Androgen and MicroRNAs in Prostate Cancer

描述（由申请人提供）：MicroRNAs是17-24核苷酸长的单链RNA分子，通过退火和抑制许多靶基因的表达，深刻地影响细胞的基因表达程序。microrna有数百种，不同的细胞类型表达不同水平的特定microrna。最近发现，microrna表达的变化与不同类型的恶性肿瘤有关。此外，还发现了新的短rna家族，如pi- rna、rasi- rna和trf，它们与microrna无关，但也会影响细胞的表型。作为弗吉尼亚大学梅隆泌尿癌症研究所的一部分，我们对研究和逆转前列腺癌的进展很感兴趣。前列腺癌的增殖高度依赖于雄激素。雄激素通过改变前列腺上皮细胞的基因表达程序作用于前列腺上皮细胞，但其对microRNA和其他短RNA表达的影响尚未被研究。此外，虽然前列腺癌是用抗雄激素治疗的，而且最初对抗雄激素非常敏感，但它们通常会复发为不依赖雄激素的癌症，最终转移并导致死亡。为了逆转这一进展，我们必须了解前列腺癌细胞成为雄激素非依赖性的机制，并获得进展的其他特征。在本提案中，我们将通过Solexa/Illumina文库克隆和测序来自前列腺癌细胞的小rna，以鉴定(a)受雄激素调节和(b)参与前列腺癌进展的microrna和其他短rna。我们还将通过锁定核酸微阵列杂交来跟踪已知microrna的变化。这些结果不仅将发现新的短rna参与前列腺癌的进展，而且还将验证雄激素抑制的短rna在前列腺癌的进展过程中通常也被抑制的假设。然后，我们将在体外和体内测试前列腺癌进展过程中抑制的microrna有助于进展表型的假设，从已经在初步结果中得到验证的四个microrna（两个家族）开始。该假设的最终检验将是确定这些在癌症进展过程中去抑制导致恶性表型恶化的microrna的靶标。我们将通过实验和计算靶标预测相结合的新方法，从已经通过实验筛选的八个靶标开始，确定参与癌症进展的两个关键microRNA家族的靶标。因此，短RNA克隆和超高通量测序等新技术以及鉴定相关靶点的新方法组合将被应用于前列腺癌进展的分子分析，并在此过程中剖析关键microrna的功能。研究结果有望为前列腺癌的研究、治疗和诊断开辟新的途径。

DESCRIPTION (provided by applicant): MicroRNAs are 17-24 nucleotide long single-stranded RNA molecules that profoundly impact the gene expression program of a cell by annealing to and suppressing the expression of many target genes. There are hundreds of microRNAs and different cell types express different levels of specific microRNAs. It has recently become apparent that changes in expression of microRNAs are involved in different types of malignancies. In addition, new families of short RNAs, like pi-RNAs, rasi-RNAs and tRFs are being discovered that are not related to microRNAs but also impact the phenotype of a cell. As part of the Mellon Urological Cancer Research Institute at the University of Virginia we are interested in studying and reversing prostate cancer progression. Prostate cancers are highly dependent on androgens for their proliferation. Androgens act on prostate epithelial cells by changing the gene expression program, but their effects on microRNA and other short RNA expression have not been studied. In addition, although prostate cancers are treated with and initially very responsive to anti-androgens, they usually recur as androgen-independent cancers that eventually metastasize and lead to death. To reverse this progression, we have to understand the mechanism by which prostate cancer cells become androgen-independent and acquire other features of progression. In this proposal we will clone and sequence by Solexa/Illumina libraries of small RNAs from prostate cancer cells to identify microRNAs and other short RNAs that are (a) regulated by androgens and (b) involved in prostate cancer progression. We will also follow the changes in known microRNAs by hybridization to locked-nucleic acid microarrays. These results will not only identify new short RNAs involved in prostate cancer progression, but also test the hypothesis that androgen-repressed short RNAs are often also repressed during progression of prostate cancer. We will then test the hypothesis that microRNAs repressed during prostate cancer progression contribute to the phenotype of progression, both in vitro and in vivo, starting with four microRNAs (in two families) that have already been validated in preliminary results. The final test of the hypothesis will be the identification of targets of these microRNAs whose de-repression during cancer progression leads to worsening of the malignant phenotype. We will identify the targets of two key microRNA families involved in cancer progression by a novel combination of combination of experiments and computational target prediction, starting with eight targets that have already passed the experimental filters. Thus new technologies, short RNA cloning and ultra-high throughput sequencing, and new combinations of assays for identification of relevant targets will be applied to the molecular analysis of prostate cancer progression and the function of key microRNAs in this process dissected. The results are expected to open new avenues of research, therapy and diagnosis of prostate cancers. PUBLIC HEALTH RELEVANCE: MicroRNAs and short RNAs that are repressed by androgens will open a new chapter in how androgens regulate gene expression. Specific microRNAs and short RNAs that are also repressed during prostate cancer progression can be increased for therapeutic purposes. Studying the effects of these microRNAs (and short RNAs) on target genes will reveal how progression of prostate cancer is accompanied by repression of microRNAs to de-repress specific target genes, rendering the cancer cells less dependent on androgens, more proliferative, migratory and invasive and more metastatic. Thus, these androgen-repressed and progression-repressed microRNAs and non-micro-short RNAs will be useful both as biomarkers of cancer progression and as tools to identify genes whose de-repression is critical for cancer progression. Two methods will be developed here that will improve the study of microRNAs in cancer: (1) The combination of cloning/sequencing with locked-nucleic acid microarrays to obtain a well validated list of microRNAs that are changed during cancer progression. (2) The combination of bioinformatics, microarrays of mRNAs and polyribosome fractionation of mRNAs to produce a list of targets with a high true-positive rate of being direct targets of the microRNAs.