前列腺癌是美国男性发病率最高的恶性肿瘤。除了既有的330万存活的前列腺癌患者，2018年全美约有164690的新发前列腺癌病例。近年来，随着我国人口老龄化加剧，生活习惯的西化，我国成年男性前列腺癌的发病率与死亡率均超过了膀胱癌，呈逐年上升的趋势。尽管前列腺癌相较于其他肿瘤进展缓慢，同时伴随前列腺癌早期筛查与诊断率的提高，患者的预后相对较好。但是前列腺癌仍然长期影响患者的健康并成为全球范围内男性第三大致死性肿瘤。目前前列腺癌根治性切除术及根治性放疗术是局限性前列腺癌的标准治疗方式，而转移性前列腺癌，无法根治性手术切除或放射治疗及术后生化复发的病人，则应用内分泌治疗。遗憾的是，在经历18-24个月的中位敏感期后，多数患者转归为去势抵抗性前列腺癌 (castration-resistant prostate cancer, CRPC）。既往研究表明，发展到CRPC阶段的患者，中位生存期仅有18-24个月，是前列腺癌患者死亡的主要原因。因此进一步阐明前列腺癌进展的分子机制，寻找新的生物学治疗靶点，对延缓前列腺癌进展及提高患者生存率具有重要意义。
近年来非编码RNA在前列腺癌进展及转移当中的作用已经得到了广泛的研究。其中microRNA (miRNA)是长度为18-25个核苷酸构成的高度保守单链非编码RNA，成熟体的miRNA依赖碱基互补配对的方式和靶基因mRNA 的3’非编码区 (3’-untranslated regions，3’UTR) 结合，影响下游基因的蛋白表达水平，从而间接调控生物学功能。大量研究表明miRNA参与了细胞的生长、分化、凋亡及细胞周期的调控，异常表达的miRNA则可能与肿瘤的增殖、转移、分化、复发及治疗抵抗密切相关。现有研究表明大量的miRNA在前列腺癌的恶性进展中扮演了重要的角色。
而长链非编码RNA (lncRNA)是长度大于200个核苷酸的一组非编码RNA，在人类多种疾病中都发现了其异常表达，它能够促进疾病的进展或者维持疾病的异常状态。lncRNA的功能调控主要依赖于其生物学结构，它能特异性结合相应的蛋白、DNA或者RNA从而通过表观沉默或者转录后调控影响下游基因的表达。近年来，多种lncRNA被证实参与了前列腺癌的恶性进展，如新型前列腺癌抗原3 (Prostate Cancer Antigen 3，PCA3) 参与了前列腺癌上皮间质转化 (Epithelial-Mesenchymal Transition ，EMT)，人肺腺癌转移相关转录本1 (Metastasis-Associated Lung Adenocarcinoma Transcript-1，MALAT1)和核富含丰富的转录本 1 (nuclear-enriched abundant transcript 1, NEAT1)能诱导前列腺癌的去势抵抗。其中lncRNA通过招募多梳抑制复合物2 (polycomb repressor complex 2, PRC2)调节染色质的状态及甲基化水平是其参与肿瘤进展的重要机制之一，而癌基因EZH2作为PRC2的重要催化亚基也因此受到研究者越来越多的关注。lncRNA通过表观修饰调控前列腺癌恶性进展成为了近年来研究的热点。
lncRNA HOTAIR (HOX transcript antisense RNA，HOX转录反义RNA)是一个确定的肿瘤相关基因，在乳腺癌、结直肠癌、肺癌及肝癌中均显著高表达，并促进肿瘤的远处转移。最新的研究显示HOTAIR亦在CRPC中高表达，并直接与雄激素受体(AR)结合降低其泛素化水平和蛋白降解从而促进前列腺癌的增殖与侵袭。而HOTAIR通过招募PRC2表观调控下游基因表达是其发挥癌基因作用的重要机制。本课题组前期分析了APDC与CRPC组织的miRNA差异表达谱，筛选出CRPC组织中低表达的miRNAs；另一方面，结合既往发表文献我们进一步筛选出在前列腺癌细胞系中EZH2能负调控的miRNAs；再利用miRcode (www.mircode.org)、DIANA TOOLS (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=lncBase/indexbio)及starBase v2.0（http://starbase.sysu.edu.cn/mirLncRNA.php）生物学预测网站找到与HOTAIR有靶向关系的miRNAs群。将三者miRNAs群交叉发现16个miRNAs中，进一步分析发现miR-193a-3p (miR-193a) 在转移性前列腺癌显著低表达。MiR-193a在口腔癌、肺癌、结直肠癌、肝癌、恶性胸膜间皮瘤、急性骨髓性白血病患者的组织中均发现其异常表达。然而其在前列腺癌中的表达水平及生物学功能尚未见报道。基于这样的研究现状，本研究首先通过分析纪念斯隆-凯特琳癌症中心 (Memorial Sloan-Kettering Cancer Center, MSKCC) 和癌症基因组图谱(The Cancer Genome Atlas, TCGA) 数据库及本中心的前列腺癌组织标本库验证miR-193a的表达水平，并通过EZH2/miR-193a生物轴表达失调的体内外功能实验，探讨EZH2/miR-193a轴在前列腺癌中的生物学功能。并利用染色质免疫共沉淀实验 (Chromatin Immuoprecipitation, ChIP)、荧光素酶报告实验等探讨HOTAIR联合EZH2表观沉默miR-193a的分子机制。最后通过生物信息学及荧光素酶实验验证miR-193a靶向负调控HOTAIR的表达。本研究拟探讨HOTAIR/EZH2/miR-193a环路在前列腺癌进展中发挥的分子机制，为前列腺癌的早期筛查及靶向治疗提供坚实的理论依据。
