MiR-200b-3p在雄激素非依赖性前列腺癌细胞增殖中的作用及其分子机制研究
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摘要
前列腺癌(Prostate cancer)是男性泌尿生殖系常见恶性肿瘤之一,在美国位居男性恶性肿瘤之首,占男性死亡原因的第二位。随着我国人民的饮食结构和生活习惯与西方差别的逐渐缩小,前列腺癌在我国的发病率也逐年增加。
前列腺癌细胞的生长依赖于雄激素的存在。雄激素阻断疗法(去势疗法)治疗前列腺癌,可以抑制肿瘤,且绝大多数患者大约可无病生存1.5-3年,但此后,肿瘤往往复发,获得雄激素非依赖性增殖的能力。通常,临床上将雄激素完全阻断后仍有进展的前列腺癌称为雄激素非依赖性前列腺癌(androgen-independent prostate cancer, AIPC)或雄激素抵抗性的前列腺癌(castration-resistant prostate cancer, CRPC)。目前对于AIPC的定义一般认为应同时具备以下几点：血清睾酮达去势水平;抗雄激素撤退治疗4周以上；间隔2周,连续3次前列腺特异性抗原升高；二线内分泌治疗期间前列腺特异性抗原继续进展；骨或软组织转移病变有进展。对于这些AIPC病例,当前的治疗手段有限,预后很差。为这些AIPC患者找出新的治疗方案有重要的社会效益,而深入揭示AIPC的发生机制,是预防和治疗AIPC的关键。
AIPC发生发展的机制并不清楚。长期以来,对前列腺癌雄激素非依赖性演进发生机制的研究主要集中在雄激素受体(androgen receptor, AR)。认为其可能机制主要包括：(1)AR过表达或扩增；(2)AR突变；(3)前列腺细胞雄激素局部产物增加；(4)生长因子/细胞因子对AR信号通路的活化；(5)辅助活化因子(共激活子/共抑制子)表达异常；(6)AR蛋白水解为雄激素非依赖型；(7)基质细胞AR表达丢失。但是AR自身的表达调控及AR下游的信号机制研究并不清楚。
近年,随着前列腺癌的发病率增加,以及新技术的应用,人们发现microRNA (miRNA)对其靶基因表达的调控,在AIPC的发生发展中起到重要的调控作用。对miRNAs在AIPC发生发展中的作用进行深入探讨,有助于全面理解AIPC的发生机制,为AIPC提供良好的诊断和治疗靶标。
miRNA参与AIPC的发生,也有相关报道。Sung等发现几种：miRNA,尤其miR-221和miR-222,在AIDC细胞株中相对于雄激素依赖性(androgen-dependent prostate cancer, ADPC)细胞株明显高表达,提示miRNA在AIPC的发生发展中起作用。White等发现在AIPC细胞株中有一小部分miRNA异常表达。在恶性前列腺组织中存在miR-616过表达。在体外,LNCaP细胞过度表达,miR-616可以促进AI细胞增殖。更重要的是,体内miR-616过表达的LNCaP (ADPC)细胞能够克服去势抵抗,即双侧睾丸切除术后仍可提高增殖能力。miR-616抑制剂用于AI细胞可以产生相反作用。另外还证实miR-616靶基因是TF通道抑制蛋白TFPI-2mRNA。在AIPC发展中,miR-616过表达发挥重要作用。
信息生物学预测及实验研究表明,一个miRNA可调控多个靶基因；这些靶基因并不是随机分布的,而是富集在同一个或某几个信号通路。这提示一个miRNA可同时调控同一信号通路中上下游靶蛋白的表达,或通过调控多个信号通路,从而对基因调控网络实施更有效的调节,最终对细胞功能和表型进行更有力的调控。
因此,必然还有其它的miRNAs参与AIPC的发生,并且,这些miRNAs影响前列腺癌细胞对雄激素依赖性的分子机制研究尚处于起步阶段。
由此可见,虽然已发现一些miRNA在AIPC发生发展中发挥很重要的作用,但尚有许多问题仍不清楚：如是否还有其它的：miRNA分子参与了AIPC发生发展过程?这些miRNA表达水平的变化与AIPC发生发展之间有何关系?这些miRNA调控AIPC的分子机制是什么?它们是否可以作为AIPC的治疗靶标?对这些问题的深入研究必将促进我们对AIPC发生发展机制的深入认识,为AIPC的临床治疗提供新的理论依据和新的miRNA靶点。
基于以上分析,本课题主要进行以下研究：
1、验证前列腺癌细胞系LnCap、PC3对雄激素的依赖性。
2、Q-PCR方法筛选LnCap、PC3细胞间差异表达的miRNA和肿瘤相关基因。
3、生物信息学分析功能相关的差异miRNA和基因。
4、验证3中miRNA和基因表达改变、二者调控关系及其对AIPC增殖的调控作用。
5、采用蛋白质组学鉴定AIPC中受上述3、4中关键miRNA调控的蛋白,探讨该miRNA调控AIPC细胞增殖的分子机制。
我们首先用Q-PCR方法筛选了一些niRNA和肿瘤相关基因在PC-3细胞和LnCap细胞中的表达差异。在差异表达的miRNA中miR-200b-3p(t=23.575,P=0.002)在PC-3中表达显著较低。这与Xu等先前的报道相一致。然而,miR-200b-3p在AIPC中的作用并不清楚。我们的结果表明：无论过表达或抑制表达miR-200b-3p,都不会影响LnCap细胞在含雄激素培养环境中的增殖,F值=1.088,P=0.375。撤除雄激素后,过表达miR-200b-3p不会影响LnCap-Ad的增殖,P=0.814, P=0.348, P=0.083, P=0.162;但是抑制miR-200b-3p表达则会促进LnCap-Ad增殖,P=0.023,P=0.003。
我们还检测了一些肿瘤相关基因的表达,发现TP73(编码蛋白p73)表达(t=127.000,P<0.001)变化趋势与miR-200b-3p一致。p73参与DNA损伤引起的凋亡,可能是一种肿瘤抑制蛋白。我们的western-blot实验及免疫组化实验表明2例ADPC组织中有微弱p73表达,而AIPC组织中没有p73表达。
我们进一步检测了p73表达变化对雄激素撤除培养环境下前列腺癌细胞增殖的影响,结果表明过表达p73既可以抑制含雄激素环境培养的LnCap细胞增殖P=0.004, P=0.005, P=0.024, P=0.016,也可以抑制雄激素撤除环境培养的PC-3-Ad细胞增殖,P-0.026, P=0.026, P=0.004, P=0.006;但是抑制p73表达并不影响这两种细胞系的增殖。这可能是由于这两种细胞增殖速度都很快,因此难以进
步增加其增殖速度。在撤除雄激素培养环境中,p73过表达对LnCap-Ad细胞增殖没有影响,P=0.814, P=0.348, P=0.083, P=0.162;但是p73抑制表达可以促进该细胞增殖,P=0.023,P=0.003。这可能是由于在雄激素撤除培养环境中p73的高表达和LnCap-Ad细胞的低增殖率。在撤除雄激素培养环境中,p73表达抑制对PC-3-Ad细胞增殖没有影响,而p73过表达能够抑制该细胞增殖。这可能是由于在雄激素撤除环境中PC-3-Ad细胞p73的低表达和细胞的高增殖率。因此,p73的低表达可能在AIPC增殖中发挥重要作用。
我们的结果还显示：p73的在前列腺癌细胞中表达与miR-200b-3p呈正相关。我们试图阐明二者在AIPC中的调控关系。我们采用转染pcDNA3/HA-TP73真核表达载体的策略使PC3-Ad细胞过表达p73,发现：miR-200b-3p表达随之增高,t值分别为=-14.322,t=-34.773,P值均<0.001。而当采用RNAi技术瞬时抑制p73在LnCap-Ad细胞中表达时,miR-200b-3p表达也随之降低,t值分别为=10.852,t=10.767,P值均<0.001。miR-200b-3p表达受到p73调控也得到了Knouf等在其它肿瘤研究中的支持。
由于miRNA的功能研究很大程度上与其所调控的靶基因有关,鉴定miRNA的靶基因是该领域研究的重要内容。
综合各种策略优缺点,本课题采用蛋白质组学筛选结合生物信息学预测的策略,鉴定miR-200b-3p的靶基因及表达受其调控的蛋白质。经过蛋白质组分析,筛选出16个表达量具有显著差异的蛋白质斑点。经过质谱鉴定以及生物信息学分析后得到蛋白14个。其中miR-200b-3p过表达组上调的蛋白包括Stathmin (STMN1), POTE ankyrin domain family member E (POTEE), Inosine triphosphate pyrophosphatase (ITPA), UV excision repair protein RAD23homolog B (RAD23B), Importin subunit alpha-4(KPNA4);下调的蛋白包括：Thioredoxin (TXN),Cytochrome c oxidase subunit5A, mitochondrial (COX5A), Peroxiredoxin-2(PRDX2), Protein Drl (DR1), polypeptide-associated complex subunit alpha (NACA), Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA), Elongation factor1-delta (EEFID), Nucleophosmin (NPM1), Vimentin (VIM)。
