The Inhibition Effect Of The RNA Interfere Targeting PCNA On The Growth Of Cervical Carcinoma Cell In Vitro And In Vivo

Cervical carcinoma is one of the highest causes of mortality in female cancer patients worldwide. Improved treatment options for this type of malignancy are highly needed. PCNA, being a cell cycle regulated nuclear protein, remains an attractive target for cancer gene. According to Bravo'research, it plays a fundamental role in the regulation of DNA synthesis and also over-expressed in most malignant cells including ovary, lung, salivary gland, and particularly in cervical cancer. Previous studies have suggested that over-expression of PCNA proteins could induce cell cycle progression, and several intervention trials have been used to block the action of PCNA proteins to inhibit DNA synthesis, which emphasized the importance of PCNA in cellular DNA synthesis and cell cycle progression. Nowadays, alternative strategies have been developed for inhibiting proliferation and inducing apoptosis in cancer and other human disease by counteracting PCNA expression in tumor cells. It was reported that using various PCNA molecular antagonists, such as antisense oligonucleotides, dominant-negative mutants, could induce cell division defects, accelerate cells apoptosis, reduce tumor growth potential.Small-interfering RNAs have been recently shown to be the most powerful tools to specifically silence gene expression by degrading mRNA in a sequence-specific manner. Extensive research on the development of therapeutic siRNA has been carried out since these small RNA molecules were shown to possess RNA interfering (RNAi) activity in mammalian cells in 2001. Further, recent in vitro and in vivo experiments have successfully demonstrated that RNAi is a potential application for human cancers .In the present study we develop and optimize shRNA targeting activated PCNA and find that circular shRNA effectively inhibit proliferation of cancer cells.ObjectiveUterine cervix carcinoma was one disease that threated people's health seriously, on the basis of molecular biology and immunological properties of PCNA, we constructed eukaryotic expression plasmid of small hairpin RNA of PCNA , then expressed plasmid in HeLa cells and investigated the inhibitory effect of shRNA of PCNA on HeLa cell carcinoma cell proliferation and detected if it could inhibit HeLa cells tumour growth in a xenograft murine modle.Methods1. To study its role in stress response, we designed and used short hairpin RNA to inhibit PCNA expression in HeLa cells and validated its effect on cell proliferation. In this study, the prime of proliferating cell nuclear antigen was designed according to cDNA sequence of PCNA, the prime fragment was inserted into downstream of the vector pGenesil-1 to obtain the recombinant eukaryotic expression plasmid pGenesil-PCNA1-4 which we identified with the methods of enzyme digestion,and sequence analysis, the results were consistent with what we expected. We transfected the recombinant plasmid pGenesil-PCNA1-4 into eukaryotic HeLa cells. The inhibition of the HeLa cell carcinoma cell proliferation was estimated colony formation test ,the protein expression was analysed by immunohistochemistry and western blot method, the cell cycle was analysed by flow cytometric. Apoptotic cell is detected by single cell gel electrophoresis assay (comet assay) and caspase cleavage is studied also. Expression of PCNA mRNA is assessed by real-time reverse transcription-PCR (RT-PCR).2. HeLa cells were injected in nude mice to gengerate xenograft than transfected with PCNA siRNA or lipofectamine 2000,respectively. The stable HeLa cell clone which expresses PCNA shRNA-plasmid or non-specific-plasmid and HeLa cell without transfection were injected in nude mice respectively, to evaluating their biologic effects.Results1. Upon transient transfection with plasmid encoding shRNA, it was found that expression of PCNA decreased in shRNA-transfected cells, down-regulation of PCNA inhibited cell growth and induced apoptosis in HeLa cells. PCNA down-regulation also increased cell population in the G0-G1 phase.2. After a certain period of injection with HeLa cells into the axilla of nude mice visible tumors had developed .Transient-transfection of HeLa shRNA-plasmid in tumour the with lipofectamine 2000 or injection sites showed on effect on tumour growth or PCNA in tumour content. Stable-transfection HeLa cells of nude mice xenograft model showed tumour formation was 100%. Analysis of the growth curves showed that PCNA shRNA reduced tumor volume from day 45 after injection compared with non-silencing group or control group. The logarithmic regression analysis of the growth curves showed that PCNA shRNA significantly reduced the growth rate of the tumors compared with non-silencing shRNA group or control group. We compared PCNA shRNA group with non-silencing shRNA group, showed that the mean tumor volume was reduced. The time for xenografts reached 50mm~3 after injection of the tumor cells in PCNA shRNA group was delayed compared with non-silencing shRNA group. ELISA of PCNA in tumor contents showed reduction in the PCNA expression of tumors from PCNA shRNA group as compared with non-silencing shRNA group. Conclusion1. We successfully constructed recombinant eukaryotic expression vector and found that specific shRNA of PCNA might be effective in the behavior of HeLa cell lines and inhibited the proliferating cell nuclear antigen by inbiting expression of PCNA2. Transient-transfection of PCNA shRNA-plasmid in vivo with lipofectamine2000 showed on effect on tumour growth or PCNA in tumour content.3. Stable- transfection HeLa cell of PCNA shRNA in a xenograft murine modle showed time for xenografts reached 50mm~3 was delayed and tumour growth rate was inhibited.