内皮祖细胞源性外泌体传递LINC00944促进肾透明细胞癌恶性进展的机制研究

The molecular mechanisms underlying and the therapeutic strategies against the malignant transformation of clear cell renal cell carcinoma (ccRCC) remain the focus and difficulty in urology. Endothelial progenitor cell (EPC) can contribute to the development and progression of ccRCC via promoting angiogenesis; however, the direct impacts of EPC on ccRCC cells remain far from understood. Our preliminary experiments have found that: 1. EPC can promote the proliferation and invasion of ccRCC cells via transferring exosomes; 2. EPC-derived exosomes (EPC-Exo) can transmit LINC00944 to ccRCC cells; 3. LINC00944 can promote the malignant development of ccRCC, and might function as competing endogenous RNA (ceRNA) for miR-143. Here comes our work hypothesis: EPC might promote the malignant development of ccRCC via transmitting LINC00944, which might function as ceRNA by competitively binding miR-143. In this study, we will examine the influence of LINC00944 transmitted by EPC-Exo on the proliferation, migration and invasion of ccRCC both in vitro and in vivo, explore the exact oncogenic mechanisms of LINC00944 transmitted by EPC-Exo with the aid of RNA-Seq, Dual-Luciferase reporter gene assay, and RNA immunoprecipitation, which will elucidate the new mechanisms underlying the malignant development of ccRCC, and provide novel insights and potential targets for the therapy against ccRCC.

肾透明细胞癌（ccRCC）恶性进展的分子机制和应对策略是泌尿外科的重点和难题。内皮祖细胞（EPC）可促进血管新生从而间接调控ccRCC的发生发展，但是其对ccRCC的直接作用尚不清楚。我们前期预实验发现：1、EPC可分泌外泌体（EPC-Exo）促进ccRCC增殖转移；2、EPC-Exo可向ccRCC细胞传递LINC00944；3、LINC00944可促进ccRCC恶性进展，其机制可能是作为竞争性内源RNA吸附miR-143。据此提出科学假说：EPC通过分泌外泌体向ccRCC细胞传递LINC00944吸附miR-143从而促进ccRCC发生发展。本项目拟在体内外水平观察EPC-Exo传递的LINC00944对ccRCC细胞增殖转移的影响，采用RNA-Seq、双荧光素酶实验、RIP等探索LINC001944的作用机制，以期阐明EPC促进ccRCC恶性进展的新机制，为其治疗提供新的思路和靶点。

肾透明细胞癌（clear cell renal cell carcinoma, ccRCC）侵袭转移的分子机制及应对策略是泌尿肿瘤学的难点及重点。既往研究发现内皮祖细胞（endothelial progenitor cell, EPC）可以促进血管新生从而间接调控ccRCC的发生发展；我们在前期预试验中发现EPC可以传递外泌体（EPC-Exo）直接调控ccRCC的恶性进展，进一步利用高通量测序及后续验证发现其主要通过传递长链非编码RNA：LINC00944发挥作用。在本项目中，我们进一步论证了LINC00944在ccRCC恶性进展中的作用及可能机制。研究发现：1、LINC00944在ccRCC细胞系中高表达，核质分离及FISH实验发现其在细胞核及胞质中均有所表达；2、细胞平板克隆及Edu实验提示LINC00944可促进细胞增殖；3、流式细胞术发现LINC00944可促进ccRCC周期进程、抑制细胞凋亡；4、Transwell提示过表达LINC00944可显著提高ccRCC细胞的侵袭、转移能力，western blot证实其可调控增殖转移相关蛋白；5、皮下荷瘤及肺转移模型在体内水平证实过表达LINC00944可显著提高ccRCC细胞的侵袭转移能力；6、机制探索发面，利用Pull-down及质谱检测等技术，发现LINC00944可与SRSF7蛋白结合调控下游靶基因MYC的表达水平，体内外实验发现SRSF7可促进ccRCC的增殖转移。以上试验证明LINC00944可促进ccRCC的增殖、转移，最终有望为ccRCC的早期诊断、预后评估和治疗靶点提供新的理论依据。