方法：对MSKCC、TCGA数据库中获得的关于前列腺癌miRNA表达的基因芯片数据进行二次分析，比较miR-193a在转移及非转移性前列腺癌以及不同临床分期的前列腺癌的表达差异，通过原位杂交 (ISH) 检测其在不同Gleason评分前列腺癌组织中的表达。在去势抵抗性前列腺癌细胞系中 (PC3和DU145)，通过干预EZH2和miR-193a的表达量 (过表达或敲低)，利用CCK-8和低密度细胞集落形成实验验证EZH2/miR-193a轴前列腺癌细胞增殖能力的影响；流式细胞计数及TUNEL实验检测EZH2/miR-193a生物轴对前列腺细胞凋亡能力的影响；Transwell迁移和侵袭实验明确miR-193a对前列腺癌细胞转移的作用；过表达miR-193a的PC3和DU145细胞提取RNA后，测序获得全转录组RNA数据，并通过基因探针富集分析 (GSEA)分析miR-193a对前列腺癌细胞增殖及转移相关信号通路的影响。裸鼠皮下成瘤实验进一步验证miR-193a对去势抵抗性前列腺癌细胞系PC3在体内成瘤能力的影响，并通过ISH和免疫组化 (IHC) 等检测两组皮下瘤中Ki-67、CD31、CD34等增殖及血管形成的相关基因表达水平，进一步验证miR-193a对前列腺癌增殖及侵袭能力的影响。
结果：对MSKCC及TCGA数据库资料的二次分析表明，miR-193a在转移前列腺癌中的表达低于非转移前列腺癌。接受者操作特性曲线(Receiver-operating characteristic curve, ROC)分析表明miR-193a能够用于转移前列腺癌的诊断，其曲线下面积（Area under the curve, AUC）为0.8709，95%置信区间为0.7390-1.0003，P<0.0001。miR-193a在T3-4临床分期的前列腺癌中的表达低于T1-2分期前列腺癌，ROC曲线表明miR-193a可用于T3-4分期前列腺癌的诊断，AUC值为0.6705，95%置信区间为0.6074-0.7336，P<0.0001。原位杂交显示，miR-193a在Gleason < 7 、Gleason = 7及Gleason > 7的前列腺癌组织中表达水平依次降低 (P<0.05)。CCK-8实验和低密度细胞集落形成实验结果均表明在去势抵抗性前列腺癌细胞中 (PC3和DU145) 过表达EZH2能够促进细胞的增殖能力，而在此基础上同时过表达miR-193a，EZH2的促进增殖作用显著受抑制；而在前列腺癌细胞系中敲低EZH2，细胞增殖活性明显降低，而在此基础上同时抑制miR-193a的表达，细胞的增殖活性得到部分恢复 (P < 0.05)。GSEA分析提示miR-193a过表达后，细胞有丝分裂 (NES = ?1.64, FDR = 0.02, P = 0.069) 及G2/M细胞周期检查点(NES = ?2.0, FDR < 0.001, P < 0.001) 两个基因簇呈现负向富集。流式细胞仪术及TUNEL实验发现过表达EZH2前列腺癌细胞凋亡与NC组无明显差异，而在此基础上同时过表达miR-193a细胞凋亡明显增加（P<0.05）；另一方面敲低EZH2，前列腺癌细胞凋亡率明显增加，而在此基础上同时抑制miR-193a的表达，细胞的凋亡率明显降低 (P < 0.05)。在Transwell小室实验中，过表达miR-193a能抑制PC3和DU145细胞的迁移及侵袭的能力（P<0.05）。GSEA分析提示miR-193a过表达后，促进前列腺癌转移相关的信号通路TGF-β (NES = ?1.79, FDR = 0.01, P = 0.015)、TNF-α/NF-kB (NES = ?1.77, FDR = 0.005, P = 0.017)及KRAS (NES = ?1.69, FDR = 0.08, P = 0.203)基因簇呈现负向富集。裸鼠皮下成瘤实验表明，过表达miR-193a表达组的细胞瘤体大小和体积明显小于对照组（P<0.05），同时在皮下移植瘤体的病理切片中，免疫组化实验结果表明细胞增殖相关基因Ki-67、血管生成影响因子CD31、CD34的表达均显著降低。
目的：明确HOTAIR能通过联合EZH2通过诱导组蛋白H3第27位赖氨酸三甲基化 (H3K27me3) 表观沉默前列腺癌中miR-193a的表达。
方法：通过实时荧光定量PCR（quantitative real time PCR，qRT-PCR）检测过表达/敲低EZH2后前列腺癌细胞中miR-193a表达水平。在PC3及DU145过表达EZH2的基础上进一步敲低HOTAIR，qRT-PCR检测miR-193a表达水平变化。Western Blotting检测过表达/敲低EZH2或敲低HOTAIR后，PC3与DU145中EZH2和H3K27me3表达水平变化。通过既往文献明确miR-193a的启动子区域，并对高通量基因表达数据库（Gene Expression Omnibus, GEO）数据二次分析PC3与DU145细胞中miR-193a启动子H3K27me3蛋白的富集区域，从而利用该区域设计相应的ChIP引物。接着通过启动子荧光素酶报告基因及ChIP实验验证HOTAIR联合EZH2能诱导miR-193a启动子区的H3K27的三甲基化。
