OLA1通过HSP70降解p53蛋白促进结直肠癌细胞生长的机制研究

Colorectal cancer (CRC) is one of the most malignant tumors in China, which threatening the health of citizens. Illustrate the biological features of cancer cells by detecting cancer associated gene is good for the diagnosis and cure of cancer. The expression of OLA1 is heavily elevated in CRC than intestinal mucosa. Higher expression of OLA1 predicts poor survival. Besides, OLA1 knock out CRC cells represent growth retardation. These data indicated that OLA1 is a cancer associated gene in the process of colorectal cancer growth. To further investigation, we found p53 protein elevated in OLA1 knock out CRC cells, which ascribed to the prolonged degradation. Previous studies have proved that OLA1 interacts with HSP70, meanwhile, HSP70 promoted the degradation of p53. So, we hypothesize that OLA1 interacts with HSP70 to promote the growth of CRC by degrading p53.This proposal focuses on 3 parts: First, verify the role of OLA1 in the process of CRC growth by OLA1 overexpression or knock out CRC cells and OLA1 conditional knock out mice; Then, clarify p53 is central for OLA1 promoting CRC growth by gene knock down and small molecular inhibitor; Finally, explore the direct interactions between OLA1 and HSP70. The study will enrich the mechanism of CRC formation and development, and provide potential target for clinical treatment.

结直肠癌是严重威胁国民健康的恶性肿瘤，寻找肿瘤相关基因阐明其生物学特征有重要意义。前期研究发现：OLA1在结直肠癌中表达升高，且与不良预后相关；OLA1敲除的肠癌细胞生长受抑，提示OLA1参与结直肠癌细胞的生长过程。机制探索发现OLA1敲低的肠癌细胞中p53蛋白表达上调，蛋白半衰期延长。结合文献报导：OLA1与HSP70直接结合；HSP70敲除后p53蛋白降解减少，由此假设: OLA1通过HSP70降解p53蛋白促进结直肠癌细胞生长。本研究拟构建OLA1差异表达的肠癌细胞和肠道OLA1特异性敲除的小鼠经体内外实验明确OLA1在结直肠癌细胞生长中的作用，并通过小分子抑制剂、基因敲降/过表达技术鉴定p53是OLA1影响结直肠癌细胞生长的关键分子，最后验证OLA1与HSP70的相互作用及其调控p53促进结直肠癌细胞生长的具体机制。该研究可丰富结直肠癌发生发展机制，为临床干预提供潜在靶点。

研究背景：.结直肠癌是人类最常见的恶性肿瘤之一，期发病率和死亡率均占所有恶性肿瘤第5位，2015年我国新增结直肠癌患者36万例，严重威胁国民健康。结直肠癌的发生、发展是一个多因素、多阶段的复杂过程，常伴有遗传和表观遗传的变化，其中包括原癌基因的激活、抑癌基因的失活等。对肿瘤相关基因的深入认识对结直肠癌的临床转化有重要的意义。.研究内容：.1..在前期预实验的基础上从细胞水平、动物水平和人体组织水平挖掘OLA1的表达与结直肠癌的生长及长期预后的关系.2..通过RNAseq检测OLA1不同表达的肠癌细胞中基因表达的差异.3..验证并鉴定OLA1通过HIF1a/CA9信号轴调控肠癌细胞的生长。.研究结果：.1..OLA1在结直肠癌中表达升高且高表达的OLA1提示不良预后：通过分析TCGA和GEO数据库以及本中心手术的肠癌及正常配对黏膜组织显示：在结直肠癌中，OLA1的mRNA表达及蛋白表达均高于正常黏膜，OLA1表达越高，预后越差。.2..沉默OLA1抑制肠癌细胞的增殖和成瘤能力：通过Crispr-caspase9构建OLA1敲除的肠癌细胞系。体外实验和体裸鼠皮下成瘤实验表明OLA1敲除后实验组细胞的增殖减慢，克隆形成能力下降，皮下成瘤能力减弱。.3..OLA1敲除的肠癌细胞系中HIF1信号通路受抑制：通过Affymetrix Human Genome U133 Plus 2.0 Array分析两组细胞mRNA，进通过基因富集分析及KEGG通路分析发现，在OLA1敲除后HIF1a下游通路相关分子的表达收到抑制.4..通过免疫组化在组织水平验证OLA1敲除的肠癌细胞中HIF1α、CA9表达降低。.科学意义：.该研究表明OLA1是结直肠癌发生发展相关基因，与结直肠癌的不良预后直接相关，且初步对其信号通路进行了证明，为后续进行深入的机制研究及药物靶点的开发提供了基础。