The Construction Of Mutant Plasmid On Phosphorylation Sites Of P27^Kip1 And Research Of Expression Of P-p27^Kip1 Protein In Hepatocellular Carcinoma And Cirrhosis

Objective:We constructed the p27kip1 phosphorylation site mutant plasmids and observed the expression of p-p27kip1 protein in HCCs, paratumor cirrhosis,cirrhosis and normal liver tissues,to clarify the function of phosphorylation and the change of expression and localization of p27kip1 protein in occurrence and development of HCC.Methods:Part one Construction of Mutant Plasmid1. Extract the target gene:（1）To get p27kip1 mRNA sequence in Pubmed Genebank, and design primers containing restriction enzyme cutting site （2） To extract total mRNA in human normal hepatocytes （3） To amplify p27kip1 （wt） by RT-PCR 1.5% agarose gel electrophoresis （4） To sequence the p27kip1 （wt） gene products and to detect with Blast. （5） To purify the p27kip1 （wt） PCR product, and link its A-side with linear the prokaryotic expression vector plasmid pUCm-T. （6） To transform bacterial, to screening, amplifiy, and extract the plasmid then sequence. （7） To digest pUCm-T-p27kip1（wt） and eukaryotic expression vector pIRES2-EGFP with double enzymes（BgⅢI/SaiI） （8） To link p27kip1 （wt） with eukaryotic vector pIRES2-EGFP, and sequence the product and do Blast testing.2. Construct a mutant eukaryotic expression vector:（1） To design mutagenic primers （2） To make gene point mutations with pIRES2-EGFP-p27kip1 （wt） （3） To transform bacterial, to screening, amplifiy, and extract the plasmid then sequence. （4） To sequence the product and do Blast testingPart two Immunochemistry We have selected 92 wax blocks of human’ HCC （36）, paratumor cirrhosis （36）..cirrhosis （38） and normal liver tissue （18） from the First Affiliated Hospital of Kunming Medical University, the Second Affiliated Hospital of Kunming Medical University and Ganmei Affiliated Hospital of Kunming Medical University.Immunohistochemistry was used to detect the expression of p-p27kipT157, p-p27kiP1T187,p-p27kip1T198 and p-p27kipS10 in HCCs, paratumor cirrhosis,cirrhosis and normal liver tissues.Results:Part one Construction of Mutant Plasmid1. Extraction and identification of target gene p27kip1 （wt） in human normal hepatocytes（1） Agarose gel electrophoresis showed good quality of total RNA extracted in normal human normal hepatocytes （HL-7702）（2） Agarose gel electrophoresis of RT-PCR production gene p27kip1 （wt） showed specific banding.（3） The test of Blast showed DNA sequence was the same to the DNA fragment（NM₀04064.4） announced by Geneballk,no mutation and no frame shif,and the target gene amplified was just the same with coding sequences of p27kip1 （wt）.2. Gene sequencing and Blast text of pIRES2-EGFP conenection products showed: DNA sequence of the target gene which inserted pIRES2-EGFP was the same to the DNA fragment（NM₀04064.4） announced by Genebank by the test of Blast, and was the same with coding sequences of p27kipl （wt）,no frame shif and no mutation.3.After digested using BgⅢ/SalI,these 8 kinds of pIRES2-EGFP-p27kip1 （muts） all showed 5.3kb and 600bp two straps.4.Gene sequencing and Blast text of these 8 kinds of pIRES2-EGFP-p27kip1 （muts） showed,4 kinds of analog form phosphoylation eukaryotic plasmids pIRES2-EGFP-p27kiP1（S10A）,pIRES2-EGFP-p27kip1（T1 57A）,pIRES2-EGFP-p27kip1（Tl 87A）,pIRES2-EGFP-p27kip1（T198A） and 4 kinds of dephosphorylation eukaryotic plasmids pIRES2-EGFP-p27kip1（S10E）,pIRES2-EGFP-p27kip1（T157E）,pIRES2-EGFP-p27kiP1（T 187E）, pIRES2-EGFP-p27kiP1（T198E） were all same to the DNA fragment（NM₀04064.4） announced by Genebank by the test of Blast,except for the specific mutant site.Part two Immunochemistry1. p-p27kip1T157 was mainly expressed in nuclear, and its expression can be observed as the strongest in HCC group,which was significantly higher than those in corresponding paratumor cirrhosis group （P< 0.001） and normal liver tissue group （P < 0.001）.2. p-p27kiP1T1 87, p-p27kiP’S10 and p-p27k1P1T198 were expressed in HCCs and paratumor cirrhosis tissues, and the first two mainly located in cytoplasm, while the third in nuclei.Conclusion:1. This experiment extracted p27kip1 （wt） gene in human normal hepatocyte by genetic engineering techniques, and constructed pIRES2-EGFP-p27kip1 （wt） eukaryotic expression plasmids, and then constructed 4 kinds of analog form phosphoylation eukaryotic plasmids pIRES2-EGFP-p27kiP1 （S10A）,pIRES2-EGFP-p27kiP1 （T157A）,pIRES2-EGFP-p27kiP1 （T187A）,pIRES2-EGFP-p27kiP1 （T198A） and 4 kinds of dephosphorylation eukaryotic plasmids pIRES2-EGFP-p27kip1 （S10E）,pIRES2-EGFP-p27kip1（T157E）,pIRES2-EGFP-p27ki1（T187E）,pIRES2-EGFP-p27kip1 （T198E）,which would expressed as activation （E） and inactivation （A） separately in S10, T157, T187 and T198 phosphorylation sites of p27kiP1 protein. We have prepared for subsequent experiments,provided important information for further and made a more comprehensive study of the role of the four p27kip1 phosphorylation sites in the process of hepatocellular carcinoma.2. The phosphorylation of p27kiP1 protein and the expression and the change of localization of p-p27kip1protein had close association with the occurrence of HCC. The p-p27kip1T157 expression of paratumor cirrhosis was different from HCC and cirrhosis,which will need to be further gone into.