Prostate cancer (PCa) is the most common malignancy among men in the USA. In 2018, approximately 164690 men will be diagnosed with prostate cancer, adding to 3.3 million existing survivors. In recent years, with the economy development, the incidence and mortality rates of PCa have also been steadily elevated in China which both exceeds that of bladder cancer. PCa is the most common indolent course of many tumors, and the prognosis of PCa patients is improving regarding the utility of screening and early detection. Even so, prostate cancer can threaten long-term health and remains the third-leading cause of cancer death in men. Radical prostatectomy and radical radiotherapy are the standard therapies for localized prostate cancer. And androgen deprivation therapy (ADT) is needed when prostate cancer can not been radically resected or biological recurrence is occurred. The majority of PCa patients initially respond quite well for ADT, however, the biggest challenge is that most PCa patients will inevitably progress to the castration-resistant prostate cancer (CRPC) within two years. Previous study demonstrated that median overall survival time for CRPC patients is only two years and CRPC is the main cause of cancer-related death. Thus, it is urgent need to understand the molecular mechanisms underlying PCa progression and develop the novel promising therapeutic approaches.
Non-coding RNAs have been widely studied in the area of PCa progression and metastasis. MicroRNAs (miRNAs) are small (18-25 nucleotides) endogenous non-coding RNAs that regulate gene expression through a sequence-specific manner via the degradation of target mRNAs, thereby modulating the biological activities of cells. It is reported that miRNAs are involved in the regulation of cell proliferation, apoptosis, differentiation and cell cycle. Hundreds of studies found that aberrantly expressed miRNAs contributed to the proliferation, metastasis, differentiation, recurrence and therapeutic resistance of cancers. Recent studies have revealed that large amount of miRNAs played a vital role in the malignant progression of prostate cancer.
