AGGF1-SIRT2轴通过调控自噬抑制肾癌发生的分子机制研究

Renal cell carcinoma (RCC) is the most common malignant tumor of renal parenchyma. It becomes particularly important to find new markers for early diagnosis of RCC. It has been reported that angiogentic factor AGGF1 could promote tumor angiogenesis, but the role of AGGF1 in tumor cells remains unclear. The applicant found a specifically low expression of AGGF1 in 96 cases of RCC through immunohistochemistry assay, and result turned out that AGGF1 could inhibit cell cycle and proliferation, and thus promote apoptosis of A498 cell line. Our previous studies showed that under starvation, AGGF1 could interact with SIRT2 promoting alpha-tubulin acetylation. AGGF1 might affect the activity of SIRT2. Further study showed that AGGF1 could increase the expression of LC3B, a protein essential for autophagy initiation, presenting for huge possibility for AGGF1 to inhibit RCC through autophagy pathway. Suggesting that AGGF1 can interact with SIRT2 and inhibit SIRT2 activity so as to regulate alpha-tubulin acetylation, and further activate autophagy by increasing the expression of autophagy gene LC3B, thus inhibiting RCC. Based on our preliminary results, in Aim 1 we propose to map the interaction domain between AGGF1 and SIRT2 using Co-IP assay, and clarify the relationship between AGGF1, SIRT2 and Ace-tubulin through Western blot and ELISA with SIRT2 deacetylase detection kit. In Aim 2 we propose to investigate the molecular mechanism and signaling pathway of LC3B expression and autophagy initiation, after that the role of AGGF1/SIRT2/Ace-tubulin in autophagy pathway was evaluated by autophagy activation and inhibition agent. And in Aim 3 we propose to exam the effect of AGGF1/SIRT2/Ace-tubulin/LC3B pathway to the autophagy activation and RCC occurrence. Successful completion of experiments outlined in this proposal will enrich our current understanding of the role of AGGF1 in tumor progression, and may provide novel therapeutic strategies and targets for renal cell carcinoma.

肾细胞癌是最常见的肾实质恶性肿瘤，因此早期诊断和治疗显得尤为重要。血管生成因子AGGF1可促进肿瘤血管生成，但其在肾癌细胞中的作用尚不清楚。前期研究发现AGGF1在肾癌中低表达，且发现AGGF1导致肾癌细胞周期阻滞、增殖抑制和凋亡。进一步研究发现，AGGF1与SIRT2相互作用，促进alpha-tubulin乙酰化以及自噬基因LC3B的表达。据此推测AGGF1-SIRT2轴通过激活自噬抑制肾癌发生。本项目拟从3个方面进行研究：①AGGF1调节SIRT2活性及alpha-tubulin乙酰化的具体机制；②AGGF1促进LC3B表达并激活自噬的信号通路；③体内和体外实验验证AGGF1/SIRT2/Ace-tubulin/LC3B通路激活自噬影响肾癌的发生。本项目如获资助并顺利实施，将揭示AGGF1在肿瘤发生发展中的新功能，为肾癌的预防与治疗提供新的思路。

肾癌是最常见的肾实质恶性肿瘤，起病隐匿，一经发现就是晚期。血管生成因子在肿瘤的发生发展中起着极为重要的作用，是肾癌临床治疗的靶点。血管生成因子AGGF1被报道与肿瘤血管生成密切相关，然而其在肿瘤细胞中的作用未见报道。在本课题中，我们通过生物信息学分析和肾癌样本免疫组化分析，发现AGGF1在肾癌中低表达，且低表达的AGGF1预示患者较低的生存率，推测AGGF1在肾癌发生发展过程中起重要作用。进一步体外细胞实验研究发现，过表达AGGF1可以抑制肾癌细胞周期和增殖，促进凋亡，体内小鼠荷瘤实验发现，过表达AGGF1抑制肿瘤生长，而敲低AGGF1表达促进肿瘤生长。自噬作为一种抑制肿瘤的机制已被广泛证实。我们前期研究发现，在肾癌细胞中，在饥饿条件下过表达 AGGF1，促进自噬分子标记 LC3B 的表达。进一步研究发现，AGGF1通过调控SIRT2/Ace-tubulin/JNK通路激活自噬， 抑制肾癌发生。微管是细胞骨架的重要组成部分, 微管蛋白乙酰化影响微管的稳定性，而自噬聚集体的形成与动力蛋白介导的自噬体转运都依赖于动态微管转运支架。本研究发现，AGGF1同SIRT2相互作用并抑制Sirt2活性，促进微管alpha-tubulin乙酰化。alpha-tubulin 乙酰化激活 JNK，P-JNK磷酸化 Bcl-2 释放 Beclin1 从而诱导自噬体形成。同时，我们还发现，在肿瘤细胞中，AGGF1同抑癌因子P53相互作用，并介导P53的磷酸化和乙酰化修饰，从而阻断MDM2介导的P53泛素化降解，进而稳定P53蛋白表达，抑制肿瘤细胞生长。本研究详述了AGGF1 在肿瘤细胞中作为“肿瘤抑制因子”抑制肿瘤发生发展的具体分子机制，将为肾癌的防治策略和药物靶点提供新的重要线索和理论依据。

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