【作者】 曲发军；

【导师】 徐丹枫；

【作者基本信息】 第二军医大学 ， 外科学， 2012， 博士

【摘要】 前列腺癌（cancer of prostate）是欧美国家最常见的恶性肿瘤之一，占第二位。在美国，前列腺癌的发病率占首位，死亡率仅次于肺癌。中国等亚洲国家前列腺癌远低于欧美，但是其发病率呈现显著增长的趋势。前列腺癌的病因至今尚未阐明，激素假说获得了较为广泛的支持。前列腺的生长依赖雄激素的作用，前列腺癌细胞的增殖也有赖于雄激素的生物学作用。因此，目前对于前列腺癌的治疗措施中除根治性前列腺癌切除术等手术治疗外，最大阻断雄激素（MAB）为最主要的方法，即手术切除睾丸+氟他胺+促黄体激素释放激素促效剂（LHRH-A）联合应用，以期达到雄激素的最大阻断，从而抑制前列腺癌细胞的增长。尽管MAB能够达到很好的抑制前列腺癌细胞生长的目的，仍有部分前列腺癌会进展为另一阶段：激素非依赖性前列腺癌。激素非依赖性前列腺癌不依赖雄激素的生物学作用，在此阶段前列腺癌细胞的生长通过其他方式增殖、迁移和侵袭。由激素依赖性前列腺癌进展为激素非依赖性前列腺癌的具体机制尚未阐明，目前主要存在四种观点：雄激素受体表达上调、雄激素受体基因突变、配体非依赖性激活和共调节因子发挥作用。由此可见，雄激素受体在前列腺癌的进展过程中发挥着重要的作用。雄激素作用的发挥主要是通过与雄激素受体结合来启动下游信号通路。雄激素受体信号通路的下游分子多种多样，其生物学功能涉及细胞增殖、细胞周期和细胞凋亡等等。最常见的例子便是其下游分子PSA的高表达可以促使前列腺细胞的增殖。研究发现，雄激素受体参与了前列腺癌的进展过程，因此如果能够采取干预措施抑制雄激素受体的生物学作用，则可以有效抑制前列腺癌的发展。进一步讲，通过有效干预雄激素受体的功能，有助于抑制前列腺癌由激素依赖性前列腺癌进展为激素非依赖性前列腺癌。MicroRNAs为内源性、非编码性的小的核苷酸片段，长度一般为22~25nt。近年来，有关microRNA对细胞生物学特性的调控作用一直是医学界研究的热点。MicroRNAs能够与mRNA的3’UTR区结合，从而阻遏mRNA的翻译功能，甚至直接降解mRNA，这两方面作用都能够抑制蛋白的合成，从而发挥相应的生物学调节作用。研究发现，microRNAs在肿瘤的发生发展过程中同样发挥着重要的调节作用，可以表现为癌基因发挥促进肿瘤的作用，也可以表现为抑癌基因发挥抑制肿瘤的作用。由此可见，如若采取相应的干预手段上调或者下调相应microRNAs的水平，可以达到抑制肿瘤生长的目的。研究发现，microRNA-185（缩写为miR-185）在诸多肿瘤中发挥抑制肿瘤生长的作用，例如直肠癌、卵巢癌等等，但是有关其在前列腺癌中的作用至今未见报道。鉴于雄激素受体在前列腺癌的发生发展和激素非依赖性前列腺癌的进展过程中发挥着重要的作用，我们拟研究miR-185与雄激素受体是否存在相应的调控关系以及miR-185是否能够干预前列腺癌细胞的生长。一、 miR-185在临床PCa组织中的低表达以及靶点预测和靶点验证我们首先检测了miR-185在临床前列腺癌标本和癌旁正常组织中的表达情况。首先，microRNA芯片分析发现miR-185在前列腺癌组织中呈现低表达。然后取前列腺癌和前列腺癌旁正常组织各25例，部分冻存，部分用中性福尔马林固定。病理切片HE染色证实所取组织为前列腺癌或正常前列腺上皮组织。冻存标本经RNA抽提、逆转录扩增和qRT-PCR检测，结果发现miR-185在前列腺癌组织中的表达量显著低于癌旁正常组织。这一结果提示miR-185可能参与了前列腺癌的肿瘤发展过程。在此基础上，我们利用TargetScan5.2algorithm (www.targetscan.org)预测miR-185的作用靶点。结果令人兴奋的是，雄激素受体的3’UTR存在miR-185的潜在作用靶点，其作用位点为173-179nt。之后，我们利用双荧光报告载体，验证了miR-185与雄激素受体的靶向作用关系，结果证实miR-185确实能够结合雄激素受体的3’UTR区，导致荧光强度的降低，从而证实了预测结果。化学合成miR-185mimics（miR-185m）和对照microRNA negative control（miR-NC），利用INTERFERin将化学合成物转染入LNCaP细胞，之后检测雄激素受体AR和前列腺特异性抗原PSA的表达水平。结果发现，转染miR-185m促使AR蛋白的表达显著降低。同时，PSA的表达也显著降低，表现在mRNA和蛋白两个水平。上述结果从基因和蛋白水平证实了miR-185对雄激素受体的靶向作用。二、 miR-185高表达对前列腺癌细胞增殖、迁移和侵袭的影响在本部分实验中，我们重点研究miR-185对前列腺癌细胞肿瘤生物学特性的影响。我们选择了雄激素受体表达强阳性的LNCaP细胞株作为研究对象。首先进行细胞的培养、传代，其中总结了关于LNCaP培养过程中的经验。之后化学合成miR-185mimics（miR-185m）和对照microRNA negative control（miR-NC），利用转染试剂INTERFERin将化学合成物转染入LNCaP细胞。PCR证实转染效率足够研究要求。在细胞增殖实验中，利用日本同仁化学研究所的CCK-8试剂盒，连续检测测转染后5天的LNCaP细胞生长情况。结果发现，转染miR-185m后LNCaP细胞的增殖受到显著抑制。在细胞迁移实验中，我们采用划痕实验证实转染miR-185m显著削弱了LNCaP细胞的肿瘤迁移能力。在细胞侵袭实验中，我们发现转染miR-185m后，穿过8微米细孔的LNCaP细胞显著减少，提示其肿瘤侵袭能力显著降低。为探求miR-185对LNCaP细胞周期的影响，我们利用流式细胞检测技术检测了转染miR-185m后细胞各周期的的比率，结果发现转染miR-185m后细胞被阻滞在G1期，S期的细胞数量显著减少，说明miR-185能够起到G1/S阻滞的作用。上述研究证实：miR-185能够抑制前列腺癌细胞的增殖，主要是通过细胞周期G1/S阻滞实现；miR-185能够减弱前列腺癌细胞迁移和侵袭的能力，提示其显著的肿瘤抑制作用。三、 miR-185高表达对裸鼠皮下前列腺癌肿瘤生长和转移的影响在本部分实验中，我们重点研究经miR-185m转染的LNCaP细胞在裸鼠体内的生长情况。为使得转染能够稳定传代，我们将miR-185m进行2’ome修饰。转染后将106个LNCaP细胞用100μL的PBS溶液重悬，注入裸鼠皮下。经过6周的生长，测定实验组和对照组肿瘤的重量、大小以及肿瘤中雄激素受体的表达情况。结果发现，经过miR-185m转染后，LNCaP细胞的致瘤作用显著减弱，主要表现为肿瘤的大小和重量较对照组显著降低，另外western-blotting证实肿瘤中AR的表达也显著降低。8周后解剖发现，裸鼠肝脏出现了转移性结节，比较发现，高表达miR-185的LNCaP细胞所形成的转移性肝结节显著少于对照组。上述研究证实miR-185在裸鼠体内同样具有肿瘤抑制作用。综合本实验研究结果，我们得出以下结论：miR-185可以作为“tumor suppressor”抑制前列腺细胞的肿瘤生物学特性，此作用的发挥依靠miR-185直接作用于雄激素受体。由miR-185过表达导致的雄激素受体低表达可以显著抑制前列腺癌细胞的增殖、侵袭和迁移能力。鉴于雄激素受体在前列腺癌发生发展过程中发挥重要的作用，我们认为可以将miR-185设计为有效地前列腺癌抑制工具，并有助于激素非依赖性前列腺癌的治疗。更多还原