生物信息学(Targetscan, http://www.targetscan.org/)的分析表明：Peroxiredoxin-2可能是miRNA-200b-3p的靶蛋白。为了对双向电泳的结果进行验证,除了Peroxiredoxin-2,我们又随机选择了Drl进行Q-PCR和western-blot检测。结果显示相对于对照组细胞,这两种蛋白在miRNA-200b-3p过表达的PC3细胞中表达显著下调,t值分别为=-16.000, t=-13.856, P=0004, P=0005。这与双向电泳的结果一致。
Peroxiredoxin-2在肿瘤发生发展中发挥双重作用：一方面,癌变前期,体内存在衰老细胞,有可能转化为癌细胞,Prdxs家族成员通过清除活性氧,能抑制细胞衰老,降低癌变的可能。另一方面,一旦肿瘤细胞已经生成,则对肿瘤细胞具有保护作用。结合本课题双向电泳筛选结果和Prdx2保护肿瘤细胞作用,我们推测：是miR-200b-3p抑制Prdx2表达,从而抑制细胞清除活性氧的能力,导致细胞衰老甚至死亡这可能是miR-200b-3p过表达抑制AIPC细胞增殖的可能机制之一。
为了全面探讨miR-200b-3p抑制AIPC细胞增殖的作用机制。我们对蛋白质组学筛选得到的蛋白质进行了相互作用分析(STRING, http://string-db.org/)。结果给了我们重要的提示：在miR-200b-3p过表达的PC-3细胞中随之发生表达变化的蛋白质中,11种蛋白包括PRDX2、TNX、VIM、RAD23B、POTEE、 SGTA、STMN1、NACA、NPM1、KPNA4都通过泛素(Ubiquitin)联系起来,形成蛋白质相互作用网络。
泛素是一个由76个氨基酸组成的高度保守的多肽链。它存在于所有真核细胞中,以游离形式存在或与细胞膜、胞浆及核内的各种蛋白共价结合而存在,是泛素——蛋白酶体系统(ubiquitin proteasome system, UPS)的核心蛋白。正常细胞蛋白质代谢是一个持续降解和再合成的动态过程。UPS是细胞内ATP依赖的蛋白质选择性降解的主要途径,是真核细胞内重要的蛋白质质控系统,主要降解细胞内80%--90%泛素化的蛋白质,并调节炎症、细胞增生与分化、信号转导、细胞周期进程、转录调控、抗原提呈、免疫应答、细胞凋亡和DNA修复等各种细胞生物学功能。泛素分子在一系列酶作用下,对靶蛋白进行特异性修饰的过程称为泛素化。泛素化主要的作用就是使其靶蛋白降解。
上述与泛素存在直接相互作用的差异蛋白中,TNX、VIM、EEF1D、SGTA、 NACA、NPM1在miR-200b-3p过表达后是下调的；而RAD23B、POTEE、STMN1、 KPNA4是上调的。
下调的蛋白中,大多数(TNX、VIM、EEF1D、SGTA、NPM1)都具有促进肿瘤增殖的作用,与肿瘤发生发展呈正相关。Thioredoxin与Prdx相似,在肿瘤细胞中的功能随着肿瘤发展阶段的不同而不同。在早期阶段,Trx可以抵抗由多种致癌物引起的氧化应激,从而在一定程度上阻止肿瘤的发展。而一旦细胞发生恶性变异,高浓度的Trx则表现为促生长及抗凋亡功能,加速肿瘤细胞进展。在肿瘤发生的后期阶段,Trx则可能与肿瘤血管形成和转移扩散相关。vimentin调节细胞骨架蛋白、细胞粘附分子等蛋白间的相互作用,参与肿瘤细胞和肿瘤相关内皮细胞、巨噬细胞的粘附、迁移、侵袭和细胞信号转导。Elongation factor-1(EF-1) delta也被认为是一种癌基因。Alpha-SGT可以作为AR的分子伴侣,调控AR信号通路,从而影响肿瘤进展。nucleophosmin是一种多功能的蛋白质,参与核糖体的生物合成,控制中心体复制,具有分子伴侣作用,并可通过多种信号通路调节细胞增殖和凋亡,参与多种肿瘤的发生,它在多种肿瘤中过表达,有人提出它可作为胃、结肠、卵巢和前列腺肿瘤的标志物。
miR-200b-3p过表达后上调而与泛素存在直接相互作用的差异蛋白中,RAD23B直接参与泛素-蛋白酶体蛋白降解过程的调控。
因此我们推论：miR-200b-3p除了抑制其靶基因PRDX2编码的Peroxiredoxin-2蛋白抑制AIPC细胞增殖外,还可能通过激活泛素-蛋白酶体降解蛋白通路,降解其底物TNX、VIM、EEF1D、SGTA、NPM1等肿瘤发生发展相关蛋白,使其功能抑制,从而达到抑制蛋白增殖的目的。但是在这个过程中泛素活性为何增强了呢?我们又利用生物信息学分析发现,miR-200b-3p的潜在靶基因中,有5个泛素特异性蛋白酶(ubiquitin specific peptidase, USP),包括：ubiquitin specific peptidase27, X-linked; ubiquitin specific peptidase25; ubiquitin specific peptidase31; ubiquitin specific peptidase46; ubiquitin specific peptidase47。而USP的主要作用是抑制泛素化过程。
因此,我们提出miR-200b-3p过表达后其下游事件可能是：miR-200b-3p抑制其靶基因Peroxiredoxin-2和一些USP; Peroxiredoxin-2表达下调后其保护肿瘤细胞作用减弱,细胞增殖受到抑制；USP表达受到抑制后,其负调控泛素化的作用减弱,同时一些促进泛素化的因素出现(包括RAD23B的表达上调),导致泛素选择性降解一些能够促进肿瘤发生发展的蛋白质降解,细胞增殖受到抑制。
综合本课题研究结果,我们得出如下结论：
1. AIPC细胞中miR-200b-3p受到p73调控,过表达p73可以增加miR-200b-3p表达。
2. miR-200b-3p表达增高抑制AIPC的Peroxiredoxin-2表达,抑制细胞增殖。
3. miR-200b-3p表达增高抑制一些USP (ubiquitin specific peptidase27, X-linked; ubiquitin specific peptidase25; ubiquitin specific peptidase31; ubiquitin specific peptidase46; ubiquitin specific peptidase47)表达。
4. miR-200b-3p表达增高使UV excision repair protein RAD23homolog B蛋白表达增高。
5.USP表达下调以及UV excision repair protein RAD23homolog B上调,选择性地使一些具有促进肿瘤发生发展作用的蛋白质(TNX、VIM、EEF1D、SGTA. NPM1)泛素化,使它们降解,从而抑制细胞增殖。
综上所述,我们认为：在AIPC中,miR-200b-3p的低表达使降解TNX、VIM、 EEF1D、SGTA、NPM1等促癌基因的泛素化通路受到抑制,因此这些促癌基因高表达,这可能是AIPC增殖的原因之一。
Prostate cancer is one of the common malignant tumors of the male genitourinary system and accounts for the second most common cause of death in males. Prostate cancer cell growth is dependent on the presence of androgens. Androgen hormone-blocking treatments (castration therapy) of prostate cancer, can inhibit tumor growth, and improve disease-free survival for the majority of patients by1.5to3years. However, the tumors tend to relapse following this disease free period with androgen independent proliferation ability. This kind of prostate cancer is named as androgen-independent prostate cancer (AIPC) or castration resistant prostate cancer (CRPC). For these AIPC cases, the current treatment is limited and the prognosis is poor.
