miR-651和miR-708作为肾细胞癌分子标志物的价值及其作用机制研究

The incidence of renal cell carcinoma (RCC) is increasing annually and is getting younger, however, no specific diagnostic marker has been discovered. Our previous studies found that the levels of miR-651 and miR-708 were significantly downregulated in RCC tissues, cell lines and sera samples from patients with RCC; Results from bioinformatics analysis suggested that miR-651 and miR-708 might be involved in the regulatory network of RCC, and RAP1B and several other oncogenes have been confirmed to be the direct target genes of miR-651 and miR-708 by using luciferase reporter system. Thus, we hypothesize that miR-651 and miR-708 are potential molecular biomarkers of RCC and they may act as tumor suppressors in RCC. In order to verify this hypothesis, we will adopted clinical samples, RCC cell lines and tumor-bearing mouse model and use some techniques such as TaqMan Low Density Chip assay, qRT-PCR, western blot, co-immunoprecipitation, liposomes and lentivirus transfection, RNA interference, etc. to define the diagnostic and prognostic values of miR-651 and miR-708 for RCC; to explore the roles and the mechanisms of miR-651 and miR-708 in the development and progression of RCC; and to construct the regulatory networks between miR-651, miR-708 and their upstream transcription factors and their down target gens. The purpose of our study is to open up a new way to improve the diagnosis and monitoring of RCC and to provide a new approach and theoretical foundation for the pathogenesis of RCC.

肾细胞癌（RCC）发病率逐年增高，呈年轻化趋势，但无理想的分子诊断标志物。我们发现，miR-651和miR-708在RCC血清、组织及肾癌细胞株均低表达；生物信息学预测它们参与RCC的调控网络，荧光素酶实验证实了RAP1B等原癌基因是直接靶标。据此提出假设：miR-651和miR-708是RCC潜在的分子标志物，它们作为抑癌miRNA参与RCC的发生发展。为验证假说，我们拟通过临床标本、肾癌细胞株及荷瘤小鼠模型，采用低密度芯片、qRT-PCR、Western blot、免疫共沉淀、脂质体及慢病毒载体转染、RNA干扰等手段，确认miR-651和miR-708对RCC的诊断和预后价值；探明它们在RCC发生发展中的作用及分子机制；构建它们与上游转录因子和下游靶基因间的调控网络。力图为实现RCC无创伤性检出和治疗效果适时监控开辟一条新途径，同时也为进一步阐明RCC的发病机制，为临床诊疗提供新思路。

肾细胞癌（RCC）早期多无症状且复发率高，发病机制不详, 且缺乏RCC 特异的血清学标志物。本项目运用高通量低密度芯片技术初筛和RT-qPCR验证相结合的方法发现12种miRNA在RCC患者血清中水平显著变化，其中miR-651、miR-708、miR-28-5p等6种miRNA显著降低，miR-194、miR-324-5p、miR-362等6种miRNA显著增加。miR-651、miR-708对RCC具有较高的诊断准确性。miR-193a-3p、miR-572、miR-362、miR-28-5p和miR-378等五种miRNA组合在一起，对早期RCC具有较高的诊断准确性。miR-651、miR-708和miR-28-5p在RCC组织和肾癌细胞株中的表达下调，且可降低肾癌细胞的生长、迁移和侵袭，在肾癌中起抑癌miRNA作用。体外细胞实验和体内动物模型实验揭示，miR-651、miR-708和miR-28-5p通过介导调控RAP1B及其下游基因的表达抑制肾癌的生长、迁移和侵袭。miR-520/372/373家族（miR-520c-3p、miR-372-3p和miR-373-3p）也在RCC组织中显著下调，且与原癌斑点型POZ蛋白（SPOP）的蛋白水平负相关。此外，体外细胞实验和多种动物模型实验揭示， E2F1-miR-520/372/373家族-SPOP轴调控RCC的发生发展。结论：本研究我们寻找到了可作为RCC潜在分子标志物的血清miR-651、miR-708和miR-28-5p等，它们在诊断、早期诊断方面具有较高的临床应用潜能。同时我们明确了miR-651、miR-708、miR-28-5p以及miR-520/372/373家族在RCC病理过程中的作用，阐明了它们的调控基因，构建了它们参与RCC的分子网络。本研究结果为RCC寻找到了新的分子标志物和治疗靶标，也为阐明RCC发生发展机制提供了新思路。.本项目累计发表13篇SCI论文（6篇影响因子大于5）；获得省部级奖二项；参加国内外学术交流13次；培养博士后1名、博硕士生3名。项目投入经费80万元，支出70.9433万元，各项支出基本与预算相符；剩余经费9.0567万元，计划用于本项目研究后续支出。