Constructed eukaryotic expression plasmid of small hairpin RNA of PCNA and investigated the inhibitory effect on HeLa carcinoma cells. Hao. Huang¹, Xin. Tu², Nancai. Yu¹, Wenli. Wu¹, Qian. Liu¹, Wei. Ma¹, Jianjun. Hao¹, Yandong. Yi¹. 1) Center of Medicine, Wuhan first hospital, Wuhan, Hubei, China; 2) Human Genome Research Center, Huazhong University of Science & Technology.
   Objective: To evaluate a new plasmid mediated RNA interference (RNAi) system and nvestigate whether knock-down of PCNA by short hairpin RNA (shRNA) can inhibited proliferation of human on HeLa cell carcinoma ( HeLa) line in vitro. Methods: The pGenesil-1 plasmid containing mU6 promoter was digested by Bam HIand HindIII enzyme ,recombinant plasmid expressing shRNA targeting PCNA gene was designed and constructed, and were co-transfected cells with green fluorescence protein expressing plasmid. Flow cytometry was used to analyse the cell cycle and Western blot were applied to analyze PCNA mRNA and protein levels, and MTT test were applied to estimate the inhibition of growth, Single cell gel electrophoresis was used to analyse apoptosis ,respectively. Results: The shRNA expressed by the recombinant plasmid efficiently suppressedPCNA gene expression and induced apoptosis of HeLa cell carcinoma cell in vitro Conclusion: The recombinant plasmid can sufficiently mediate RNAi in HeLaarcinoma cells, and knock-down of the PCNA expression by shRNA significantlyinhibited the proliferation and induced apoptosis in HeLa cells. The results suggest this new system, mediated RNAi can be used as a tool for the study of gene function and gene therapy.