Over the years, the research on AIPC mechanisms focuses on the androgen receptor (androgen receptor, AR). However, the mechanism of AIPC development remains unclear. Such mechanisms are further complicated with the recent discovery of microRNA (miRNA), a class of non-coding RNAs that regulate gene expression at the post-transcriptional level.
Accumulating evidence indicates that microRNAs play critical roles in multiple biological processes, including cell cycle control, cell growth and differentiation, apoptosis, and embryological development. Some miRNAs involved in the occurrence of AIPC have been reported. White et al., found a small part of miRNAs abnormally expressed in AIPC cell lines. Sung et al., found a significantly high expression of miR-221/222in AIPC cell lines relative to the ADPC cell lines, suggesting its role in the occurrence and development of AIPC. Ma et al., found that miR-616is overexpressed in malignant prostate tissue. Over expression of miR-616in LNCaP cells (androgen dependent prostate cancer, ADPC) in vitro, can promote cell proliferation in a non-androgen environment.
Studies have shown that one miRNA regulates more than one target gene, and one gene can be regulated by more than one miRNAs. Thus, there remain many questions. Are there any other miRNAs invoved in AIPC regulation?
What function of these miRNAs in AIPC progress?
How these miRNAs regulate AIPC progress?
Can these miRNAs be candidate treatment target of AIPC?
In order to reveal the role of miRNAs played in AIPC, We conducted the following experiments.
First, the dependce of prostate cancer cell lines LnCap and PC3was verified.
Second, the differential miRNAs and cancer related genes were screened by Quantitative real-time PCR (Q-PCR).
Third, the correlation between miRNAs and genes was analysed by literature search and bioinformatics method.
Fourth, the expression level of differential miRNA and gene was verified. The expression of interested miRNA regulated by the specific gene and the role of this regulation played in AIPC proliferation were invested.
Fifth, the proteins regulated by the interested miRNA were identified by proteomics method, and the molecular mechanism of the proliferation regulation by interested miRNA was explored.
We compared several relevant miRNAs and cancer related genes between the ADPC cell line (LnCap) and the AIPC cell line (PC3) using Q-PCR and western-blot, and found p73and miR-200b-3p were co-downregulated in the PC3cell line. The present study will investigate the regulation between miR-200b and p73, and whose role in the androgen-independence of prostate cancer cells.
We first screened several miRNAs by Q-PCR. Among the differential microRNAs, miR-200b-3p was significantly repressed in PC3-Ad. This is consistent with the findings of Xu et al. Several studies have shown the function of miR-200b in cancer. Kong et al. reported that miR-200b regulates PDGF-D-mediated epithelial-mesenchymal transition, adhesion, and invasion of prostate cancer cells. However, the function of miR-200b-3p in AIPC is riot clear. Our results indicated that down-regulation of miR-200b-3p increased the proliferation of ADPC cells cultured with androgen-free medium, while up-regulation of miR-200b-3p decreased the proliferation of AIPC cells. Thus, low expression of miR-200b-3p may contribute to the androgen-independent growth of prostate cancer.
We detected several proteins which correlated with cancer, and found p73expression showing a positive correlation to miR-200b-3p. p73participates in the apoptotic response to DNA damage, and may be a tumor suppressor protein. There is also some evidence indicating p73expression decreases in AIPC. We further detected the effect of p73expression changes on the proliferation of prostate cance cells cultured with androgen-free medium. Our results showed that inhibition of p73expression promoted prostate cancer cell proliferation without androgen, while over-expression of p73inhibited prostate cancer cell proliferation without androgen. Thus, p73inhibited androgen-independent prostate cancer growth.
Our results demonstrated that the expression and function of p73strongly correlated to miR-200b-3p. We wanted to clarify the relationship between p73and miR-200b-3p in AIPC. p73was over-expressed in the PC3-Ad cell subline, while miR-200b-3p expression increased accordingly. While p73was inhibited in LnCap-Ad cell subline, miR-200b-3p expression also decreased significantly. The regulation of miR-200b-3p by p73is also supported by Emily C et al. for other cancers. They have proved that p73directly regulates miR-200transcription by chromatin immunoprecipitations method.
The regulation of p73by miR-200b-3p in AIPC cells was also explored because the bioinformatic resource TargetScan (http://www.targetscan.org/) predicted TP73may be a potential miR-200b-3p target. Our results indicated that neither over-expression nor inhibition of miR-200b-3p affected the expression of p73in prostate cancer cell lines with androgen-free culture medium.
Next, we identified the proteins regulated by miR-200b-3p by proteomics. The result showed14differential expressed proteins. Among the proteins,5up regulated in miR-200b-3p over-expressed PC3cells and9down regulated. The former includes: Stathmin (STMN1), POTE ankyrin domain family member E (POTEE), Inosine triphosphate pyrophosphatase (ITPA), UV excision repair protein RAD23homolog B (RAD23B), Importin subunit alpha-4(KPNA4). The latter includes:Thioredoxin (TXN), Cytochrome c oxidase subunit5A, mitochondrial (COX5A), Peroxiredoxin-2(PRDX2), Protein Drl (DR1), polypeptide-associated complex subunit alpha (NACA), Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA), Elongation factor1-delta (EEF1D), Nucleophosmin (NPM1), Vimentin (VIM).
PRDX2was identified as a target gene of miR-200b-3p by TargetScan, http://www.targetscan.org/. The expression of Peroxiredoxin-2and another protein Drl in the cells miR-200b-3p over-expressed or not were verified by Q-PCR and western-blot. The results were accordance with which of proteomics.
Peroxiredoxin-2plays bifunction in cancer cells. This protein may have a proliferative effect and play a role in cancer development or progression. But this protein can prevent cancer cell transformation from normal cell. Thus, we speculated that peroxiredoxin-2inhibited by miR-200b-3p, is one of the mechanism of miR-200b-3p's anti-cancer function.
The proteins interaction among the proteins identified by proteomics was analysed by informatics STRING (http://string-db.org/). The result indicated that most proteins down-regulated in the PC3cells with miR-200b-3p over-expression were connected by ubiquitin, including PRDX2、TNX、VIM、EEF1D、SGTA、NACA、 NPM1. Almost al of these proteins directly or indirectly can promote the cell proliferation. Furthermore, we found some ubiquitin-specific proteases (USPs) may be the target gene of miR-200b-3p by Targetscan. Thus, we deduced that miR-200b-3p can inhibit the expression of USPs, then ubiquitin pathway increased, many proteins including Thioredoxin, Vimentin, Elongation factor1-delta, Small glutamine-rich tetratricopeptide repeat-containing protein alpha, Nucleophosmin were degraded by ubiquitin, the AIPC cells proliferation was inhibited. In sum up, we concluded:
1. miR-200b-3p were regulated by p73in AIPC. miR-200b-3p was increased by p73over-expression.
2. miR-200b-3p can inhibit the expression of Peroxiredoxin-2, then inhibited the proliferation of AIPC.
3. miR-200b-3p can inhibit the expression of some USPs including ubiquitin specific peptidase27, X-linked; ubiquitin specific peptidase25; ubiquitin specific
peptidase31; ubiquitin specific peptidase46; ubiquitin specific peptidase47.
4. miR-200b-3p can increase the expression of UV excision repair protein RAD23homolog B.
