【作者】 张晓光；

【导师】 徐勇； 张志宏；

【作者基本信息】 天津医科大学 ， 外科学， 2009， 博士

【摘要】 目的目前，前列腺癌(Prostate Cancer，PCa)的发病率已跃居我国男性泌尿、生殖系统恶性肿瘤的第三位。对于晚期PCa以及雄激素非依赖型PCa，基因治疗可以作为一种很有前途的治疗手段。Pim-1是一种丝氨酸／苏氨酸蛋白激酶，已有研究显示Pim-1在细胞凋亡、增殖、分化和肿瘤发生过程中起重要作用，并证实其在Pca中有高表达。本研究旨在利用RNA干扰(RNAi)技术下调Pim-1基因的表达水平，研究其对PCa细胞在增殖、凋亡及侵袭等方面的影响，并确定Pim-1基因是否可作为PCa基因治疗的靶基因，进而为PCa基因治疗的临床应用奠定实验基础。方法利用RT-PCR和Western-blot筛选Pim-1表达水平最高的前列腺癌细胞系作为研究对象。利用相关软件设计、筛选出若干Pim-1 mRNA干扰序列，再通过BLAST及干扰靶点mRNA的二级结构进一步筛选出3条靶向Pim-1mRNA的干扰序列，长度均为19nt，分别靶向674-692、707-725和1080-1098位点。人工合成靶向Pim-1基因的siRNA转录模板，与shRNA表达载体相连接，成功构建针对PIM-1 mRNA上述位点的RNAi重组质粒pRNAT-U6．1／Neo-Pim1-shRNA-1，-2，-3。重组质粒在脂质体介导下转染前列腺癌细胞，利用RT-PCR和Western-blot检测Pim-1基因沉默效果，并筛选稳定转染的细胞克隆。利用流式细胞技术检测Pim-1基因沉默对前列腺癌细胞的细胞周期及凋亡的影响，利用Transwell实验检测细胞侵袭能力的变化。同时利用RT-PCR和Western-blot检测Pim-1表达下调后前列腺癌细胞中原癌基因c-Myc表达的变化情况。制备前列腺癌裸鼠移植瘤模型，肿瘤局部注射针对Pim-1基因的RNAi重组质粒和脂质体，观察前列腺癌组织细胞内Pim-1基因的沉默效果及RNAi重组质粒对前列腺癌在动物体内生长的抑制作用。RT-PCR和Western-blot检测中均以GAPDH的表达水平作内参照。结果RT-PCR检测显示3个RNAi重组质粒转染PC-3细胞后Pim-1 mRNA表达的相对量分别为0．324±0．025、0．136±0．013、0．125±0．016，与PBS对照组(表达量为1．078±0．132)相比均明显下降(P＜0．05)。与重组质粒-1相比，RNAi重组质粒-2，-3具有更强的Pim-1基因沉默效应(P＜0．05)。Western-blot检测显示，与PBS对照组相比这2个RNAi重组质粒均可以显著降低Pim-1蛋白的表达水平。将靶向1080-1098位点的RNAi重组质粒-3以脂质体为介导转染PC-3细胞，并筛选出稳定转染细胞。与PBS对照组相比，Pim-1基因沉默PC-3细胞的增殖速度、克隆形成能力及致瘤能力均显著下降，细胞周期中G1期细胞的比例升高而S期细胞的比例降低，说明G1期→S期进程受阻。Pim-1基因沉默对前列腺癌细胞的侵袭能力并无显著影响。而且，Pim-1基因沉默前列腺癌细胞中c-Myc蛋白水平显著降低(沉默组和PBS对照组c-Myc蛋白相对量分别为0．384±0．041和0．866±0．079，P＜0．05)，而其mRNA水平却无明显变化(沉默组和PBS对照组c-MycmRNA相对量分别为0．986±0．158和1．027±0．105，P＞0．05)。与PBS对照组相比，种植瘤组织内注射Pim-1 RNAi重组质粒-3可以有效降低前列腺癌细胞内Pim-1 mRNA水平(Pim-1 mRNA的相对量分别为1．113±0．048和0．437±0．013，P＜0．05)和Pim-1蛋白水平(Pim-1蛋白相对量分别为0．948±0．097和0．378±0．048，P＜0．05)。另外，注射重组质粒可以显著抑制裸鼠皮下种植瘤的生长，重组质粒组和PBS对照组种植瘤的最后重量分别为0．33±0．08g和0．68±0．14g(P＜0．05)。PBS对照组和空质粒对照组在各项指标检测中均无显著差异(P＞0．05)。结论本实验成功构建了针对Pim-1 mRNA的RNAi重组质粒表达载体，并验证了靶向不同位点重组质粒载体的基因沉默效果。重组载体在脂质体的协助下能够高效的转染PC-3细胞，靶向Pim-1 mRNA 1080-1098位点的RNAi重组质粒能够有效的在转录后水平抑制前列腺癌细胞Pim-1基因的表达，并使PC-3细胞的细胞周期受到阻滞，抑制了细胞的增殖，同时诱发细胞凋亡。Pim-1可以在蛋白水平调控c-Myc的表达，对抑制肿瘤生长可能产生协同效应。裸鼠种植瘤模型的动物体内抑瘤实验显示，Pim-1可以作为前列腺癌基因治疗的有效靶点，具有潜在的临床应用价值。更多还原

【Abstract】 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.更多还原