The Influence Of Taurine On Autophagy And Apoptosis Of Cervical Cancer Cells

Objective:1.Observing the effect of Tau on apoptosis in human cervical cancer cells and its preliminary mechanism.2.Observing the effect of Tau on autophagy in human cervical cancer cells and its preliminary mechanism.Methods:1.HeLa and SiHa cells were treated with different concentrations of Tau(0 mM,10 mM,20 mM,40 m M,80 mM,160 mM,320 mM)for 24 h,48 h,72 h,the viability rate was detected in order to make clear the dose-effect relationship by MTT assay.2.HeLa and Si Ha cells were treated with different concentrations of Tau(0 mM,20 mM,40 mM,80 mM,160 mM)and CQ 20 μM for 48 h.The expression level of apoptosis-related proteins MST1,Bax,Bcl-2 and autophagy related proteins LC3-II,LC3-I,Beclin1 was detected by Western blot,and evaluate the effect of Tau on apoptosis and autophagy.3.HeLa and SiHa cells were treated with different concentrations of Tau(0mM,20 mM,40 mM,80 mM,160 mM)and CQ 20 μM for 48 h,RT-qPCR assay was used to explore the expressions of apoptosis-related mRNA MST1,Bax,Bcl-2,Caspase-3 and autophagy-related mRNA AKT,Beclin1 and evaluate the effect of Tau on apoptosis and autophagy.4.The logarithmic growth phase of HeLa cells was treated with Tau(0 mM,40mM)for 48 h,and the formation of autophagic vesicles was observed by staining with monodansy cadaverine(MDC).5.GFP-RFP-LC3 was transfect in to HeLa and SiHa cells.fluorescence microscope was employed to observe fluorescence aggregation of green puncta and red puncta to detect autophagy after treated with different concentrations of Tau(0mM,20 mM,40 m M,80 mM,160 mM)and CQ 20 μM for 48 h.Results:1.The cell viability was measured with an MTT assay after incubation of HeLaand SiHa cells with Tau for various periods.The result showed Tau(0 mM,10 mM,20 mM,40 mM,80 mM,160 mM,320 mM)reduced HeLa and SiHa cells growth in a time-dose dependent manner,(P<0.01).Calculating the IC50 values of two kinds of cells with various concentration Tau treating for 24 h,48 h,72 h,we found that the capacity of inhibiting proliferation in HeLa cell is greater than SiHa cell.2.Western Blot showed that compared with other concentration groups,the Tau40 mM group significantly increased the expression of MST1 protein in HeLa cells(P<0.01).The expression of MST1 protein increased with the increase of Tau concentration in SiHa cells.The Bcl-2 protein expression in two cell lines were gradually decreased as the concentration increase Tau(P<0.01).The expression of autophagy marker proteins LC3 and Beclin1 decreased with the increase of Tau concentration in both cellls.The conversion of cytoplasmic LC3(LC3-I)to autophagic membrane type LC3(LC3-II)decreases with increasing Tau concentration,LC3-II\LC3-I ratio decreased(P<0.01).3.RT-qPCR showed that the expression of Bax and MST1 m RNA are significantly increased(P<0.01)in HeLa and SiHa cells,and Caspase-3 mRNA is slightly increased(P<0.05),while the expression of Bcl-2 mRNA was significantly decreased(P<0.01).The expression of AKT mRNA was significantly increased(P<0.01).The expression of Beclin1 mRNA was significantly increased(P<0.01).4.High concentration of the Tau(80 mM,160 mM,320 mM)induced apoptosis in a time-dose-dependent effect.p-EGFP-MST1 plasmid was mediated into SiHa cell in vitro,when MST1 gene is used in combination with Tau,the apoptosis rate of cells was higher(P <0.01).5.MDC can be absorbed by the cells and selectively bound to the autophagic vesicle membrane.Autophagic vacuoles appear as yellowish green or dark green dotted structures under fluorescence microscopy.Compared with the Control cells,the cells with Tau 40 mM showed decreased MDC-positive cells and autophagosome formation.These results implied that Tau can inhibit autophagy.6.HeLa and SiHa cells treated with Tau had decrease in green puncta and red puncta in comparison with the Control and CQ 20 μM.These results implied that Tau can inhibit autophagy.Conclusion:1.we can draw a conclusion that Tau can significantly inhibit proliferation in cervical cancer HeLa and SiHa cells in a dose-time dependent manner.2.Tau can promote apoptosis in cervical cancer HeLa and SiHa cells,and its mechanism is achieved by up-regulating the expression of MST1,Bax protein and down-regulating Bcl-2 protein expression.3.Tau can inhibit autophagy in cervical cancer cells HeLa and SiHa,and the mechanism of Tau inhibits the autophagy of human cervical cancer cells is achieved by down-regulation of Beclin1 protein expression and LC3-II\LC3-I ratio and up-regulating AKT.