5. The USPs inhibition and RAD23homolog B elevation increased the ubiquitination of some proteins including Thioredoxin, Vimentin, Elongation factor1-delta, Small glutamine-rich tetratricopeptide repeat-containing protein alpha, Nucleophosmin, then the proliferation of AIPC cells wes inhibited.
前列腺癌细胞的生长依赖于雄激素的存在。雄激素阻断疗法(去势疗法)治疗前列腺癌,可以抑制肿瘤,且绝大多数患者大约可无病生存1.5-3年,但此后,肿瘤往往复发,获得雄激素非依赖性增殖的能力。通常,临床上将雄激素完全阻断后仍有进展的前列腺癌称为雄激素非依赖性前列腺癌(androgen-independent prostate cancer, AIPC)或雄激素抵抗性的前列腺癌(castration-resistant prostate cancer, CRPC)。目前对于AIPC的定义一般认为应同时具备以下几点：血清睾酮达去势水平;抗雄激素撤退治疗4周以上；间隔2周,连续3次前列腺特异性抗原升高；二线内分泌治疗期间前列腺特异性抗原继续进展；骨或软组织转移病变有进展。对于这些AIPC病例,当前的治疗手段有限,预后很差。为这些AIPC患者找出新的治疗方案有重要的社会效益,而深入揭示AIPC的发生机制,是预防和治疗AIPC的关键。
AIPC发生发展的机制并不清楚。长期以来,对前列腺癌雄激素非依赖性演进发生机制的研究主要集中在雄激素受体(androgen receptor, AR)。认为其可能机制主要包括：(1)AR过表达或扩增；(2)AR突变；(3)前列腺细胞雄激素局部产物增加；(4)生长因子/细胞因子对AR信号通路的活化；(5)辅助活化因子(共激活子/共抑制子)表达异常；(6)AR蛋白水解为雄激素非依赖型；(7)基质细胞AR表达丢失。但是AR自身的表达调控及AR下游的信号机制研究并不清楚。
近年,随着前列腺癌的发病率增加,以及新技术的应用,人们发现microRNA (miRNA)对其靶基因表达的调控,在AIPC的发生发展中起到重要的调控作用。对miRNAs在AIPC发生发展中的作用进行深入探讨,有助于全面理解AIPC的发生机制,为AIPC提供良好的诊断和治疗靶标。
miRNA参与AIPC的发生,也有相关报道。Sung等发现几种：miRNA,尤其miR-221和miR-222,在AIDC细胞株中相对于雄激素依赖性(androgen-dependent prostate cancer, ADPC)细胞株明显高表达,提示miRNA在AIPC的发生发展中起作用。White等发现在AIPC细胞株中有一小部分miRNA异常表达。在恶性前列腺组织中存在miR-616过表达。在体外,LNCaP细胞过度表达,miR-616可以促进AI细胞增殖。更重要的是,体内miR-616过表达的LNCaP (ADPC)细胞能够克服去势抵抗,即双侧睾丸切除术后仍可提高增殖能力。miR-616抑制剂用于AI细胞可以产生相反作用。另外还证实miR-616靶基因是TF通道抑制蛋白TFPI-2mRNA。在AIPC发展中,miR-616过表达发挥重要作用。
信息生物学预测及实验研究表明,一个miRNA可调控多个靶基因；这些靶基因并不是随机分布的,而是富集在同一个或某几个信号通路。这提示一个miRNA可同时调控同一信号通路中上下游靶蛋白的表达,或通过调控多个信号通路,从而对基因调控网络实施更有效的调节,最终对细胞功能和表型进行更有力的调控。
因此,必然还有其它的miRNAs参与AIPC的发生,并且,这些miRNAs影响前列腺癌细胞对雄激素依赖性的分子机制研究尚处于起步阶段。
由此可见,虽然已发现一些miRNA在AIPC发生发展中发挥很重要的作用,但尚有许多问题仍不清楚：如是否还有其它的：miRNA分子参与了AIPC发生发展过程?这些miRNA表达水平的变化与AIPC发生发展之间有何关系?这些miRNA调控AIPC的分子机制是什么?它们是否可以作为AIPC的治疗靶标?对这些问题的深入研究必将促进我们对AIPC发生发展机制的深入认识,为AIPC的临床治疗提供新的理论依据和新的miRNA靶点。
基于以上分析,本课题主要进行以下研究：
1、验证前列腺癌细胞系LnCap、PC3对雄激素的依赖性。
2、Q-PCR方法筛选LnCap、PC3细胞间差异表达的miRNA和肿瘤相关基因。
3、生物信息学分析功能相关的差异miRNA和基因。
4、验证3中miRNA和基因表达改变、二者调控关系及其对AIPC增殖的调控作用。
5、采用蛋白质组学鉴定AIPC中受上述3、4中关键miRNA调控的蛋白,探讨该miRNA调控AIPC细胞增殖的分子机制。
我们首先用Q-PCR方法筛选了一些niRNA和肿瘤相关基因在PC-3细胞和LnCap细胞中的表达差异。在差异表达的miRNA中miR-200b-3p(t=23.575,P=0.002)在PC-3中表达显著较低。这与Xu等先前的报道相一致。然而,miR-200b-3p在AIPC中的作用并不清楚。我们的结果表明：无论过表达或抑制表达miR-200b-3p,都不会影响LnCap细胞在含雄激素培养环境中的增殖,F值=1.088,P=0.375。撤除雄激素后,过表达miR-200b-3p不会影响LnCap-Ad的增殖,P=0.814, P=0.348, P=0.083, P=0.162;但是抑制miR-200b-3p表达则会促进LnCap-Ad增殖,P=0.023,P=0.003。
我们还检测了一些肿瘤相关基因的表达,发现TP73(编码蛋白p73)表达(t=127.000,P<0.001)变化趋势与miR-200b-3p一致。p73参与DNA损伤引起的凋亡,可能是一种肿瘤抑制蛋白。我们的western-blot实验及免疫组化实验表明2例ADPC组织中有微弱p73表达,而AIPC组织中没有p73表达。
我们进一步检测了p73表达变化对雄激素撤除培养环境下前列腺癌细胞增殖的影响,结果表明过表达p73既可以抑制含雄激素环境培养的LnCap细胞增殖P=0.004, P=0.005, P=0.024, P=0.016,也可以抑制雄激素撤除环境培养的PC-3-Ad细胞增殖,P-0.026, P=0.026, P=0.004, P=0.006;但是抑制p73表达并不影响这两种细胞系的增殖。这可能是由于这两种细胞增殖速度都很快,因此难以进
步增加其增殖速度。在撤除雄激素培养环境中,p73过表达对LnCap-Ad细胞增殖没有影响,P=0.814, P=0.348, P=0.083, P=0.162;但是p73抑制表达可以促进该细胞增殖,P=0.023,P=0.003。这可能是由于在雄激素撤除培养环境中p73的高表达和LnCap-Ad细胞的低增殖率。在撤除雄激素培养环境中,p73表达抑制对PC-3-Ad细胞增殖没有影响,而p73过表达能够抑制该细胞增殖。这可能是由于在雄激素撤除环境中PC-3-Ad细胞p73的低表达和细胞的高增殖率。因此,p73的低表达可能在AIPC增殖中发挥重要作用。
我们的结果还显示：p73的在前列腺癌细胞中表达与miR-200b-3p呈正相关。我们试图阐明二者在AIPC中的调控关系。我们采用转染pcDNA3/HA-TP73真核表达载体的策略使PC3-Ad细胞过表达p73,发现：miR-200b-3p表达随之增高,t值分别为=-14.322,t=-34.773,P值均<0.001。而当采用RNAi技术瞬时抑制p73在LnCap-Ad细胞中表达时,miR-200b-3p表达也随之降低,t值分别为=10.852,t=10.767,P值均<0.001。miR-200b-3p表达受到p73调控也得到了Knouf等在其它肿瘤研究中的支持。
由于miRNA的功能研究很大程度上与其所调控的靶基因有关,鉴定miRNA的靶基因是该领域研究的重要内容。
综合各种策略优缺点,本课题采用蛋白质组学筛选结合生物信息学预测的策略,鉴定miR-200b-3p的靶基因及表达受其调控的蛋白质。经过蛋白质组分析,筛选出16个表达量具有显著差异的蛋白质斑点。经过质谱鉴定以及生物信息学分析后得到蛋白14个。其中miR-200b-3p过表达组上调的蛋白包括Stathmin (STMN1), POTE ankyrin domain family member E (POTEE), Inosine triphosphate pyrophosphatase (ITPA), UV excision repair protein RAD23homolog B (RAD23B), Importin subunit alpha-4(KPNA4);下调的蛋白包括：Thioredoxin (TXN),Cytochrome c oxidase subunit5A, mitochondrial (COX5A), Peroxiredoxin-2(PRDX2), Protein Drl (DR1), polypeptide-associated complex subunit alpha (NACA), Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA), Elongation factor1-delta (EEFID), Nucleophosmin (NPM1), Vimentin (VIM)。