【Abstract】 Prostate cancer (CaP) is the most frequently diagnosed carcinoma and the secondleading cause of death among men in the Western World. Androgen receptor (AR), whichcontrols the growth and differentiation of the normal prostate, plays a pivotal role in thedevelopment and progression of CaP. AR blockage is the first-line initial treatment for CaP.The mechanisms of such fatal transition remain largely unknown. AR is expressed in bothandrogen dependent and independent CaP. Therefore, AR may play a crucial role in theprogression of androgen independence of CaP.Unfortunately, some advanced cancers will still develop into castration-resistantdisease even with maximum androgen blockade (MAB) therapy. Androgen receptor canregulate various target genes, including prostate specific antigen (PSA), which isup-regulated by androgen in the prostate and is thought to contribute to the progression ofCaP. AR and its target genes are consistently regarded as important points for revealing theprocess of castration-resistant CaP. Recent studies have shown that AR is involved in theprogression of androgen-independent CaP via several mechanisms. AR is known to beassociated with various stages of CaP progression, and therefore it is essential to know howthe expression and function of AR could be inhibited effectively so as to control theprogression of CaP. In fact, AR has been regarded as an important therapeutic target toCaP.MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs with20~25nucleotides and play important negative regulatory roles through binding to the3’untranslated regions (3’ UTR) of target transcripts. MiRNAs can lead to protein translationarrest, and mRNA degradation in some cases. These effects of miRNAs would furtherinfluence the activity of downstream signal pathways. Studies have demonstrated thatmiRNAs play a role in tumorigenesis, acting as oncogenes or tumor suppressor genes.Among microRNAs, miR-185is reported to have the ability to suppress tumor growthof some cancer cell lines, such as rectal cancer, ovarian cancer and so on. However, there is no report about the effect of miR-185on the proliferation and invasion of CaP. To seewhether miR-185has correlations with CaP development, we performed a series of trial todetermine the effect of miR-185on the proliferation, migration, invasion of LNCaP cell.Additionally, we also detected whether over-expression of miR-185could inhibit tumorformation in nude mice in vivo. These trials suggest that miR-185has tumor-suppressivefunction during the development of CaP.Part Ⅰ: miR-185is down-regulated in clinical CaP tissues and miR-185directly targets androgen receptor.To compare the expression levels of miR-185in clinical CaP tissues and adjacentnon-tumorous tissues,25CaP samples and their adjacent nontumorous samples werechosen for qRT-PCR examination. The results showed that the expression level of miR-185in CaP samples was significantly lower as compared with the non-tumorous specimens. Atargets prediction