Construction of an inducible vector for human p27~(KIP1) gene and its expression

Objective To elucidate the effect of p27 KIP1 on cell cycle and proliferation in hepatocellular carcinoma (HCC) cells.Methods We constructed an inducible vector pMD KIP1 containing 0.6?kb p27 KIP1 cDNA,which allowed controlled expression of protein upon addition of 100?μmol/L Zinc as an external inducer.The whole length of p27 KIP1 cDNA was transfected into human HCC cells HCC 9204 by the method of lipofectin transfection.Expression of p27KIP1 in protein or mRNA level were analyzed by Western blotting and reverse transcription polymerase chain reaction (RT PCR) respectively.Effect of p27 KIP1 on cell growth was observed by typan blue exclusion assay.Flow cytometry was applied to assess the effect of p27 KIP1 on cell cycle.Results Expression of p27 KIP1 in protein or mRNA increased evidently in HCC 9204 cells transfected with p27 KIP1 .The cell growth was reduced by 35% 48?h post induction with zinc as determined by cell viability assay.p27 KIP1 over expression caused cell arrest with 33% (from 35.21% to 68.28%,P0.01) increase in G 1 population.Conclusion The inducible expression system pMD KIP1 prepared in this study can efficiently transfer p27 KIP1 gene into HCC 9204 cells and over express p27 KIP1 protein in the infected cells.p27 KIP1 may cause cell cycle arrest in G1 phase.