The Study On Roles Of Pokemon Targeted Regulation Of STRN4 In The Progression Of Prostate Cancer

Objective:In this study,we aimed to investigate the role of Pokemon and its regulation mechanism on cell proliferation,apoptosis and cell cycle in prostate cancer,to explore a new target in treating the prostate cancer.Methods:1.Firstly,immunohistochemical staining was to usded to detecd the pokemon expression in human prostate cancer tissues and human normal prostate tissues.Secondly,RT-PCR and western blot was used to detecd the pokemon expression in human prostate cancer tissues and human normal prostate tissues.2.We transfected prostate cancer cell lines PC3 and DU 145 by artificial shRNA,screened the stable pokemon silenced cell lines,and confirmed the knockdown of pokemon gene,then determined the the effect of pokemon on cell proliferation,apoptosis and cell cycle of prostate cancer cell lines PC3 and DU 145 via MTT assay,PI single staining and Annexin V-FITC/PI double staining.3.To determine the underlying mechanisms of Pokemon in promoting prostate cancer cell growth,we used cDNA microarray analysis to identify genes regulated by Pokemon in PC3 cancer cells.Then used the bioinformatics website to classify the downstream genes and screen the suitable downstream target genes.Via the dual fluorescein reporter assay to detecd the mechanism of the interaction between Pokemon gene and STRN4.The real-time quantitative PCR and western blot were used to further verify the interaction between Pokrmon gene and STRN4 Gene.4.Quantitative real time PCR and immunohistochemical staining were used to determine the expression of STRN4 in prostate cancer tissues and normal prostate tissues.Prostate cancer cell lines PC3 and DU 145 transfected with a synthesized shRNA,in which knocked down by STRN4.Then we observed the cell proliferation,apoptosis and cell cycle via MTT assay,PI single staining and Annexin V-FITC/PI double staining.We overexpressed Pokemon and STRN4 by transfecting the pLKO.1-puro vector.A rescue experiment was performed by co-transfecting with pLKO.1-Pokemon(versus the negative control)and pLKO.1-STRN4(versus the negative control)into PC3 and DU 145 cells.Knockdown of STRN4 and Pokemon by shRNA in PC3 and DU 145 cells.Then we observed the cell proliferation,apoptosis and cell cycle via MTT assay,PI single staining and Annexin V-FITC/PI double staining.5.The effects of Pokemon and STRN4 on the growth of prostate cancer in vivo were observed by nude mice model.Results:1.Using immunohistochemical staining,we found that Pokemon was expressed at low levels in normal tissues,but overexpressed in prostate cancer tissues(65.8%).western blotn showed elevated protein expression of Pokemon was confirmed in prostate cancer tissues,and PCR showed the mRNA levels of Pokemon were also elevated in prostate cancer tissues vs normal tissues(P<0.05).Similarly,Pokomon is similarly highly expressed in prostate cancer cell lines PC3 and DU145(P<0.01).2.RT-PCR and western blotn showed Pokemon was clearly knocked down by short hairpin RNA(shRNA)in PC3 and DU 145 cells(P<0.01).MTT assay showd The OD570 value of sh-Pokemon group began to decrease significantly compared with that of sh-Control group on day 2 after 96-well plates were transfected with PC3 and DU 145 cells,and decreased most significantly on day 4.Cell cycle was analyzed by flow cytometry,the result showed the number of GO/G1 phase cells in sh-Pokemon group were significantly more than those in sh-Control group and the number of S phase cells were significantly lower(P<0.01),blocking at GO/G1 phase in cell cycle.The results of flow cytometry showed that the apoptosis rate of sh-Pokemon group was significantly higher than that of shRNA-Control group(P<0.01).3.The results of cDNA microarray analysis showed that there were altogether 121 genes which could be related to Pokemon.Analysis by the Kyoto Encyclopedia of Genes and Genomes and the Gene Ontology databases,we found that 121 Pokemon-related genes which included STRN4 gene were mainly involved in signal transduction,biosynthesis metabolism,apoptosis,cell cycle and differentiation.Dual luciferase reporter gene assay showed sh-Pokemon significantly reduced the luciferase activity of the wild-type STRN4 3’UTR but had no significant effect on the mutant STRN4 3’UTR.Meanwhile,knockdown of Pokemon and overexpression of Pokemon in PC3 and DU 145 cells resulted in low and high expression of STRN4.4.STRN4 is highly expressed in prostate cancer tissues.RT-PCR and western blot showed the expression of sh-STRN4 in sh-STRN4 group was significantly lower than that of sh-Control STRN4 after knockdown of STRN4 by shRNA(P<0.01).Knockdown of STRN4 by shRNA in PC3 and DU145 cells attenuated cell growth(P<0.01),induced G0/G1 cell-cycle arrest(P<0.01),promoted cell apoptosis(P<0.01),similarly to knockdown of Pokemon.A rescue experiment showed the roles of pokemon knockdown on cells proliferation,cells cycle,cells apoptosis in PC3 and DU 145 cells was effectively reversed by STRN4 overexpression.5.By injected subcutaneously sh-Control,sh-Pokemon and sh-STRN4 PC3 cells into the nude mice,we found that the average volume of tumor formation of the sh-Pokemon or sh-STRN4 cells was significantly reduced compared with the tumor formation of the sh-Control cells.Conclusion:Pokemon can be used as a potential marker in prostate cancer diagnosis.Pokemon can significantly increase tumor cell proliferation and inhibit tumor cell apoptosis in prostate cancer.Pokemon exerts a carcinogenic on prostate cancer by targeting the STRN4 gene.