The Study Of Beclin-1 Gene Silencing On The Autophagy And Apoptosis Of The Prostatic Hyperplasia Epithelial Cells

Objective: In previous studies,we found that deprivation of androgen and inhibition of autophagy can promote the apoptosis of prostatic epithelial cells,and the apoptosis is further strengthened after combined treatment.This experiment will be under the conditions of androgen deprivation and inhibition of autophagy.Objective to study the effect of silencing Beclin-1 gene on the autophagy level and apoptosis level of prostate epithelial cells,and to study the regulation of BPH-1 gene silencing on PI3K-AKT-mTOR signaling pathway,and explore the possible pathogenesis of BPH from the perspective of autophagy and apoptosis.Methods: Through cloning construction,lentivirus packaging,lentivirus infection and puromycin screening,we obtained the BPH-1 cell line with the empty vector(sh-RNA)and with low expression of Beclin-1gene(sh-Becclin-1),and to evaluate the transfection efficiency by Western blot and qRT-PCR.After the construction of LC3 cell autophagic adenovirus Adv-LC3-GFP,normal cells,empty carrier cells(sh-RNA)and sh-Beclin-1 cells were incubated overnight to make cell adherence(8-12 hours),and Adv-LC3-GFP infection for 48 hours.Photograph observation.Then,remove the old culture medium,the different cells were cultured under androgen deprivation(AD,Treatment of fetal bovine serum with 10% activated carbon)and autophagy inhibition(AI,50μM chloroquine)for 24 hours.Photograph observation.Cells of each group were incubated overnight to make cell adherence(8-12 hours).Removing the old culture medium,PSC cleaning three times.The different cells were cultured under androgen deprivation(AD)and autophagy inhibition(AI)for 24 hours.The apoptosis level of each group was detected by flow cytometry,and the total protein of each group was extracted.The expression of LC3 II,PARP-1,Caspase-3,BAX,BCL-2,AKT,P-AKT,m-TOR and beta-actin protein were detected by Western blot.Results: Western blot and qRT-PCR methods were used to detect the expression of Beclin-1 protein and Beclin-1 RNA for evaluating the transfection efficiency.The results show that normal control group,sh-RNA-BPH-1 group were no significant difference,the expression of sh-Beclin1-BPH-1 group was significantly reduced.Cells of each group were incubated overnight to make cell adherence(8-12 hours).Every groups were infected with Adv-LC3-GFP for 48 hours,Photograph observation,the results show: The expression of GFP-LC3 in the sh-Beclin-1 group was significantly lower than in the normal control group and the sh-RNA-BPH-1 group(P<0.001).Then,the different groups were cultured under androgen deprivation(AD)and autophagy inhibition(AI)for 24 hours.Photograph observation,the expression of GFP-LC3 was significantly lower in the sh-Beclin-1 group compared with the normal control group and the sh-RNA-BPH-1 group(P<0.001).Flow cytometry was used to detect the level of cell apoptosis in each group.The result show: the apoptosis rate of the sh-Beclin1-BPH-1 group was significantly higher than that in the normal control group and the sh-RNA-BPH-1 group(P<0.01).The protein expression levels of different groups was detected by Western blot.The different groups were cultured under androgen deprivation(AD)and autophagy inhibition(AI)for 24 hours,extract protein.Western blot results,the expression level of LC3 II protein in sh-Beclin1-BPH-1 group was obviously lower than that in normal cell and sh-RNA group.The expression level of PARP-1 protein in the sh-Beclin1-BPH-1 group decreased and produced the fragmentation fragment(89KDa)and the expression level of Caspase-3 protein was down.The expression of BCL-2 in group sh-Beclin1-BPH-1 was down regulated and the expression of Bax protein was up-regulated,and the ratio of BCL2/BAX decreased.There was no difference in protein AKT expression between normal control group,sh-RNA-BPH-1 group and sh-Beclin1-BPH-1 group,but the expression of P-AKT in group sh-Beclin1-BPH-1 was significantly lower than that in the first two groups.The expression of m-TOR protein was upregulated.Conclusion: chloroquine can effectively inhibit the autophagy level of BPH-1 cell.Under the conditions of autophagy inhibition and androgen deprivation,beclin-1 gene silencing can reduce the level of autophagy in BPH-1.In addition,the apoptotic proteins of PARP-1,Caspase-3,Bcl-2 and Bax were activated by AKT pathway,and to promote the apoptosis of BPH-1 cell.