生物信息学(Targetscan, http://www.targetscan.org/)的分析表明：Peroxiredoxin-2可能是miRNA-200b-3p的靶蛋白。为了对双向电泳的结果进行验证,除了Peroxiredoxin-2,我们又随机选择了Drl进行Q-PCR和western-blot检测。结果显示相对于对照组细胞,这两种蛋白在miRNA-200b-3p过表达的PC3细胞中表达显著下调,t值分别为=-16.000, t=-13.856, P=0004, P=0005。这与双向电泳的结果一致。
Peroxiredoxin-2在肿瘤发生发展中发挥双重作用：一方面,癌变前期,体内存在衰老细胞,有可能转化为癌细胞,Prdxs家族成员通过清除活性氧,能抑制细胞衰老,降低癌变的可能。另一方面,一旦肿瘤细胞已经生成,则对肿瘤细胞具有保护作用。结合本课题双向电泳筛选结果和Prdx2保护肿瘤细胞作用,我们推测：是miR-200b-3p抑制Prdx2表达,从而抑制细胞清除活性氧的能力,导致细胞衰老甚至死亡这可能是miR-200b-3p过表达抑制AIPC细胞增殖的可能机制之一。
为了全面探讨miR-200b-3p抑制AIPC细胞增殖的作用机制。我们对蛋白质组学筛选得到的蛋白质进行了相互作用分析(STRING, http://string-db.org/)。结果给了我们重要的提示：在miR-200b-3p过表达的PC-3细胞中随之发生表达变化的蛋白质中,11种蛋白包括PRDX2、TNX、VIM、RAD23B、POTEE、 SGTA、STMN1、NACA、NPM1、KPNA4都通过泛素(Ubiquitin)联系起来,形成蛋白质相互作用网络。
泛素是一个由76个氨基酸组成的高度保守的多肽链。它存在于所有真核细胞中,以游离形式存在或与细胞膜、胞浆及核内的各种蛋白共价结合而存在,是泛素——蛋白酶体系统(ubiquitin proteasome system, UPS)的核心蛋白。正常细胞蛋白质代谢是一个持续降解和再合成的动态过程。UPS是细胞内ATP依赖的蛋白质选择性降解的主要途径,是真核细胞内重要的蛋白质质控系统,主要降解细胞内80%--90%泛素化的蛋白质,并调节炎症、细胞增生与分化、信号转导、细胞周期进程、转录调控、抗原提呈、免疫应答、细胞凋亡和DNA修复等各种细胞生物学功能。泛素分子在一系列酶作用下,对靶蛋白进行特异性修饰的过程称为泛素化。泛素化主要的作用就是使其靶蛋白降解。
上述与泛素存在直接相互作用的差异蛋白中,TNX、VIM、EEF1D、SGTA、 NACA、NPM1在miR-200b-3p过表达后是下调的；而RAD23B、POTEE、STMN1、 KPNA4是上调的。
下调的蛋白中,大多数(TNX、VIM、EEF1D、SGTA、NPM1)都具有促进肿瘤增殖的作用,与肿瘤发生发展呈正相关。Thioredoxin与Prdx相似,在肿瘤细胞中的功能随着肿瘤发展阶段的不同而不同。在早期阶段,Trx可以抵抗由多种致癌物引起的氧化应激,从而在一定程度上阻止肿瘤的发展。而一旦细胞发生恶性变异,高浓度的Trx则表现为促生长及抗凋亡功能,加速肿瘤细胞进展。在肿瘤发生的后期阶段,Trx则可能与肿瘤血管形成和转移扩散相关。vimentin调节细胞骨架蛋白、细胞粘附分子等蛋白间的相互作用,参与肿瘤细胞和肿瘤相关内皮细胞、巨噬细胞的粘附、迁移、侵袭和细胞信号转导。Elongation factor-1(EF-1) delta也被认为是一种癌基因。Alpha-SGT可以作为AR的分子伴侣,调控AR信号通路,从而影响肿瘤进展。nucleophosmin是一种多功能的蛋白质,参与核糖体的生物合成,控制中心体复制,具有分子伴侣作用,并可通过多种信号通路调节细胞增殖和凋亡,参与多种肿瘤的发生,它在多种肿瘤中过表达,有人提出它可作为胃、结肠、卵巢和前列腺肿瘤的标志物。
miR-200b-3p过表达后上调而与泛素存在直接相互作用的差异蛋白中,RAD23B直接参与泛素-蛋白酶体蛋白降解过程的调控。
因此我们推论：miR-200b-3p除了抑制其靶基因PRDX2编码的Peroxiredoxin-2蛋白抑制AIPC细胞增殖外,还可能通过激活泛素-蛋白酶体降解蛋白通路,降解其底物TNX、VIM、EEF1D、SGTA、NPM1等肿瘤发生发展相关蛋白,使其功能抑制,从而达到抑制蛋白增殖的目的。但是在这个过程中泛素活性为何增强了呢?我们又利用生物信息学分析发现,miR-200b-3p的潜在靶基因中,有5个泛素特异性蛋白酶(ubiquitin specific peptidase, USP),包括：ubiquitin specific peptidase27, X-linked; ubiquitin specific peptidase25; ubiquitin specific peptidase31; ubiquitin specific peptidase46; ubiquitin specific peptidase47。而USP的主要作用是抑制泛素化过程。
因此,我们提出miR-200b-3p过表达后其下游事件可能是：miR-200b-3p抑制其靶基因Peroxiredoxin-2和一些USP; Peroxiredoxin-2表达下调后其保护肿瘤细胞作用减弱,细胞增殖受到抑制；USP表达受到抑制后,其负调控泛素化的作用减弱,同时一些促进泛素化的因素出现(包括RAD23B的表达上调),导致泛素选择性降解一些能够促进肿瘤发生发展的蛋白质降解,细胞增殖受到抑制。
综合本课题研究结果,我们得出如下结论：
1. AIPC细胞中miR-200b-3p受到p73调控,过表达p73可以增加miR-200b-3p表达。
2. miR-200b-3p表达增高抑制AIPC的Peroxiredoxin-2表达,抑制细胞增殖。
3. miR-200b-3p表达增高抑制一些USP (ubiquitin specific peptidase27, X-linked; ubiquitin specific peptidase25; ubiquitin specific peptidase31; ubiquitin specific peptidase46; ubiquitin specific peptidase47)表达。
4. miR-200b-3p表达增高使UV excision repair protein RAD23homolog B蛋白表达增高。
5.USP表达下调以及UV excision repair protein RAD23homolog B上调,选择性地使一些具有促进肿瘤发生发展作用的蛋白质(TNX、VIM、EEF1D、SGTA. NPM1)泛素化,使它们降解,从而抑制细胞增殖。
综上所述,我们认为：在AIPC中,miR-200b-3p的低表达使降解TNX、VIM、 EEF1D、SGTA、NPM1等促癌基因的泛素化通路受到抑制,因此这些促癌基因高表达,这可能是AIPC增殖的原因之一。
Prostate cancer is one of the common malignant tumors of the male genitourinary system and accounts for the second most common cause of death in males. Prostate cancer cell growth is dependent on the presence of androgens. Androgen hormone-blocking treatments (castration therapy) of prostate cancer, can inhibit tumor growth, and improve disease-free survival for the majority of patients by1.5to3years. However, the tumors tend to relapse following this disease free period with androgen independent proliferation ability. This kind of prostate cancer is named as androgen-independent prostate cancer (AIPC) or castration resistant prostate cancer (CRPC). For these AIPC cases, the current treatment is limited and the prognosis is poor.
