Research On Survivin Gene In Cytarabine Induced Apoptosis Mechanism Of K562Cells

Objective:To discuss the expression change of Survivin in Cytarabine induced apoptosis of K562cells and the expression correlation between Survivin and the mRNA of Caspase-8,Caspase-9and Caspase-3.To clarify the molecular mechanism of Cytarabine induced apoptosis of K.562cells and the role of Survivin in this process.Method:MTT was used to test the sensibility of K562cells to Cytarabine, FCM was used to test apoptosis rate of K.562cells. Real time-PCR (qRT-PCR) was applied to detect the mRNA expression of Survivin, Caspase-8, Caspase-9, Caspase-3. Western blot (WB) was provided to check the expression of Survivin protein.Results:1. MTT shows:K562cells had varying degrees of inhibition after treated with different concentration of Ara-C for24h.48h,72h.With the increase of drug concentration, the inhibition rate of K.562cells induced by Ara-C gradually enhanced, presenting the concentration dependence. The highest inhibition rate of K562cells induced by Ara-C was at48h. According to the concentration-inhibition ratio curve, we took the IC50of Cytarabine at48h (1×10-4mol/L) as the optimal concentration.2. Flow cytometry shows:Compared with control group, the level of apoptosis rates were increased after exposed to Cytarabine for24h.48h and72h (P<0.05). respectively1.85,2.51.1.48times higher than that of control group, and got the highest effect at48h, the apoptosis rate of72h was lower than that at48h (P<0.05).3. qRT-PCR shows:Compared with control group, the mRNA expression of Survivin was decreased after exposed to Cytarabine for24h,48h and72h (P<0.05),and was lowest at48h. The mRNA expression of Survivin was Obviously higher at72h than that at48h (P<0.05), which was negatively correlated with apoptosis rate of K562cells(r=-0.933,P<0.01). Compared with control group, the mRNA expression of Caspase-8,Caspase-9.Caspase-3were higher after exposed to Cytarabine for24h,48h,72h (P<0.05) and got the higest level at48h,which were positively correlated with apoptosis rate of K562cells (r=0.917, r=0.850. r=0.833, P<0.01).4.W B shows:Compared with control group, the level of Survivin protein was decreased after exposed to Cytarabine for24h.48h,72h (P<0.05) and got the lowest at48h, respectively0.873,0.739,0.798times lower than that of control group, the level of Survivin protein at72h was Obviously higher than that at48h (P<0.05). The expression of Survivin protein was negatively correlated with apoptosis rate of K562cells (r=-0.833.P<0.01).Conclusions:1. The down-regulation of Survivin gene and the up-regulation of mRNA expression of Caspase-8, Caspase-9and Caspase-3,which were the one of main mechanisms that induced apoptosis of K562cells effectively.2. The mRNA expression of Survivin was negatively correlated with the mRNA expression of Caspase-8.Caspase-9and Caspase-3(r=-0.883, r=-0.817, r=-0.867.P<0.01)after exposed to Cytarabine. We hypothesized that Survivin may have inhibitory effect on the transcription of Caspase-8. Caspase-9, Caspase-3, and this effect was one of the main anti-apoptosis mechanisms of Survivin gene.