| Objective: To study the effects of epigallocatechin gallate(EGCG)on the proliferation and cell cycle of prostate cancer PC-3 cells after acting on prostate cancer PC-3 cells,and further discuss the effect of EGCG on the proliferation of prostate cancer PC-3 cells Possible mechanism of action.Method:Select the best drug concentration by CCK8 method to understand the inhibition of EGCG on the proliferation of PC-3 cells,and then use the selected gradient concentration of EGCG(0、12.5、25、50、100μg/ml)for prostate cancer PC-3 Cell intervention;Summarize the expression and differential expression levels of EGCG in inhibiting transcriptome genes in prostate cancer PC-3 cells through transcriptome gene sequencing,select genes and further verify them;perform immunofluorescence on P27kip1 protein and PI3K/AKT protein Semi-quantitative determination;the influence of the control group and the EGCG experimental group on the expression of P27kip1,PI3K/AKT and other influencing factors was analyzed by Western blot;the control group and the EGCG experimental group on the cell cycle of prostate cancer PC-3 cells were detected by the Attune Nx T flow cytometer influences.Results:(1)The results of CCK8 showed that EGCG inhibited prostate cancer PC-3 cells in a dose-dependent manner.The inhibition rates of PC-3 cells were(5.3±0.33)%、(12±1.53)%、(26±2.31)%、(62±0.33)%(as shown in figure1),the differences between the groups were statistically significant(P<0.05).Compared with the control group in each experimental group of EGCG,the expression of AKT and its activation level did not change with the increase of drug concentration.(2)Analysis of the transcriptome gene sequencing results showed that the P27kip1 gene expression FPKM of the control group and the EGCG experimental group were 7.284 ± 0.954、7.520±0.603、7.032 ± 0.0972、8.357 ± 0.801、13.480±0.320,the difference was statistically significant,F=53.936,P<0.05,FC>1.5,The difference between the 100μg/ml EGCG experimental group and the other groups was statistically significant(P<0.01).The FC valuesbetween EGCG100μg/ml and the control group,andthe remaining EGCG experimental groups were all>1.5.By analyzing the expression of PI3 K gene PIK3CD(FPKM)and the difference between the groups,the following results are obtained: the control group and the EGCG experimental group PIK3 CD gene expression are respectively:1.750 ± 0.060、1.888 ± 0.194、1.817 ± 0.122、1.473 ± 0.099、0.704±0.016,the difference is statistically significant,F=53.366,P<0.05.The difference between the 100 or 50μg/ml EGCG experimental groups and other groups is statistically significant Academic significance,P<0.05.The FC values between EGCG 100 μg/ml and the control group,and the remaining EGCG experimental groups were all less than 0.67.(3)The results of immunofluorescence showed that PI3 K was expressed in the cytoplasm and its expression level decreased with the increase of EGCG concentration.AKT is expressed in the cytoplasm and the expression level does not change with the change of EGCG concentration.P27kip1 is expressed in both the cell cytoplasm and the nucleus,and the expression level and nuclear expression level increase with the increase of EGCG concentration.(4)The results of Western blot showed that the ratio of PI3 K proenzyme optical density(corrected byβ-actin)in the control group and the EGCG experimental group were:2.000±0.324 、 1.752±0.204 、 1.728±0.117 、 1.520±0.070 、1.133±0.184(as shown in Figure 7B),F=7.884,P<0.01,indicating that different concentrations of EGCG have different inhibitory effects on the expression of PI3 K protein in PC-3 cells.The pairwise comparison results between the groups showed that,except for the P>0.05 between the control group and the 12.5μg/ml EGCG experimental group,the25μg/ml and 50μg/ml EGCG experimental groups,the P<0.05 between the other groups.The ratio of the P27kip1 proenzyme optical density value(corrected by β-actin)in the control group and the EGCG experimental groupis: 0.970±0.113 、 1.152±0.160 、 1.519±0.141 、1.634±0.094、1.713±0.092(as shown in Figure 10 C,the F value is20.489,P<0.01.The protein expression level of AKT does not change with the concentration of EGCG.(5)The results of flow cytometry showed that the proportions of S phase cells in the control group and EGCG experimental groups were(27.21 ± 2.43)%、(14.78 ± 1.41)%、(12.04 ± 0.66)%、(10.76 ± 0.74)%、(9.47±0.18)%(as shown in Figure11B),F=86.84,P<0.01.EGCG can inhibit prostate PC-3 cells from stagnating in the S phase.After the intervention of different doses of EGCG solution,the ratio of PC-3 cells in the S phase increased significantly.Conclusion:The mechanism of EGCG inhibiting the proliferation of PC-3 cells may be related to the up-regulation of P27kip1 expression and nuclear expression and down-regulation of the PI3K/AKT proteinpathway,and inhibits the S phase of the PC-3 cell cycle. |
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