生长抑制特异性转录因子5与苹果酸脱氢酶1相互调控抑制膀胱癌侵袭转移的分子机制

Invasion and metastasis remains as the leading cause of mortality in patients with bladder transitional cell carcinoma (BTCC). We previously demonstrated that one of the long non-coding RNAs, Growth Arrest Specific transcript 5 (GAS5), plays a crucial role in preventing proliferation and invasion of tumor cells. Our preliminary results in vitro also showed that GAS5 can bind and degrade Malate Dehydrogenase 1 (MDH1) through protein ubiquitination. MDH1 is one of the rate-limiting enzymes in Krebs cycle and Reverse Krebs cycle, involving in cellular energy metabolism. Recent studies suggest that upregulation of MDH1 may increase intracellular glutamine, which actives mTOR signal pathway and leads to GAS5 suppression. We therefore, hypothesize that reciprocal regulation of GAS5 and MDH1 may be recognized as a GAS5-MDH1-Glutamine-GAS5 loop, which in turn affects energy and material metabolism, preventing BTCC invasion and metastasis. The goal of our current study is to identify the mechanisms of the interaction between GAS5 and MDH1. Moreover, this study will further characterize the effect and illustrate the molecular mechanisms of GAS5-MDH1-Glutamine-GAS5 regulation loop that supresses BTCC invasion and metastasis. Specifically, we will be utilizing laser capture microdissection technology, gene transefection and interference and mass spectrometry analysis, etc. It will help further understanding the mechanism for tumor metastasis, identifying potential therapeutic targets for BTCC.

膀胱尿路上皮癌（BTCC）侵袭转移是患者死亡的主要原因。申请人前期研究发现一种lncRNA——生长抑制特异性转录因子5（GAS5）是抑制BTCC增殖、侵袭的重要分子。预实验显示GAS5与苹果酸脱氢酶1（MDH1）结合，通过泛素化修饰降解MDH1蛋白，抑制BTCC侵袭转移。MDH1是三羧酸循环和三羧酸循环反向途径中重要的代谢酶，不但参与细胞能量代谢，还影响谷氨酰胺（Gln）代谢，进而激活mTOR通路，抑制GAS5表达。因此推测，在BTCC中存在GAS5-MDH1-Gln-GAS5调控环路，GAS5与MDH1相互调控可影响肿瘤细胞物质和能量代谢，从而抑制BTCC侵袭转移。本项目拟通过激光捕获显微切割技术、基因转染和干扰以及质谱分析等方法，阐明GAS5与MDH1相互调控的分子机制；探讨上述调控环路抑制BTCC侵袭转移的作用和机制，为深入理解膀胱癌转移机制和寻找新的治疗靶点提供理论依据。

膀胱移行细胞癌（BTCC）的治疗存在明显个体差异，近25%的患者在手术和化疗后肿瘤转移，预后不佳。生长抑制特异性转录因子5（GAS5）是抑制BTCC增殖、侵袭的长链非编码RNA（lncRNA）。本课题通过外泌体miRNA与lncRNA靶向结合预测分析技术寻找到与GAS5互作的exo-miR-491-5p，结合运用生物信息学、扫描电镜技术、二代测序技术、免疫荧光技术、活体成像技术等，对exo-miR-491-5p与GAS5相互作用进行了进一步验证。粒样髓系抑制细胞（G-MDSCs）是来源于骨髓的一种免疫抑制细胞。我们在临床中发现，G-MDSCs能够促使膀胱癌肿瘤微环境发生“免疫重塑”，吉西他滨化疗后能够促进这一现象。通过流式细胞术、体外共培养技术、体内细胞过继传输技术，我们验证了吉西他滨作为一种免疫调节因素招募G-MDSCs的作用，且G-MDSCs能够通过其外泌体对膀胱癌细胞产生“免疫训导”，即通过exo-miR-491-5p与GAS5相互结合，促进膀胱癌进展与转移。综合上述结果，本研究为GAS5/exo-miR-491-5p/G-MDSCs介导的BTCC进展奠定了较为扎实的理论基础。同时对于认识吉西他滨对BTCC肿瘤微环境发生免疫重塑作用提供了重要的启示性线索。项目资助发表SCI论文4篇，培养硕士生1名，其中1名已经顺利毕业并获得学位。项目投入经费57万元，支出52.3049万元，各项支出与预算相符。剩余经费4.6951万元，剩余经费计划用于本项目研究后续支出。