靶向FOXO3a的microRNA在肾透明细胞癌中的作用及机制研究

Clear cell renal cell carcinoma(ccRCC) is the most common seen histological subtype of all renal cell carcinomas (RCC). No reliable molecular biomarker for the diagnosis of ccRCC has been identified. Previous studies showed that FOXO3a promoted cell cycle arrest, induced tumor cell apoptosis, and inhibited cellular migration and invasion in RCC cell lines. Downregulation of FOXO3a was an independent prognosis factor for ccRCC metastasis. However, the mechanisms which suppressed FOXO3a expression in ccRCC remain unclear. Based on our previous experimental findings, we hypothesized that certain microRNAs (miRNAs) were deregulated in ccRCC, which suppressed FOXO3a expression thereby promoted tumorigenesis and progression of ccRCC. In order to screen these miRNAs, fourteen paired ccRCC tumor tissues and corresponding adjacent normal tissues were detected by miRNA arrays. Combined with bioinformatics analysis, 5 miRNAs were finally selected for further study. To test our hypothesis, firstly, we plan to validate these miRNA levels in ccRCC tissues. Afterwards the biological functions of these miRNAs, including cell cycle, apoptosis, proliferation, migration, and invasion, in RCC cell lines will be studied. Then, whether FOXO3a was directly targeted by these miRNAs will be determined in RCC cell lines. Moreover, the influence of these miRNAs on tumor growth and metastasis will be surveyed in a nude mice tumor model. Finally, the relations of the miRNAs levels and the prognosis of the patients will be analyzed. Our study will clarify the mechanism which suppressing FOXO3a, promoting ccRCC tumorigenesis and progression and provide a theoretical basis for searching for biomarkers of ccRCC.

肾癌最主要的病理类型是肾透明细胞癌(ccRCC)，目前缺少可靠的诊断标志物。前期研究表明，转录因子FOXO3a在ccRCC中表达下调并因此促进肿瘤发生发展，其表达水平与患者预后呈负相关。目前FOXO3a的下调机制尚不明确。本项目结合前期试验结果，推测ccRCC中存在异常增高的microRNA（miRNA）靶向抑制FOXO3a的表达而促进ccRCC发生发展。为了筛选出这些miRNA，我们对14组ccRCC及对应瘤旁组织进行了miRNA芯片检测与生物信息学分析，最终筛选出5个目的miRNA。本项目拟验证这些miRNA在ccRCC中的表达水平，通过体外细胞功能实验与裸鼠荷瘤实验观察其对肾癌细胞系生物学行为的影响。此外在肾癌细胞系中证明miRNA和FOXO3a之间的靶向关系。最后通过随访分析研究miRNA与ccRCC患者预后之间的关系，验证我们的推测，为寻找ccRCC的分子标志物提供理论依据。

项目背景：microRNA在肾癌中发挥重要作用，但其作用机制仍未完全明确。结合我们前期芯片结果，对miRNA数据进行综合分析,有利于关键miRNA的筛选。越来越多的研究表明miRNA-122（miR-122）在许多癌症中失调，然而其在透明细胞肾细胞癌（ccRCC）中的作用仍不清楚。.研究内容：在这项研究中，我们对四个ccRCC miRNA组织芯片进行综合分析与筛选。对筛选出的miR-122在细胞系中进行模拟物和抑制剂转染后，测定体外细胞增殖，集落形成，迁移和侵袭等实验的结果。并使用荧光素酶报告基因测定法确认miR-122的靶基因，最后用小鼠皮下成瘤模型来研究miR-122在体内的致瘤作用。.关键数据：miR-122水平在ccRCC显著升高，并且与患者无转移生存时间负相关。 在786-O细胞中过表达miR-122显著加快了细胞增殖和集落的形成，加快了迁移和侵袭;而在SN12-PM6细胞中敲低miR-122则抑制了细胞生长和集落的形成减弱了迁移和侵入。Western印迹和荧光素酶报告实验直接验证了FOXO3是miR-122作用的直接靶标。.科学意义：我们的研究结果表明miR-122通过靶向抑制FOXO3蛋白的表达在ccRCC中发挥致瘤作用。