Long non-coding RNAs (lncRNAs) are a cluster of non-coding RNAs with more than 200 nucleotides, which are aberrantly expressed in a variety of human diseased, likewise contributing to pathogenesis or maintaining diseased conditions. The function of lncRNAs has been tied to the molecular structure, which allows them to interact specifically with DNA, RNA and proteins. Thus, lncRNAs can regulate target gene expression through transcriptional or post-transcriptional ways. Recent studies demonstrated that many lncRNAs are involved in the progression of prostate cancer. For example, PCA3 is closely associated with the regulation of EMT while MALAT1 and NEAT1 are critical for the promotion of castration resistance in prostate cancer. One of the most important mechanisms is that lncRNA could recruit polycomb repressor complex 2 (PRC2) which methylates lysine27 of histone H3 (H3K27) to promote transcriptional silencing of many tumor suppressive genes. Enhancer of zeste homolog 2 (EZH2) is one of the core enzymatic subunit of histone methyltransferase PRC2 which has attracted considerable scientific attention from researchers these days. In a word, it has been a research hotspot that lncRNA modulates prostate cancer progression via epigenetic silencing of tumor suppressor genes.
It has recently become apparent that there is interesting cross-regulation between miRNAs and lncRNAs during malignant process of tumors. On one hand, lncRNAs are targeted by miRNAs to reduce lncRNA stability. On the other hand, lncRNA could also function as molecular decoys or sponges of miRNAs. In addition, lncRNAs can also compete with miRNAs for bindng to shared target mRNAs to silence target mRNAs. The cross-regulation between miRNAs and lncRNAs is an important topic in the progression of cancers.
Accumulating evidence indicates that HOTAIR serves as an oncogene in the malignant process of various cancers including prostate cancer. It is highly expressed and correlated with metastasis in a variety of cancer types, including pancreatic, lung, colorectal, breast and pancreatic cancers. HOTAIR could directly bind to the AR protein to prevent its ubiquitination and degradation, thereby promoting PCa cell growth and invasion. And HOTAIR is well known for its interacting with epigenetic regulators PRC2 to induce gene silencing, which appears to be misregulated in a variety of cancers. In our previous microarray analysis, we have detected a cluster of miRNAs which were downregulated in CRPC compared with that in ADPC (androgen dependent prostate cancer). Furthermore, we reanalyzed the GEO dataset (GSE26996) to identify EZH2 negatively-regulated miRNAs in DU145. In addition, to reveal the potential miRNAs that can regulate HOTAIR in prostate cancer, we searched miRcode algorithm (http://www.mircode.org) ，DIANA Tools (http://diana.imis.athena-innovation.gr/DianaTools/index.php?r=lncBase/indexbio) and starBase v2.0 (http://starbase.sysu.edu.cn/mirLncRNA.php). A disciplinary overlap was showed by intersecting these three groups of miRNAs. 16 miRNAs were shared. By aligning these miRNAs to MSKCC prostate cancer dataset (GSE21032), microRNA-193a-3p (miR-193a) was found to be one of the most significantly downregulated miRNAs in metastatic PCa tissues compared with primary cancer. MiR-193a was initially discovered in 2008, and it was identified as a tumor suppressive miRNA in oral carcinoma, lung cancer, colorectal cancer, and malignant pleural mesothelioma. However, the exact biological function of miR-193a in tumorigenesis of PCa remains largely unknown. In the present study, we retrieved and reanalyzed the original miRNAs expression and clinical data from the Memorial Sloan Kettering Cancer Center (MSKCC) (www.mskcc.org) and The Cancer Genome Atlas (TCGA) (http://cancergenome.nih.gov) databases to investigate clinical relevance of miR-193a with the pathological traits of patients. Prostate cancer cell functional experiment was further performed to verify the effect of EZH2/miR-193a biological axis on prostate cancer cells. Moreover, we explored that EZH2 coupled with HOTAIR to silence miR-193a through introducing trimethylation of H3K27 at miR-193a promoter region by using ChIP and luciferase reporter assays. Finally, we proved that HOTAIR was a direct target of miR-193a in PCa by using bioinformatics and molecular biology techniques. This study aims to investigate the role of HOTAIR/EZH2/miR-193a feedback loop in the progression of PCa which could provide solid theoretical basis for target therapy and early detection of PCa.
