miR-141与let-7抑制前列腺癌干/祖细胞及前列腺癌生长和转移的功能与机制研究

Background: miRNAs are key regulators of tumor development and differential miRNA expression has been reported in various tumor types. By screening a library of ～310 miRNAs, we observed that several tumor suppressive miRNAs including miR-34a, let-7b, and miR-141 were under-expressed in prostate cancer(PCa) stem/progenitor cell populations. My published work has demonstrated PCa-suppressive functions of both miR-34a and let-7. However, whether miR-141 also exerts tumor suppressive functions in tumorigenic PCa cells remains unclear. Moreover, during my studies of let-7, I observed that the let-7 miRNAs appear to be rapidly degraded in PCa cells but how this happens is unknown. Objective/Hypothesis: My overarching hypothesis is that tumor-suppressive miRNAs distinctively and concertedly regulate the key biological properties of prostate cancer stem cells(CSCs) and restrain PCa development and metastasis. The main objective of this NSFC project is: 1) To determine the functions and mechanisms of miR-141 in regulating tumorigenic PCa cells and PCa metastasis; and 2) To investigate the molecular mechanisms underlying the fast turnover of let-7 in PCa cells. Study Design: For Specific Aim 1, firstly I will use the newly developed "miRNA sensor" and FISH/IHC techniques to pinpoint miR-141 expression in phenotypically identifiable prostate CSCs. Secondly, I will investigate the biological functions of miR-141 in PCa cells. Lastly, I plan to identify novel direct targets that might mediate the biological functions of miR-141 in PCa cells by whole-genome RNA sequencing. For exploratory Aim 2, I will further confirm the fast turnover of let-7 in both cultured/xenograft and primary tumor derived PCa cells by examining the let-7 levels at different time point. Then several post-transcriptional regulators of let-7 including Lin-28, XRN2 and other exoribonuclease will be examined in prostate CSCs and relevant samples. Significance & Impact: My current project, which is both conceptually daring and technically innovative, has great significance as it addresses a fundamental question with clinical importance, i.e., how do two tumor suppressive miRNAs (i.e., miR-141 and let-7) exactly exert their PCa-inhibitory effects? My project also aims to fill a critical gap in our knowledge in PCa. Finally, elucidating the mechanisms of action for the key tumor-suppressive miRNAs that I'm studying has obvious translational significance in helping develop novel therapeutics that target prostate CSCs and CRPC cells, which will in turn impact PCa patient treatment and survival. The proposed work also has a great impact on advancing our knowledge of how specific miRNAs impinges on tumorigenic PCa cells and in turn regulates tumor development and metastasis. The success with the second Aim may have an unexpected impact on establishing a new paradigm for mechanisms that PCa cells exploit to shut down a powerful tumor-suppressive miRNA, i.e., let-7.

前列腺癌中存在具有明显成瘤性的肿瘤干/祖细胞。我的前期研究表明：①miR-34a和let-7、miR-141等抑癌miRNA在多个前列腺癌干/祖细胞亚群中表达下调；②miR-34a可抑制前列腺癌转移；③let-7也有前列腺癌抑制作用，却易快速降解（turnover）。但是，miR-141的功能尚未研究，let-7快速降解的机制尚不清楚。本课题将从以下两方面分别回答上述两问题：⑴采用"miRNA示踪"和FISH/IHC这两项新技术准确定位miR-141在前列腺癌干/祖细胞中的表达，结合其功能获得/缺失实验和全基因组RNA测序技术系统地研究miR-141在前列腺癌中的功能和机制，并鉴定其作用靶基因。⑵用前列腺癌细胞、异种移植瘤和临床病例分析不同时间点let-7的降解情况及转录后调节因子Lin28、XRN2等的表达变化，探讨let-7的快速降解机制。本课题立项新颖、技术前沿、临床转化价值重大。

miRNA在肿瘤发生发展和转移中发挥重要的作用。我们的研究发现miR-200家族成员之一miR-141在多个前列腺干／祖细胞群，移植瘤和前列腺癌样本中表达下调。在纯化的CD44+和未纯化的前列腺癌细胞中，过表达miR-141抑制克隆形成能力，成球能力等肿瘤干细胞的特性，还可以小鼠皮下肿瘤生长，原位肿瘤侵袭和转移。过表达miR-141引起间充质上皮细胞变。全基因组RNA测序结果发现一些新的miR-141作用靶基因，经过实验验证的靶基因包括Rho GTPase 家族基因CDC42, CDC42EP3, RAC1, ARPC5等，干细胞标志分子CD44 和EZH2。以上结果表明miR-141"雇佣"多个基因组阻碍肿瘤发生和转移，这对于小RNA的临床应用提供有意义的理论基础。