tool called TargetScanHuman (www.targetscan.org) was used to searchfor potential targets of miR-185. Excitedly, the results showed that there wereconsequential pairs between miR-185and3’UTR of AR. A sequence analysis revealed thatthe putative targeting site of miR-185was located at nt173-179of the AR3’UTR.Furthermore, we performed a dual luciferase reporter assay， and the results indicated thatthe3’UTR of AR was a target of miR-185and the binding site in the3’UTR wasresponsible for the regulatory effect of miR-185on AR and its biological functions.Additionally, qRT-PCR and Western blotting analysis revealed that the expression levels ofAR mRNA and protein were down-regulated by transfection of miR-185m.Part Ⅱ: miR-185suppresses proliferation, invasion and migration ofLNCaP cells and induces cell cycle arrest at G0/G1phase.To investigate the effect of miR-185on cell growth, cck-8assay was performed. Theresults showed that over-expression of miR-185markedly suppressed the proliferation ofLNCaP cells. Cell cycle distributions of miR-185m and miR-NC transfectants wereanalyzed by FACS. The results showed that over-expression of miR-185resulted in asignificant decrease in the number of S-phase cells and a concomitant increase in G0/G1phase cells. Furthermore, transwell invasion assay and wound healing trial indicated thatmiR-185m transfection impaired the invasive and migratory ability of LNCaP cells. These results indicate that miR-185could negatively regulate proliferation of LNCaP cells byinhibiting cell cycle conversion, and ectopic over-expression of miR-185could effectivelyreduce the invasive and migrating activities of LNCaP cells. These data suggest thatmiR-185could be regarded as a tumor suppressor of CaP cells in vitro.Part Ⅲ: miR-185inhibits tumor formation and migration of LNCaP cellsin nude mice in vivo.To further determine the effect of miR-185on tumor growth in vivo,5-week-old nudemice were injected subcutaneously with LNCaP cells transfected with miR-185m ormiR-NC which were subjected to2’-ome classification. Compared with the control group,animals injected with miR-185m-LNCaP showed a significant reduction in tumor growthby weight and volume. Western blotting revealed that the expression level of AR protein inxenografts arising from injection of miR-185m-LNCaP was significantly lower than that inthe control group. Additionally, the numbers of liver nodules arising from injection ofmiR-185m-LNCaP was lower than that in miR-NC-LNCaP group. These results suggestthat miR-185could inhibit tumor growth of CaP in vivo.In summary, our findings suggest that miR-185could function as a tumor-suppressorof CaP by directly targeting AR. Down-regulation of AR by miR-185could result in cellgrowth and cell malignant characteristics of CaP. Knowing that AR plays a crucial role inthe development CaP and progression of androgen-independent transition, we think thatmiR-185can be regarded as an “inhibitor” of AR and may prove to a useful tool forsuppressing the development of initial CaP and androgen-independent tumor growth ofadvanced CaP as well.更多还原