The biological role of EZH2/miR-193a axis in prostate cancer progression
Objective: To investigate the expression profile of miR-193a in metastatic or non-metastatic prostate cancer and prostate cancer with different clinical stages or different Gleason scores. And we further analyze the role of miR-193a expression in the diagnosis of metastatic and advanced prostate cancer. Moreover, we intend to clarify the role of EZH2/miR-193a axis in regulation of PCa cell proliferation, apoptosis, migration and invasion abilities via in vitro studies. And to further illustrate the biological effects of miR-193a on PCa tumor formation ability by using a mice xenograft tumor model.
Methods: MSKCC and TCGA datasets and our clinical samples were used to identify miR-193a expression profile in PCa. Cell Counting Kit-8 (CCK-8) assays, colony formation, invasion, migration, flow cytometry, TUNEL assay, a xenograft model and Gene Set Enrichment Analysis were used to detect and analyze the biological function of EZH2/miR-193a axis.
Results: MSKCC data indicated that the expression of miR-193a in metastatic cancer was significantly decreased compared to primary cancer (P < 0.05). ROC analysis demonstrated that the expression of miR-193a can be used to discriminate between metastatic and primary prostate cancers (AUC=0.8709, 95%CI: [0.7390 to 1.0003], P < 0.0001). We also found that miR-193a expression was significantly downregulated in higher-T stage tumors via reanalysis of TCGA dataset (P < 0.05). ROC analysis also showed that the level of miR-193a could be used to discriminate between pT3–4 and pT1–2 PCa (AUC=0.6705, 95%CI: [0.6074 to 0.7336], P < 0.0001). We evaluated the miR-193a expression in our clinical specimens via ISH (in situ hybridization) and found that miR-193a was aberrantly downregulated in high-Gleason score tumors (Gleason score: 8–10) (P < 0.05). CCK-8 and colony formation assays indicated that miR-193a significantly alleviated the promoting effect of EZH2 on cell growth. Conversely, anti-miR-193a (miR-193a inhibitor) could also partially abrogate the inhibition effect of proliferation caused by knock-down of EZH2 in PCa cells (P < 0.05). GSEA showed that a negatively enriched expression of gene sets was involved in mitotic spindle assembly (NES = ?1.64, FDR = 0.02, P = 0.069) and G2/M cell-cycle checkpoint (NES = ?2.0, FDR < 0.05, P < 0.05) in miR-193a-overexpressing cells. EZH2 depletion increased the apoptosis rate in both PC3 and DU145 cells which could be partially rescued by supplementing the cells with anti-miR-193a. On the other hand, upregulation of miR-193a in EZH2-overexpressing cells could significantly induce apoptosis in PCa cells. These results were both confirmed by flow cytometry and TUNEL assay. Transwell assay indicated that overexpression of miR-193a in PCa cell lines strongly suppressed cell migration and invasion abilities. GSEA showed that a negatively enriched expression of genes sets was involved in hallmarks of TGF-β signaling (NES = ?1.79, FDR = 0.01, P = 0.015), TNF-α via NF-kB signaling (NES = ?1.77, FDR = 0.005, P = 0.017) and KRAS prostate up signaling (NES = ?1.69, FDR = 0.08, P = 0.203) in miR-193a-overexpressing PCa cells. Overexpression of miR-193a via using lentivirus obviously suppressed tumor growth as manifested by reduced tumor size and tumor weight. Immunohistochemical staining (IHC) illustrated that reduced Ki67-positive cells, CD31-positive cells and CD34-positive cells were found in miR-193a-overexpressed xenograft tumor cells.
Conclusion: miR-193a expression was much lower in metastatic PCa, higher-T stage PCa and higher-Gleason score PCa. MiR-193a serves as a tumor suppressor via inhibiting PCa cells proliferation, migration, invasion and promoting cell apoptosis in PCa, which could partially abrogate the oncogenic function of EZH2.
