Exploration On Mechanism Of Over-expression Polo-like Kinase 1 In Hepatocellular Carcinoma By SiRNA Silencing The Gene Of BEL-7402 In Vitro And In Vivo Experiments

Objective:1.To investigate the expression difference of Polo-like kinase 1 in normal liver tissues, para-cancer tissues and hepatocellular carcinoma tissues.2.To create human hepatoma BEL-7402 Cell Substrain-Plk1siRNA251 cells whose expression of Plkl were stable silent by siRNA intervention. To analyze the difference of Plkl expression level and cell cycle between pre and post siRNA intervention.3. To establish the nude mice subcutaneous tumor models of Plk1siRNA251 and BEL-7402 cells,and observation of their growth and analysis their pathological changes.4. To explore mechanisms of over-expression Plkl in HCC by semi-quantitative RT-PCR and western blot technology.Methods:1 The distribution of Plkl protein expression in hepatocellular carcinoma tissues were examined by means of immunohistochemical analysis; the difference degree of PLK1 mRNA and protein expression among normal liver tissues para-cancerous liver tissues and neoplasm tissues were detected by semi-quantitative RT-PCR and western blot technology.2.After the synthesis, identification procedures of Plk1 siRNA plasmid, the plasmids were transfected into the human BEL-7402 cell line by electricity transfection technology. By screened with G418, Plk1siRNA251 cells were obtained with stable siRNA silencing Plkl genes expression. These two groups cells (PlklsiRNA251 and BEL-7402 cells) were detected for mRNA and protein expression levels by RT-PCR and western blot.Changes of cell cycle were detected by flow cytometry technologies.3. The subcutaneous tumors model of PlklsiRNA251 and BEL-7402 cells were established. The tumorigenicity time, tumorigenicity ratio and tumor growth curve were recorded in details; The two groups subcutaneous tumors were analyzed by hematoxylin and eosin(H.E) stain and apoptosis TUNEL assay.4 The mRNA and protein expression expression of Cdc25c,and survivin were detected by semi-quantitative RT-PCR and western blot technology.Results:1.The positive expression of Plkl were found in hepatocellular carcinoma tissues, No or sporadic expression were observed in para-cancerous and normal liver tissues. Compared to para-cancerous and normal liver tissues, over-expression Plkl mRNA and protein expression were also found in hepatocellular carcinoma tissues by semi-quantitative RT-PCR and western blot analysis.2. Successful acquisition Plkl siRNA plasmid and the plasmid electricity transfection into human hepatoma BEL-7402 cells. Screened with G418, Plk1siRNA251 cells were obtained. Compared to human hepatoma BEL-7402 cells,low expression level of Plkl mRNA and protein were detected in Plk1siRNA251 cells by RT-PCR and Western blot. And flow cytometry result showed PlklsiRNA251 cells were arrested in G2/M phase more easily than the BEL-7402 cells.3. Successfully established were the nude mice subcutaneous tumors model of Plk1siRNA251 and BEL-7402 cells. Obvious differences were observed in the tumorigenicity time, tumorigenicity ratio and the tumor growth between PlklsiRNA251 and BEL-7402 groups. Results of H.E staining and Tunel assay showed:there were no significantly cell morphology change between PlklsiRNA251 and BEL-7402 groups. But karyopyknosis and apoptotic bodies.could be observed in the groups of PlklsiRNA251, rare karyopyknosis and apoptotic bodies were found in BEL-7402 groups. It had significant difference for the counts of apoptotic bodies of these two groups under high power microscope (p<0.01).4. Low mRNA and protein expression of Cdc25c and survivin were observed in tumors of PlklsiRNA251 groups by semi-quantitative RT-PCR and western blot analysis, while the opposite results were found in BEL-7402 cells groups. In immunohistochemistry analysis,Negative expression of Plkl and Survivin were observed in PlklsiRNA251 groups while positive expression were found in BEL-7402 groups.Conclusion:1. Over-expression mRNA and protein of Plkl were found in hepatocellular carcinoma compared to para-cancerous and normal liver tissues.2. Compared to BEL-7402 cells, the expression level of Plkl mRNA and protein of PlklsiRNA251 cells were much lower, and PlklsiRNA251 cells were more easily arrested in G2/M phase.3. The growth of PlklsiRNA251 subcutaneous tumors were more slowly than that of BEL-7402 groups, Apoptosis were much more in the growth of Plk1siRNA251 than that of BEL-7402 groups.4.Low mRNA expression of Cdc25c and survivin were detected in PlklsiRNA251 groups compared to BEL-7402 groups by semi-quantitative RT-PCR and western blot analysis. Positive expression of Plkl and surviving were found in BEL-7402 groups while negative expression in PlklsiRNA251 groups. Over-expression of Plkl may be the promotion of cell proliferation, inhibition apoptosis of liver and avoidance the arrest of G2/M phase.