Study On The Correlation Of Proliferation And Apoptosis With Expression Of Cks In Human Hepatoma Cell Line BEL-7402 Induced By ATRA

Background and Objective:Hepatocellular carcinoma,HCC is one of the word's common malignant tumor with high malignancy,rapid development,average survival time only for six months if treatment is not timely or inappropriate.China is one of the regions of high incidence of liver cancer,accounting for about 55%of worldwide patients,and Tongan District, Xiamen City,is one of Chinese two major areas,which annual incidence rate is about 30/100000.Hepatocellular carcinoma is easy to miss the best timing of surgery because of difficulty to early detect.At present,the poor efficacy of conventional chemotherapy drugs resulted in lower cure rates.Therefore,studing the occurrence and development mechanism of HCC,verifying the important related genes,that will provide the new mentality to HCC diagnosis,means a great deal to conquer the HCC. This topic raked the person liver cancer BEL-7402 cell as the object,provided experimental and theoretical basis for clincal application with all-trans retinoic acid, piled up experience and data to further study of Cks1 and Cks2,through detecting effect of all-trans retinoic acid on liver cancer cell proliferation,apoptosis and Cks1, Cks2 expression,analysed the correlation between cell proliferation,apoptosis and Cks1,Cks2 expression,elementary studied the possible mechanisms that may exist.Methods:1.The method of MTT was used to examine the cell proliferation suppression activity of ATRA to cell BEL-7402.The experimental groups was cultured with ATRA in (0.1,1,5,10,20)μmol/L different concentration and control groups only adds the corresponding quantity RPMI 1640.After a further culture 24 h,48 h,72 h,96 h.The effect of different group cells' inhibition rate was evaluated with MTT method.2.Cell cycle and apoptosis rate was detected by flow cytometry(FCM) with PI and Annexin V/PI double stainings.The experimental groups was cultured with ATRA in 20μmol/L concentration and control groups with 1‰anhydrous ethanol.The Cell cycle and apoptosis rate was detected by FCM after a further culture 24 h,48 h,72 h.3.Cks1,Cks2 mRNA were detected by RT-PCR.The experimental groups was cultured with ATRA in 20μmol/L concentration and control groups with 1‰anhydrous ethanol.The differential expression of Cks1,Cks2 mRNA was detected by RT-PCR after a further culture 24 h,48 h,72 h.4.The change of protein Cks1,Cks2,P27 expression was examined by immunocytochemistry.The experimental groups was cultured with ATRA in 20μmol/L concentration and control groups with 1%0 anhydrous ethanol.The results of protein Cks1,Cks2,P27 expression was analysed by HSCORE.Results:1.Cell BEL-7402 proliferation was significantly inhibited in time-dependent and dose-dependent manners after induced by ATRA.Inhibition rate of proliferation was 17.45±1.93 after 24 h culture and 50.23±2.44 after 96 h culture.The difference of absorbance A and inhibition rate were statistically significant among different groups (P＜0.01).2.After induced by ATRA,G₀-G₁ phase ratio of BEL-7402 cells increased,and plenty of cells were blocked in G₀-G₁ phase,while S phase ratio decreased.G₀-G₁ phase ratio was 59.87±1.07 after 24 h culture and 72.78±1.13 after 72 h culture.There was no difference among experimental groups and control groups of G₂/M phase ratio(P＞0.05).3.The apoptotic rates were increased gradually along with time of culture.The ratio of apoptosis was 6.48±0.45 after 24 h and 17.31±1.00 after 72 h.there was significantly difference between experimental groups,the control groups or different time goups(P＜0.01).4.ATRA has no significant effect on the expression of Cks1,Cks2 mRNA(P＞0.05).5.Expression of protein Cks1,Cks2 decreased after induced by ATRA.HSCORE of protein Cks1 decreased from 1.88±0.06 to 1.47±0.06 and protein Cks2 from 1.58± 0.09 to 1.39±0.03,while protein P27 increased from 2.32±0.08 to 2.9±0.11. There was significant difference among groups(P＜0.01 or P＜0.05).Conclusions:1.ATRA inhibits the proliferation of human hepatoma cell line BEL-7402 significantly in time-dependent and dose-dependent manners;2.The apoptosis in BEL-7402 cells could be induced by ATRA;3.ATRA has no significant effect on the expression of Cks1,Cks2 mRNA,but down-regulation of their protein and up-regulation protein P27.4.ATRA caused the massive ceils to hinder in the G₀-G₁ phase,probably through the mechanism of down regulation of protein Cks1 and inhibition ubiquitination of the protein P27 degradation.The protein P27 accumulated in cells and played a negative regulation effect during the cell mitosis.5.The molecular mechanism of inducing apoptosis of cell BEL-7402 may be related to down-regulation of protein Cks2.