EZH2 coupled with HOTAIR to silence miR-193a through introducing trimethylation of H3K27 at miR-193a promoter region in prostate cancer
Objective: To identify whether EZH2 could couple with HOTAIR to epigenetically silence the expression of miR-193a
Methods: In order to prove that EZH2 had a direct role in repressing miR-193a in PC3 and DU145, we depleted EZH2 by stably infection of lentiviral particles of LV-shEZH2 or upregulated EZH2 with plasmid pcDNA3.1-EZH2 in PCa cells, and measured the level of miR-193a. To prove that HOTAIR was important in EZH2 mediated silencing of miR-193a, we inhibited the HOTAIR expression by siRNAs in EZH2-overexpressing PCa cells and then measured miR-193a expression level. We also measured the protein expression of EZH2 and H3K27me3 using Western Blotting after overexpression or knockdown of EZH2 or inhibition of HOTAIR in PCa cells. We cloned 2.0 kb fragments upstream of TSS (transcription starting site) of human pri-miR-193a stem-loop into pGL3-basic-vector to generate luciferase construct pGL3-193a-promoter reporter. Then we carried out luciferase reporter assay by cotransfecting pGL3-193a-promoter and si-EZH2 or pcDNA3.1-EZH2 into PC3 and DU145 cells. ChIP assays were conducted to measure the enrichment levels of EZH2 and H3K27me3 at the promoter region of miR-193a. GEO datasets (GSM1383872 and GSM1138596) were reanalyzed to determine the binding site of H3K27me3 at miR-193a promoter region in PC3 and DU145 cells.
Results: Upon inhibition of EZH2 or HOTAIR, there were upregulation of miR-193a expression in PC3 and DU145 cells (P < 0.05). And ectopic expression of EZH2 could significantly reduce the level of miR-193a expression (P < 0.05). Western blot assay showed that the enrichment of H3K27me3 significantly decreased when EZH2 was knocked down in LV-shEZH2 infected PCa cells and vice versa. WB also indicated that both expression of EZH2 and H3K27me3 largely declined in the HOTAIR knocking down PCa cells. Luciferase assay showed that co-transfection of the two cell lines with pGL3-193a promoter and si-EZH2 led to increased luciferase activity significantly (P < 0.05). In contrast, co-transfection with pGL3-193a promoter and pcDNA3.1-EZH2 markedly reduced the luciferase activity in the same cells (P < 0.05). ChIP assay revealed that silencing of HOTAIR led to reduction of EZH2 occupancy and H3K27me3 level at the miR-193a promoter region (P < 0.05).
Conclusion: EZH2 coupled with HOTAIR to induce H3K27 trimethylation at miR-193a promoter, which reduced miR-193a expression in PC3 and DU145 cells
Mir-193a directly targets HOTAIR and negatively
modulates its expression in PCa
Objective: In this part, we intend to explore whether miR-193a could directly target HOTAIR and modulate its expression in PCa.
Methods: QRT-PCR was used to measure the HOTAIR expression in PCa cells either via transfecting with miR-193a mimics or infecting with LV-miR-193a and in xenograft tumor tissues which were generated from PC3 cells stably overexpressing miR-193a. Bioinformatics tools (miRcode, DIANA Tools and starBase v2.0) were used to identify whether HOTAIR is one of putative targets of miR-193a and verified by luciferase reporter assay. Finally, we detected the expression of miR-193a, HOTAIR and EZH2 in our clinical PCa samples via ISH and IHC.
Results: We observed ectopic miR-193a expression significantly reduced HOTAIR level both in PCa cells and PCa tumor tissues (P < 0.05). The luciferase reporter assay demonstrated that miR-193a significantly reduced luciferase activity (wild type). Mutation of miR-193a binding site in HOTAIR abrogated the inhibitory effects. Spearman correlation analysis showed significant inverse correlation between expression of HOTAIR and miR-193a (r = ?0.67, P < 0.001) as well as between that of EZH2 and miR-193a (r = ?0.59, P < 0.001).
Conclusion: MiR-193a directly targets HOTAIR and negatively modulates HOTAIR expression in prostate cancer.