Preliminary Study On Effective Mechanism Of Pim-1 In Prostate Cancer

Preliminary study on effective mechanism of Pim-1 in prostatecancerObjective In our country,the incidence of prostate cancer has increasedsignificantly in recent years and the prostate cancer has been the thirdcommon malignancy of genito-urinary system in man to date.Foradvanced and/or androgen-independent prostate cancer,the gene therapyis a promising treatment selection.Pim-1 belongs to a serine/threoninekinase family and is able to phosphorylate different targets,most ofwhich are involved in cell survival,proliferation,differentiation andtumorigenesis.Many studies have showed that there is over-expressedPim-1 in prostate cancer.In our study,by utilizing the RNA inference(RNAi)technique we intend to investigate the effective mechanism ofPIM-1 in the process of proliferation,apoptosis and migration of prostatecancer,and investigate the molecular mechanism of tumorigenesis anddevelopment of prostate cancer further.Our study will provide anexperiment base for the clinical use of gene therapy targeting Pim-1 inprostate cancer.Methods RT-PCR and western-blot were used toscreen the prostate cancer cell strain expressing Pim-1 highly.siRNAoligos of Pim-1 mRNA were designed and screened through the onlinesiRNA design software,and then the secondary structure of the targetmRNA were analyzed by the RNA-structure software 3.2 edition.At lastthree siRNA oligos have been determined which target the 674-692,707-725 and 1080-1098 nucleotides of Pim-1 mRNA respectively.Byinserting the artificially-synthesized expression frame of shRNA intolined pRNAT-U6.1/Neo plasmid,three recombinant plasmids ofpRNAT-U6.1/Neo-Piml-shRNA-1,-2-,3 were constructed successfully.The recombinant plasmids were transfected into PC-3 cells by liposome.The silence effect of the 3 recombinant plasmids on Pim-1 gene were investigated by RT-PCR and Western-blot technique,and thestable-transfection cells were screened by G418.The influence of thesilence of Pim-1 on cell-cycle and apoptosis in PC-3 cells were evaluatedby FCM,and the change of the invasion capability of PC-3 cells weredetected by Trans-well.The expression of c-Myc gene in Pim-1gene-silenced PC-3 cells was observed meanwhile.To establish xenograftprostate cancer model,PC-3 cells were inoculated subcutaneously on theventro-anterior surface of nude mice.The mixture of liposome andpRNAT-U6.1/Neo-PIM1-shRNA-3 recombinant plasmids were injectedinto the implanted tumors.The silence effect of Pim-1 gene in tumor cellsand the suppression effect in tumor growth were studied then.GAPDHserves as inner control in RT-PCR and western-blot assay.Results Threerecombinant plasmids of pRNAT-U6.1/Neo-PIM1-shRNA-1,-2,-3 candown-regulate the level of Pim-1 mRNA,and the more significantsilence effect on Pim-1 expression of the latter two of them wereobserved.The shRNA-expression recombinant plasmid targeting the1080-1098 nucleotides of Pim-1 mRNA was transfected into the PC-3cell,and the PC-3 cells of stable transfection were obtained by G418screen.Compared with the PBS contrast group,the ability ofproliferation,clone formation and tumorigenesis of the PC-3 cells withsilenced PIM-1 gene had decreased remarkably.The percentage of cellsin G1 stage increased,at the same time the percentage in S stage reduced.And there was no change in the invasive capability of PC-3 cell withdown-regulation of Pim-1.Notably,we observed also that c-Myc geneexpression of those PC-3 cell were inhibited at translation orpost-translation level but not transcription level.At last,the expression ofPIM-1 gene was suppressed in the tumor cells of xenograft prostatecancer by combined-injection with recombinant plasmid and liposome.Consequently,the growth of the implanted tumors in nude mice wasinhibited obviously by the injection of recombinant plasmid and liposome and the final weight of implanted tumor in treatment group andPBS control group were 0.3 3±0.08g and 0.68±0.14grespectively(P＜0.05).Conclusion we have successfully constructedthree recombinant plasmids which can express shRNA in vivo targetingthe PIM-1 mRNA,and we have investigated their silence effect on PIM-1gene respectively.Mediated by liposome,the recombinant plasmidtargeting 1080-1098 nucleotides of PIM-1 mRNA was transfected intoPC-3 cells.The cell cycle of the PC-3 cells whose PIM-1 gene expressionwere suppressed was blocked,the proliferation was inhibited and theapoptosis was induced of those cells meanwhile.It was postulated thatthere was a synergistic mechanism of Pim-1 and c-Myc in inhibiting thegrowth of Prostate cancer,in which Pim-1 could regulate c-Mycexpression at the translation level.In conclusion,Pim-1 gene can serve asa effective target of gene therapy for prostate cancer and this strategymay have a potential value in clinical practice deserving furtherinvestigation.