Small RNA targeted gene activation for the treatment of prostate cancer

描述(申请人提供)：在美国，前列腺癌是最常见的男性癌症，是男性癌症死亡的第二大原因。这项建议的长期目标是开发基于小RNA的前列腺癌治疗策略。小双链RNA(DsRNA)被广泛用作小干扰RNA(SiRNA)，通过靶向mRNA序列来抑制基因的表达，这种现象被称为RNA干扰(RNAi)。最近，我们和其他人发现了一种新的小RNA介导的基因调控机制，即靶向基因启动子区域的dsRNA分子诱导序列特异的转录基因激活，这种现象被称为RNA诱导基因激活(RNAa)和dsRNA分子的小激活RNAs(SarNas)。我们证明了RNAa在基因激活方面是有效的，并且它的作用是长期的，这表明RNAa具有很好的治疗作用。我们假设Sarna分子在体外和体内通过靶向激活前列腺癌中的肿瘤抑制基因(TSG)而具有抗肿瘤活性。这一假设将在两个具体目标上得到检验。特定目标#1.确定激活TSG的功能性Sarna，并评估其在培养的人前列腺癌细胞中的抗生长作用。靶向激活的基因包括(1)p16、p21、p27和p53等负细胞周期调控基因，(2)抑制肿瘤细胞侵袭和转移的基因，包括E-钙粘素、KAI1和CD44，以及(3)促凋亡基因，包括par-4和caspase-7。将候选的SarNas靶向基因启动子导入人前列腺癌LNCaP和PC3细胞，然后从mRNA和蛋白质水平评估靶向基因的表达。功能性SANAS将进一步评估其抗生长活性以及对细胞周期分布、细胞凋亡、克隆形成、侵袭和迁移的影响。特定目的#2.评估Sarna在异种移植人前列腺肿瘤模型中的抗肿瘤活性。利用人前列腺癌PC-3细胞在裸鼠体内建立异种前列腺癌模型。已建立肿瘤的小鼠将通过瘤内注射使用功能性Sarna进行治疗。用Sarna治疗的小鼠的肿瘤生长将与用对照Sarna治疗的小鼠进行比较。这些研究构成了我们以前工作的合乎逻辑的进展。这项提议的成功完成将为RNAa的进一步临床试验提供理论基础。在美国，前列腺癌是最常见的男性癌症，是男性癌症死亡的第二大原因。迫切需要开发前列腺癌的新治疗策略。这个项目将研究一种新发现的使用小RNA分子进行基因治疗的方法的治疗用途。

DESCRIPTION (provided by applicant): In the U.S., prostate cancer is the most common male cancer and the second leading cause of cancer deaths in men. The long-term objective of this proposal is to develop small RNA-based therapeutic strategies for prostate cancer. Small double-stranded RNA (dsRNA) has been widely used as small interfering RNA (siRNA) to knockdown gene expression by targeting mRNA sequences, a phenomenon known as RNA interference (RNAi). Recently we and others identified a new mechanism of small RNA-mediated gene regulation in which dsRNA molecules targeting gene promoter regions induce sequence-specific transcriptional gene activation, a phenomenon termed "RNA-induced gene activation" (RNAa) and the dsRNA molecules "small activating RNAs" (saRNAs). We demonstrated that RNAa is potent in gene activation and its effects are long-lasting, suggesting a promising therapeutic role for RNAa. We hypothesize that saRNA molecules have anti-tumor activity in vitro and in vivo via targeted activation of tumor suppressor genes (TSGs) in prostate cancer. This hypothesis will be tested in two specific aims. Specific Aim #1. Identify functional saRNAs for TSG activation and evaluate their anti-growth effects in cultured human prostate cancer cells. Genes to be targeted for activation include (1) negative cell cycle regulators such as p16, p21, p27 and p53, (2) genes that inhibit tumor cell invasion and metastasis including E-cadherin, KAI1 and CD44, and (3) pro-apoptosis genes including Par-4 and caspase-7. Candidate saRNAs targeting gene promoters will be transfected into human prostate cancer cell lines LNCaP and PC3, and the expression of targeted genes will then be evaluated at both mRNA and protein levels. Functional saRNAs will be further assessed for their anti-growth activity and effects on cell cycle distribution, apoptosis, clonogenicity, invasion and migration. Specific Aim #2. Evaluate saRNA's anti-tumor activity in xenograft human prostate tumor models. Xenograft prostate tumors will be established in athymic mice using human prostate cancer PC-3 cells. Mice with established tumors will be treated with functional saRNAs by intratumoral injections. Tumor growth in saRNA-treated mice will be compared with mice treated with control saRNAs. These studies constitute a logical progression of our previous work. Successful completion of this proposal will provide rationale for further clinical trials of RNAa. In the U.S., prostate cancer is the most common male cancer and the second leading cause of cancer deaths in men. There is an urgent need to develop novel therapeutic strategies for prostate cancer. This project will investigate the therapeutic use of a newly identified method of gene therapy using small RNA molecules.