Over the years, the research on AIPC mechanisms focuses on the androgen receptor (androgen receptor, AR). However, the mechanism of AIPC development remains unclear. Such mechanisms are further complicated with the recent discovery of microRNA (miRNA), a class of non-coding RNAs that regulate gene expression at the post-transcriptional level.
Accumulating evidence indicates that microRNAs play critical roles in multiple biological processes, including cell cycle control, cell growth and differentiation, apoptosis, and embryological development. Some miRNAs involved in the occurrence of AIPC have been reported. White et al., found a small part of miRNAs abnormally expressed in AIPC cell lines. Sung et al., found a significantly high expression of miR-221/222in AIPC cell lines relative to the ADPC cell lines, suggesting its role in the occurrence and development of AIPC. Ma et al., found that miR-616is overexpressed in malignant prostate tissue. Over expression of miR-616in LNCaP cells (androgen dependent prostate cancer, ADPC) in vitro, can promote cell proliferation in a non-androgen environment.
Studies have shown that one miRNA regulates more than one target gene, and one gene can be regulated by more than one miRNAs. Thus, there remain many questions. Are there any other miRNAs invoved in AIPC regulation?
What function of these miRNAs in AIPC progress?
How these miRNAs regulate AIPC progress?
Can these miRNAs be candidate treatment target of AIPC?
In order to reveal the role of miRNAs played in AIPC, We conducted the following experiments.
First, the dependce of prostate cancer cell lines LnCap and PC3was verified.
Second, the differential miRNAs and cancer related genes were screened by Quantitative real-time PCR (Q-PCR).
Third, the correlation between miRNAs and genes was analysed by literature search and bioinformatics method.
Fourth, the expression level of differential miRNA and gene was verified. The expression of interested miRNA regulated by the specific gene and the role of this regulation played in AIPC proliferation were invested.
Fifth, the proteins regulated by the interested miRNA were identified by proteomics method, and the molecular mechanism of the proliferation regulation by interested miRNA was explored.
We compared several relevant miRNAs and cancer related genes between the ADPC cell line (LnCap) and the AIPC cell line (PC3) using Q-PCR and western-blot, and found p73and miR-200b-3p were co-downregulated in the PC3cell line. The present study will investigate the regulation between miR-200b and p73, and whose role in the androgen-independence of prostate cancer cells.
We first screened several miRNAs by Q-PCR. Among the differential microRNAs, miR-200b-3p was significantly repressed in PC3-Ad. This is consistent with the findings of Xu et al. Several studies have shown the function of miR-200b in cancer. Kong et al. reported that miR-200b regulates PDGF-D-mediated epithelial-mesenchymal transition, adhesion, and invasion of prostate cancer cells. However, the function of miR-200b-3p in AIPC is riot clear. Our results indicated that down-regulation of miR-200b-3p increased the proliferation of ADPC cells cultured with androgen-free medium, while up-regulation of miR-200b-3p decreased the proliferation of AIPC cells. Thus, low expression of miR-200b-3p may contribute to the androgen-independent growth of prostate cancer.
We detected several proteins which correlated with cancer, and found p73expression showing a positive correlation to miR-200b-3p. p73participates in the apoptotic response to DNA damage, and may be a tumor suppressor protein. There is also some evidence indicating p73expression decreases in AIPC. We further detected the effect of p73expression changes on the proliferation of prostate cance cells cultured with androgen-free medium. Our results showed that inhibition of p73expression promoted prostate cancer cell proliferation without androgen, while over-expression of p73inhibited prostate cancer cell proliferation without androgen. Thus, p73inhibited androgen-independent prostate cancer growth.
Our results demonstrated that the expression and function of p73strongly correlated to miR-200b-3p. We wanted to clarify the relationship between p73and miR-200b-3p in AIPC. p73was over-expressed in the PC3-Ad cell subline, while miR-200b-3p expression increased accordingly. While p73was inhibited in LnCap-Ad cell subline, miR-200b-3p expression also decreased significantly. The regulation of miR-200b-3p by p73is also supported by Emily C et al. for other cancers. They have proved that p73directly regulates miR-200transcription by chromatin immunoprecipitations method.
The regulation of p73by miR-200b-3p in AIPC cells was also explored because the bioinformatic resource TargetScan (http://www.targetscan.org/) predicted TP73may be a potential miR-200b-3p target. Our results indicated that neither over-expression nor inhibition of miR-200b-3p affected the expression of p73in prostate cancer cell lines with androgen-free culture medium.
Next, we identified the proteins regulated by miR-200b-3p by proteomics. The result showed14differential expressed proteins. Among the proteins,5up regulated in miR-200b-3p over-expressed PC3cells and9down regulated. The former includes: Stathmin (STMN1), POTE ankyrin domain family member E (POTEE), Inosine triphosphate pyrophosphatase (ITPA), UV excision repair protein RAD23homolog B (RAD23B), Importin subunit alpha-4(KPNA4). The latter includes:Thioredoxin (TXN), Cytochrome c oxidase subunit5A, mitochondrial (COX5A), Peroxiredoxin-2(PRDX2), Protein Drl (DR1), polypeptide-associated complex subunit alpha (NACA), Small glutamine-rich tetratricopeptide repeat-containing protein alpha (SGTA), Elongation factor1-delta (EEF1D), Nucleophosmin (NPM1), Vimentin (VIM).
PRDX2was identified as a target gene of miR-200b-3p by TargetScan, http://www.targetscan.org/. The expression of Peroxiredoxin-2and another protein Drl in the cells miR-200b-3p over-expressed or not were verified by Q-PCR and western-blot. The results were accordance with which of proteomics.
Peroxiredoxin-2plays bifunction in cancer cells. This protein may have a proliferative effect and play a role in cancer development or progression. But this protein can prevent cancer cell transformation from normal cell. Thus, we speculated that peroxiredoxin-2inhibited by miR-200b-3p, is one of the mechanism of miR-200b-3p's anti-cancer function.
The proteins interaction among the proteins identified by proteomics was analysed by informatics STRING (http://string-db.org/). The result indicated that most proteins down-regulated in the PC3cells with miR-200b-3p over-expression were connected by ubiquitin, including PRDX2、TNX、VIM、EEF1D、SGTA、NACA、 NPM1. Almost al of these proteins directly or indirectly can promote the cell proliferation. Furthermore, we found some ubiquitin-specific proteases (USPs) may be the target gene of miR-200b-3p by Targetscan. Thus, we deduced that miR-200b-3p can inhibit the expression of USPs, then ubiquitin pathway increased, many proteins including Thioredoxin, Vimentin, Elongation factor1-delta, Small glutamine-rich tetratricopeptide repeat-containing protein alpha, Nucleophosmin were degraded by ubiquitin, the AIPC cells proliferation was inhibited. In sum up, we concluded:
1. miR-200b-3p were regulated by p73in AIPC. miR-200b-3p was increased by p73over-expression.
2. miR-200b-3p can inhibit the expression of Peroxiredoxin-2, then inhibited the proliferation of AIPC.
3. miR-200b-3p can inhibit the expression of some USPs including ubiquitin specific peptidase27, X-linked; ubiquitin specific peptidase25; ubiquitin specific
peptidase31; ubiquitin specific peptidase46; ubiquitin specific peptidase47.
4. miR-200b-3p can increase the expression of UV excision repair protein RAD23homolog B.
5. The USPs inhibition and RAD23homolog B elevation increased the ubiquitination of some proteins including Thioredoxin, Vimentin, Elongation factor1-delta, Small glutamine-rich tetratricopeptide repeat-containing protein alpha, Nucleophosmin, then the proliferation of AIPC cells wes inhibited.
