肾细胞癌分泌型miRNA作为新型液体活检分子标志物价值及参与肿瘤微环境调节机制研究

Circulating miRNAs have emerged as novel potential markers for renal cell carcinoma (RCC). Secreted miRNAs in peripheral blood are a class of circulating miRNAs that were activity secreted from cells and have physiological functions, nevertheless, whether secreted miRNAs are more valuable than circulating miRNAs still currently unknown. Our newly studies found that the expression level of some RCC secreted miRNAs in serum from RCC patients were significant different from controls. Furthermore, secreted miRNAs can be delivered into fibroblasts (FB) and vascular endothelial cell (VEC) through exosome. In addition, bioinformatics analysis indicated that these dysregulated secreted miRNAs may participate in the alteration in functions of those two kinds of cells as well as involved in angiogenesis and tumorigenesis. Thus, we hypothesize that the serum levels of RCC secreted miRNAs are novel liquid biopsy molecular markers for RCC diagnosing, monitoring and prognosis, and some important secreted miRNAs can enter into FB and VEC, change their phenotype and function and be involved in the development and progression of RCC. In order to verify our hypothesis, we will adopt clinical samples, cell lines and tumor-xenograft mouse model to fully explore the potential clinical value of RCC secreted miRNAs as novel liquid biopsy molecular markers, and further choose some key secreted miRNAs to determine their biological functions and the potential molecular mechanisms in regulating tumor microenvironment. The purpose of our present study is to provide novel theoretical basis and potential molecular targeting therapeutic strategies for RCC.

循环miRNA有望成为肾细胞癌（RCC）新型标志物；外周血分泌型miRNA是细胞主动分泌的一类具备生理功能的循环miRNA，但是否较循环miRNA更具价值尚不清楚。我们新近研究发现，RCC细胞分泌的一些miRNA在RCC患者血清中水平与对照相比变化显著，能以exosome为载体进入成纤维细胞（FB）和血管内皮细胞（VEC）；生物信息学预测它们可参与FB、VEC功能调节和血管生成。据此提出假说：血清中RCC分泌型miRNA是RCC诊断、疗效监控和预后判断新型液体活检分子标志物；特定分泌miRNA以exosome为载体进入肿瘤微环境相关FB和VEC，参与细胞功能调节、血管生成和RCC发生发展。为验证假说，我们拟通过临床标本、细胞及动物研究，全面探讨RCC分泌型miRNA作为新型分子标志物潜能；明确关键分泌型miRNA功能及参与肿瘤微环境调节机制，为RCC的诊断、分子治疗提供新的策略和理论依据。

外周血中细胞外囊泡（EVs）miRNA有望成为新型液体活检分子标志物，但其对于肾细胞癌（RCC）临床价值尚不清楚。本项目通过低密度芯片和RT-qPCR技术，发现6种miRNA，包括miR-200a，miR-28-3p，miR-1826，miR-103，miR-1249和miR-640在RCC患者血清EVs中显著升高，且在术后患者血清中均显著降低；其中，6种miRNA组合临床价值最高，可作为RCC患者诊断、早期诊断、分级、疗效监控和预后判断新型液体活检分子指标；同时，RCC细胞分泌的EVs多种miRNA亦具备作为其分子标志物潜能，并可携带miRNA进入肿瘤微环境相关血管内皮细胞和成纤维细胞，调控细胞表型和功能变化。进一步通过系列临床组织样本、RCC细胞系、生物信息学分析和细胞实验，发现与RCC患者血清EVs中miR-200a表达显著上调不同，miR-200a在RCC组织、细胞系中表达均呈现出显著降低趋势，其靶基因CBL和E2F3在RCC组织和细胞系中表达显著升高；体外细胞生物学功能实验显示，miR-200a可通过调控CBL基因和E2F3的表达，参与RCC细胞迁移、凋亡、增殖和细胞周期阻滞等生物学功能，miR-200a/CBL和miR-200a/E2F3调控通路与RCC发生、发展密切相关。小鼠RCC移植瘤模型研究结果显示，过表达miR-200a可显著抑制RCC肿瘤生长，过表达CBL则可显著促进RCC肿瘤生长，而过表达CBL同时过表达miR-200a可明显减缓肿瘤生长，miR-200a/CBL调控轴可作为RCC分子治疗的潜在靶点。目前，本项目已发表11篇论文（含SCI论文5篇），协助培养博硕士7名，参加检验医学领域国内外学术交流10余人次，多次参与优秀论文汇报并获奖。项目研究成果可为RCC新型分子标志物研发、诊断、疗效监控、预后评估及疾病分子机制研究提供新的思路和理论依据。