miR-199a-3p靶向CD44+干/祖细胞对前列腺癌发生和转移的抑制作用研究

Prostate cancer (PCa) is the most commonly diagnosed malignancy in western world and also one of the fastest increasing cancers in China. It is essential to understand the molecular mechanisms underlying PCa development and metastasis. Evidence accumulated in the past decade indicates that cancer stem cells (CSCs), a population of cancer cells with certain stem cell properties, are involved in tumor maintenance, therapy resistance, tumor progression, and distant metastasis. Despite their potential clinical significance, how intrinsic CSC properties are regulated at the molecular level is poorly understood. Work from our lab (Oncogene, 2006; Cancer Res, 2007; Cancer Res, 2008; Stem Cells, 2009; Cell Stem Cell, 2012) and many others' has shown that the CD44+ PCa cell population is highly enriched in prostate CSCs or PCSCs with enhanced clonogenic, castration-resistant, tumorigenic and metastatic capacities. Therefore, it is critical to elucidate the molecular regulators of the CD44+ PCSCs. As microRNAs (miRNAs) regulate both normal stem cells and CSCs, we recently screened a library of ~310 miRNAs in CD44+ and several other PCSC subpopulations (Nat Med, 2011; Cancer Res, 2012). We found that 37 miRNAs were commonly under-expressed in both xenograft and primary patient tumor-derived CD44+ PCa cells. Two tumor suppressive miRNAs, i.e., miR-34a and miR-199a-3p, were among the most dramatically down-regulated, representing only 2% and 4%, respectively, of the level in the corresponding CD44- PCa cells. Importantly, we have shown that miR-34a acts as a critical negative regulator of PCSCs and an inhibitor of PCa metastasis by directly targeting CD44. As miR-34a-mediated inhibition of CD44 and CD44+ PCSCs is incomplete, in this NSFC Young Investigator project, I address the KEY question of whether miR-199a-3p represents another critical negative regulator of CD44+ PCa cells and PCa metastasis. My goal is to understand the functions of miR-199a-3p in highly purified CD44+ PCa cells and PCa metastasis and to test its therapeutic efficacy. I will employ a spectrum of state-of-the-art cell biological and molecular approaches to achieve my goal. Firstly, I will evaluate the relationship between miR-199a-3p expression and CD44 by employing the newly developed miRNA sensor technique, which allows separation of PCa cells expressing low and high levels of endogenous miR-199a-3p. Secondly, I will investigate the biological functions of miR-199a-3p in CD44+ PCa cells and PCa metastasis using the miR-199a-3p sensor and inducible miR-199a-3p expression systems. Lastly, the therapeutic effects of miR-199a-3p will be evaluated via intratumoral injection and systemic delivery. Accomplishment of the goals proposed herein will greatly advance my career development as an independent researcher and also lay a solid groundwork for developing miR-199a-3p based novel therapeutics targeting drug-resistant and metastasis-prone PCSCs.

肿瘤干/祖细胞的存在是肿瘤复发和转移的重要原因。唐定国教授课题组对前列腺癌的前期研究发现：①CD44+细胞具有显著成瘤性、转移性和肿瘤干/祖细胞特征；②miR-34a和miR-199a-3p在CD44+细胞中的表达仅占CD44-细胞的2%和4%；③miR-34a通过CD44部分抑制前列腺癌转移。miR-199a-3p是否是CD44+前列腺癌细胞的另一负性调控因子尚不清楚。为回答这一问题，本课题拟从以下三方面进行研究：⑴用miR-199a-3p示踪等新技术分析其内源表达与CD44的关系；⑵结合前列腺癌小鼠模型、miRNA示踪及miR-199a-3p诱导表达系统等方法研究miR-199a-3p对CD44+细胞生长和转移的影响；⑶采用小鼠尾静脉注射和瘤内注射的方法评价miR-199a-3p对前列腺癌的治疗效果。本课题将为药物抵抗和治疗后复发等难治性前列腺癌的靶向治疗药物开发提供重要的研究基础。

肿瘤的异质性表现为部分细胞具有一定的分化能力但成瘤能力有限；部分细胞具有肿瘤干细胞特点可以形成肿瘤。近期的研究表明miRNA可以调节肿瘤干细胞并影响肿瘤发生。我们以往研究对CD44+ 及其它前列腺癌干细胞和非干细胞miRNA表达谱分析发现，miR－199a－3p在肿瘤干细胞中表达明显下调。在本项目中我们研究了miR－199a－3p在CD44+ 肿瘤干细胞和前列腺癌发生中的作用。在纯化的CD44+或未纯化的前列腺癌细胞以及前列腺癌病人样本中，过表达miR-199 a－3p可以抑制细胞增殖和克隆形成，但对细胞凋亡没有影响。过表达miR-199a-3p不仅可以抑制小鼠皮下肿瘤生长，还可以通过诱导表达抑制荷瘤小鼠肿瘤生长。荧光素酶报告基因实验表示，CD44是miR-199a-3p直接作用的靶基因。我们还发现miR-199a-3p可以直接或间接地参与抑制c-MYC, cyclin D1 (CCND1)和EGFR等细胞分裂相关基因。以上结果表明，miRNA介导的抑制作用缺失可以导致肿瘤干细胞的增殖和前列腺癌的发生。这一发现对于miRNA开发制药提供了有力的理论依据。