引文
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[12]T Sung, Wang Q-B, S Balk. The role of microRNA-221 and microRNA-222 in Androgen-Independent prostate cancer cell lines[J]. Adv Cancer Res.2009, 69(8):3356-3363.
[13]RW White, RL Vinall, CG Tepper. Micrornas and their potential for translation in prostate cancertheir potential for translation in prostate cancer[J]. Urol Oncol. 2009,27(3):307-311.
[14]Ma S, YP Chan, PS Kwan. MicroRNA-616 induces androgen-independent growth of prostate cancer cells by suppressing expression of tissue factor pathway inhibitor TFPI-2[J], Adv Cancer Res.2011,71(2):583-592.
[15]M Inui, G Martello, S Piccolo. Microrna control of signal transduction[J]. Nat Rev Mol Cell Biol.2010,11(4):252-263.
[16]Chua JH, Armugam A, Jeyaseelan K. MicroRNAs:biogenesis, function and applications^. Curr Opin Mol Ther.2009,11:189-199.
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[21]Henry JY, Lu L, Adams M, Meyer B, Bartlett JB, Dalgleish AG, Galustian C. Lenalidomide enhances the anti-prostate cancer activity of docetaxel in vitro and in vivo. Prostate 2012;72:856-867.
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[25]Liu Y, He M, Sun X, Peng K, Zhao L. Alteration of nuclear protein profiling for NIH-3T3 cells exposed to H2O2. Cell Biochem Funct.2010;28(7):578-584.
[26]Xu G, Wu J, Zhou L, Chen B, Sun Z, Zhao F, Tao Z. Characterization of the small RNA transcriptomes of androgen dependent and independent prostate cancer cell line by deep sequencing. PLoS ONE 2010;5:1-8.
[27]Au SL, Wong CC, Lee JM, Fan DN, Tsang FH, Ng IO, Wong CM. Enhancer of zeste homolog 2 (EZH2) epigenetically silences multiple tumor suppressor miRNAs to promote liver cancer metastasis. Hepatology 2012;56:622-631.
[28]Kurashige J, Kamohara H, Watanabe M, Hiyoshi Y, Iwatsuki M, Tanaka Y, Kinoshita K, Saito S, Baba Y, Baba H. MiRNA-200b regulates cell proliferation, invasion, and migration by directly targeting ZEB2 in gastric carcinoma. Ann Surg Oncol 2012;19:S656-S664.
[29]Wu Y, Xiao Y, Ding X, Zhuo Y, Ren P, Zhou C, Zhou J. A miR-200b/200c/429-binding site polymorphism in the 30 untranslated region of the AP-2a gene is associated with cisplatin resistance. PLoS ONE 2011;6:e29043.
[30]Kong D, Li Y, Wang Z, Banerjee S, Ahmad A, Kim HR,Sarkar FH. miR-200 regulates PDGF-D-mediated epithelialmesenchymal transition, adhesion, and invasion of prostate cancer cells. Stem Cells 2009;27:1712-1721.
[31]Koida N, Ozaki T, Yamamoto H, Ono S, Koda T, Ando K,Okoshi R, Kamijo T, Omura K, Nakagawara A. Inhibitory role of Plkl in the regulation of p73-dependent apoptosis through physical interaction and phosphorylation. J Biol Chem 2008;283:8555-8563.
[32]Libertini SJ, Tepper CG, Guadalupe M, Lu Y, Asmuth DM, Mudryj M. E2F1 expression in LnCap prostate cancer cells deregulates androgen dependent growth, suppresses differentiation, and enhances apoptosis. Prostate 2006;66:70-81.
[33]Singh AP, Bafna S, Chaudhary K, Venkatraman G, Smith L, Eudy JD, Johansson SL, Lin MF, Batra SK. Genome-wide expression profiling reveals transcriptomic variation and perturbed gene networks in androgen-dependent and androgenindependent prostate cancer cells. Cancer Lett 2008;259:28-38.
[34]Knouf EC, Garg K, Arroyo JD, Correa Y, Sarkar D, Parkin RK, Wurz K, O'Briant KC, Godwin AK, Urban ND, Ruzzo WL, Gentleman R, Drescher CW, Swisher EM, Tewari M. An integrative genomic approach identifies p73 and p63 as activators of miR-200 miRNA family transcription. Nucleic Acids Res 2012;40:499-510.
[35]朱鹏,李建远.Prdxs蛋白家族与肿瘤.中外医学研究.2011.4(9)：122-124.
[36]曾跃平,刘洁坤,王宝玺.过氧化物酶Peroxiredoxin家族与肿瘤关系研究进展.癌症进展.2010.8(2)：164-166.
[37]王春玲,王佳,张剑梅,王明晓.泛素-蛋白酶体系统的研究进展.中国心血管病研究.2012.10(5)：394-398.
[38]吴俊兰,卢斌,肖菊香.硫氧还蛋白系统与肿瘤的关系.现代肿瘤医学.2012.20(11)：2429-2431.
[39]潘燕,韩婧,张晔,李学军.Vimentin在肿瘤转移中的作用及药物研究进展.生理科学进展.2012.41(6)：413-416.
[40]Ogawa K, Utsunomiya T, Mimori K, Tanaka Y, Tanaka F, Inoue H, Murayama S, Mori M. Clinical significance of elongation factor-1 delta mRNA expression in oesophageal carcinoma. Br J Cancer.2004.19;91(2):282-6.
[41]Dutta S, Tan YJ. Structural and functional characterization of human SGT and its interaction with Vpu of the human immunodeficiency virus type 1." Biochemistry.2008:47:10123-10131.
[42]Trotta AP, Need EF, Butler LM, Selth LA, O'Loughlin MA, Coetzee GA, Tilley WD, Buchanan G. Subdomain structure of the co-chaperone SGTA and activity of its androgen receptor client. J Mol Endocrinol.2012 Jul 25;49(2):57-68.
[43]Liu Y, Hu Y, Li X, Niu L, Teng M. The crystal structure of the human nascent polypeptide-associated complex domain reveals a nucleic acid-binding region on the NACA subunit.Biochemistry.2010;49(13):2890-2896.
[44]徐诚望,杨晓明.核磷蛋白与肿瘤的发生.军事医学科学院院刊. 2008.32(4)：382-385.
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[46]Li X., Demartino G.N. Variably modulated gating of the 26S proteasome by ATP and polyubiquitin. Biochem J.2009.421:397-404.
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[2]D Fioriti, M Mischitelli, F Monaco. Cancer stem cells inprostate adenocarcinoma:a target for new anticancer strategies[J]. Am J Physiol Cell Physiol.2008,216(3):571-575.
[3]ID Schnadig, TM Beer. Optimal timing of chemotherapy in androgen independent prostate cancer[J]. Urol Oncol.2009,27(1):97-100.
[4]黄昆,朱明.雄激素非依赖性前列腺癌的发生研究进展[J].医学综述.2011,17(3)：371-373.
[5]王潇然,王伟华.激素非依赖性前列腺癌分子机制进展[J].中国老年学杂志.2011,31(7)：1282-1284.
[6]KP Porkka, MJ Pfeiffer, KK Waltering. Microrna expression profiling in prostate cancer[J]. Adv Cancer Res.2007,67(4):6130-6135.
[7]Tong A-W, P Fulgham, C Jay. Microrna profile analysis of human prostate cancers[J]. Cancer Gene Ther.2009,16(6):206-216.
[8]TM Rana. Illuminating the silence:understanding the structure and function of small RNAS[J]. Nat Rev Mol Cell Biol.2007,8(1):23-36.
[9]Guo H, Ingolia NT, Weissman JS, Bartel DP. Mammalian microRNAs predominantly act to decrease target mRNA levels. Nature.2010.466(7308): 835-840.
[10]周珏宇,石嵘,郑文岭,马文丽.miRNA与肿瘤细胞周期的关系及其研究方法进展.基础医学与临床.2003.33(1)：9-14.
