NFBD1 expression downregulation on cell growth, cell cycle, apoptosis and ADR sensitivity.HeLa, SiHa and CaSki cells infected with retrovirus were assayed by MTT. The growth curve of NFBD1 shRNA group was slower than that of negative and without treatment group, and the same phenomenon was observed under microscope. These indicated that NFBD1 shRNA carried by retrovirus could inhibit cell growth. We also found that inhibition of NFBD1 expression caused G2/M arrest in HeLa, SiHa and CaSki cells, and induced apoptosis in these cells. HeLa, SiHa and CaSki cells were infected with retrovirus, 24 h after infection, these cells were treated with ADR, 48 h after treatment, these cells were assayed by MTT, we found that inhibition of NFBD1 expression could enhance the ADR sensitivity on HeLa, SiHa and CaSki cells.(4) Establishment of HeLa cell line expressing NFBD1 shRNA stably and study on the inhibition of xenograft tumor in nude mice caused by NFBD1 RNAi.HeLa cells were infected with retrovirus carrying NFBD1 shRNA and screened by puromycin. Clones derived from a single cell were isolated, expanded. The expression inhibitions of NFBD1 mRNA and protein were confirmed by RT-PCR and Western Blot assays respectively. HeLa cells expressing NFBD1 shRNA stably were injected subcutaneously into athymic nude mice. Tumor growth over a period of 39 days was monitored and measured, tumor volume was calculated. Sections from xenograft tumor in nude mice were assayed by TUNEL and the ultra-structures were observed under electron microscope. We found that the tumor grew slowly, tumor weight was less than that of negative control and without treatment group. Apoptosis was manifested by TUNEL assays and apoptotic bodies were observed under electron microscope. These results indicated that downregulation of NFBD1 expression could inhibit the growth of tumor in nude mice, and induce apoptosis.(5) Exploration on the mechanisms of cell cycle arrest and apoptosis caused by NFBD1 expression inhibition.Phospho-Histone H3 was a specific molecular marker of M phase in cell cycle. Phospho-Histone H3 expression was decreased in HeLa cells in which the expression of NFBD1 was downregulated by NFBD1 shRNA. This indicated that G2/M phase arrest caused by NFBD1 expression inhibition in HeLa cells was mainly G2 phase arrest. The mechanism involved in CDC25C-CyclinB1/CDC2 pathway. Apoptosis caused by NFBD1 expression inhibition was mainly mediated by P53-Cytochrome C-Caspase 3 pathway.