The Effect Of High Expression Of Recombinant Trichosanthin On P73 Methylation And Mechanism Research In Caski Cell

Objective: To study the effect of high expression of rTCS on the proliferation and cell cycle, DNMT1 expression and methylation of p73 promotor in Caski cell line. To evaluate whether high expression of rTCS could inhibit the growth of Caski cell and induce p73 re-expression by demethylation of p73 promotor.To porvide a new pathway for the therapy of cervical cancer.Methods: (1) Eukaryotic expression plasmid pcDNA3.1(-)/6xHis -TCS was constructed by restrictive endonuclease cutting, agarose gel electrophoresis and recovery, ligation by T4 ligase. (2) Reverse transcription PCR(RT-PCR), Western-blot were used to select the Caski cell lines with rTCS high expression and to determine DNMT1 expression. (3) MTT assay was used to test the cell proliferation. (4) FCM was used to assay the effect of rCTS on the cell cycle. (5) The methylation status of p73 promoter were detected by Methylation PCR.Results: (1) Eukaryotic expression plasmid pcDNA3.1(-)/6xHis–TCS was successfully constructed and stably transfected into Caski cells. The cell lines with rTCS high expression were obtained by G418 selecting, RT-PCR and Western-blot. (2) rTCS high expression decreased the cell growth rate in all chosen time point (24h,48h,72h) in time-dependent manner (p<0.01). The FCM assay revealed that rTCS high expression caused cell G1/G2 arrest but increased the cell population in S phase (p<0.01). (3) DNMT1 expression in mRNA and protein levels was lower in the rTCS high expression cells than in the control cells, with a 0.56-fold decrease for mRNA (p<0.01). (4) The methylation level of p73 promoter in the experimental group was lower than the control cells. (5) p73 expression in the experimental group was higher than thje control cells in mRNA lever.Conclusion: (1) High expression of rTCS in the Caski cervical cancer line can inhibit cell growth obviously and arrest cells in G1/G2 phase. (2) High expression of rTCS can suppress the expression of DNMT1 on mRNA and protein level. (3) High expression of rTCS can reverse the methlytion satatu of p73 promoter in the Caski cells, and induce p73's re-expression. (4) The high expression of rTCS inhibits Caski cell proliferation through suppressing the expression of DNMT1 that will results in demethylation of p73 promoter and p73's re-expression.