[11]夏伟,曹国军,邵宁生.MicroRNA靶基因的寻找及鉴定方法研究进展.中国科学C辑：生命科学.2009.39(1)：121-128.
[12]T Sung, Wang Q-B, S Balk. The role of microRNA-221 and microRNA-222 in Androgen-Independent prostate cancer cell lines[J]. Adv Cancer Res.2009, 69(8):3356-3363.
[13]RW White, RL Vinall, CG Tepper. Micrornas and their potential for translation in prostate cancertheir potential for translation in prostate cancer[J]. Urol Oncol. 2009,27(3):307-311.
[14]Ma S, YP Chan, PS Kwan. MicroRNA-616 induces androgen-independent growth of prostate cancer cells by suppressing expression of tissue factor pathway inhibitor TFPI-2[J], Adv Cancer Res.2011,71(2):583-592.
[15]M Inui, G Martello, S Piccolo. Microrna control of signal transduction[J]. Nat Rev Mol Cell Biol.2010,11(4):252-263.
[16]Chua JH, Armugam A, Jeyaseelan K. MicroRNAs:biogenesis, function and applications^. Curr Opin Mol Ther.2009,11:189-199.
[17]DR Rhodes, Yu J, K Shanker, et al. Oncomine:a cancer microarray database and integrated data-mining platform[J]. J Mammary Gland Biol Neoplasia, 2004,6(1):1-6.
[18]E Segal, N Friedman, D Koller, et al. A module map showing conditional activity of expression modules in cancer [J]. Am J Med Genet B Neuropsychiatr Genet,2004,36(10):1090-1098.
[19]A Subramanian, P Tamayo, VK Mootha, et al. Gene set enrichment analysis:a knowledge-based approach for interpreting genome-wide expression profiles[J]. Proc Natl Acad Sci USA,2005,102(43):11555-15545.
[20]T Paranjape, FJ Slack, JB Weidhaas. Micrornas:tools for cancer diagnostics[J]. Gut,2009,58(11):1546-1554.
[21]Henry JY, Lu L, Adams M, Meyer B, Bartlett JB, Dalgleish AG, Galustian C. Lenalidomide enhances the anti-prostate cancer activity of docetaxel in vitro and in vivo. Prostate 2012;72:856-867.
[22]Schmittgen TD, Livak KJ. Analyzing real-time PCR data by the comparative CT method. Nat Protoc.2008;3:1101-1108.
[23]Schmittgen TD, Livak KJ. (2008) Analyzing real-time PCR data by the comparative CT method. Nat Protoc 3:1101-1108.
[24]Livak KJ, Schmittgen TD. (2001) Analysis of relative gene expression data using realtime quantitative PCR and the 2-△△Ct method. Methods 25:402-408.
[25]Liu Y, He M, Sun X, Peng K, Zhao L. Alteration of nuclear protein profiling for NIH-3T3 cells exposed to H2O2. Cell Biochem Funct.2010;28(7):578-584.
[26]Xu G, Wu J, Zhou L, Chen B, Sun Z, Zhao F, Tao Z. Characterization of the small RNA transcriptomes of androgen dependent and independent prostate cancer cell line by deep sequencing. PLoS ONE 2010;5:1-8.
[27]Au SL, Wong CC, Lee JM, Fan DN, Tsang FH, Ng IO, Wong CM. Enhancer of zeste homolog 2 (EZH2) epigenetically silences multiple tumor suppressor miRNAs to promote liver cancer metastasis. Hepatology 2012;56:622-631.
[28]Kurashige J, Kamohara H, Watanabe M, Hiyoshi Y, Iwatsuki M, Tanaka Y, Kinoshita K, Saito S, Baba Y, Baba H. MiRNA-200b regulates cell proliferation, invasion, and migration by directly targeting ZEB2 in gastric carcinoma. Ann Surg Oncol 2012;19:S656-S664.
[29]Wu Y, Xiao Y, Ding X, Zhuo Y, Ren P, Zhou C, Zhou J. A miR-200b/200c/429-binding site polymorphism in the 30 untranslated region of the AP-2a gene is associated with cisplatin resistance. PLoS ONE 2011;6:e29043.
[30]Kong D, Li Y, Wang Z, Banerjee S, Ahmad A, Kim HR,Sarkar FH. miR-200 regulates PDGF-D-mediated epithelialmesenchymal transition, adhesion, and invasion of prostate cancer cells. Stem Cells 2009;27:1712-1721.
[31]Koida N, Ozaki T, Yamamoto H, Ono S, Koda T, Ando K,Okoshi R, Kamijo T, Omura K, Nakagawara A. Inhibitory role of Plkl in the regulation of p73-dependent apoptosis through physical interaction and phosphorylation. J Biol Chem 2008;283:8555-8563.
[32]Libertini SJ, Tepper CG, Guadalupe M, Lu Y, Asmuth DM, Mudryj M. E2F1 expression in LnCap prostate cancer cells deregulates androgen dependent growth, suppresses differentiation, and enhances apoptosis. Prostate 2006;66:70-81.
[33]Singh AP, Bafna S, Chaudhary K, Venkatraman G, Smith L, Eudy JD, Johansson SL, Lin MF, Batra SK. Genome-wide expression profiling reveals transcriptomic variation and perturbed gene networks in androgen-dependent and androgenindependent prostate cancer cells. Cancer Lett 2008;259:28-38.
[34]Knouf EC, Garg K, Arroyo JD, Correa Y, Sarkar D, Parkin RK, Wurz K, O'Briant KC, Godwin AK, Urban ND, Ruzzo WL, Gentleman R, Drescher CW, Swisher EM, Tewari M. An integrative genomic approach identifies p73 and p63 as activators of miR-200 miRNA family transcription. Nucleic Acids Res 2012;40:499-510.
[35]朱鹏,李建远.Prdxs蛋白家族与肿瘤.中外医学研究.2011.4(9)：122-124.
[36]曾跃平,刘洁坤,王宝玺.过氧化物酶Peroxiredoxin家族与肿瘤关系研究进展.癌症进展.2010.8(2)：164-166.
[37]王春玲,王佳,张剑梅,王明晓.泛素-蛋白酶体系统的研究进展.中国心血管病研究.2012.10(5)：394-398.
[38]吴俊兰,卢斌,肖菊香.硫氧还蛋白系统与肿瘤的关系.现代肿瘤医学.2012.20(11)：2429-2431.
[39]潘燕,韩婧,张晔,李学军.Vimentin在肿瘤转移中的作用及药物研究进展.生理科学进展.2012.41(6)：413-416.
[40]Ogawa K, Utsunomiya T, Mimori K, Tanaka Y, Tanaka F, Inoue H, Murayama S, Mori M. Clinical significance of elongation factor-1 delta mRNA expression in oesophageal carcinoma. Br J Cancer.2004.19;91(2):282-6.
[41]Dutta S, Tan YJ. Structural and functional characterization of human SGT and its interaction with Vpu of the human immunodeficiency virus type 1." Biochemistry.2008:47:10123-10131.
[42]Trotta AP, Need EF, Butler LM, Selth LA, O'Loughlin MA, Coetzee GA, Tilley WD, Buchanan G. Subdomain structure of the co-chaperone SGTA and activity of its androgen receptor client. J Mol Endocrinol.2012 Jul 25;49(2):57-68.
[43]Liu Y, Hu Y, Li X, Niu L, Teng M. The crystal structure of the human nascent polypeptide-associated complex domain reveals a nucleic acid-binding region on the NACA subunit.Biochemistry.2010;49(13):2890-2896.
[44]徐诚望,杨晓明.核磷蛋白与肿瘤的发生.军事医学科学院院刊. 2008.32(4)：382-385.
[45]核磷蛋白与肿瘤的发生.张群,孙自勤.中国医师进修杂志.2011.34(12)：74-76.
[46]Li X., Demartino G.N. Variably modulated gating of the 26S proteasome by ATP and polyubiquitin. Biochem J.2009.421:397-404.
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