UCH-L3对HIF-1表达的调节及其对乳腺癌生长和侵袭转移影响的体内外研究

The process of tumorigenesis and progression is dominanted by both parenchymal cells and their microenvironment in tumor, regulated by a variety of growth factors ,accompanied by several oncogen molderations and participated by many signal pathways.Over expression of HIF-1a under hypoxic condition is helpful for tumor cell to keep surviving,adapting hypoxic metabolisms, promoting angiogenesis,avoiding apoptosis and even going on invasiong and metastasis furthuer. Our previous study found that RNA interrence of HIF-1a increased apoptosis,slowed cell growth and attenuated invasion and migration of breast cancer cells. Ubiquitin and protease system mainly degrades decrepit or damaged and regulatory proteins or peptide in variety of cellular process. Deubinquitination hydrolyses and release ubinquitin from polyubinquited protein and avoids ubinquitin to be hydrolysed by protease and polyubinquited proteins over hydrolysed .HIF-1 is a transactive factor induced by hypoxia and its expression is mainly due to suppressive degradation by ubiquitin protease. UCH-L3 is a kind enzyme of deubiquitination, which dissociate Ub from polyubiquitined complex and then release Ub so that hydrolysis of Ub is avoided and degradation of ubiquitined protein is limited in a exact fashion. Our previous study found that siRNA of HIF-1α increased the expression of UCH-L3 implying that UCH-3 possible being a target of HIF-1.HIF-1 regulates the transcription of UCH-L3 negatively, and conversely UCH-L3 limits the degradation of HIF-1α. In this project, expression vector and interference RNA of UCH-L3 is constituted and tranfected into cells respectively. Western blot is used to detect the protein expression of Ub, UCH-L3, HIF-1α and HIF-1 target proteins associated with apoptosis,invasion and metastasis as well as kinase of signal pathway. Apoptosis is tested by Flow Cytometry and Hoechst stain.The capability of immigration and invasion is measured by Would healing and Matrigel transwell. Transplanted and metastatic tumor model in nude mouse is set up via injection of cells with higher or lower UCH-L3 expression cell line under skin of breast and then analyzed to verify those detections in vitro.Human breast cancer tissues are studied by immunohistochenmystry by comparing the invasive and metastatic state of tripple negative breast cancer with ER/PR or HER2 positive breast cancer.group.This project is aimd to investigate the relationship of UCH-L3 and HIF-1,provide some potential clues to decrease the expresion of HIF-1α and supress its role in tumor growth and malignant processes.

HIF-1是恶性肿瘤在缺氧时保持生存、凋亡抑制、侵袭乃至远处转移能力的主要原因。前期研究发现干扰HIF-1a，MDA-MB-231 乳腺癌细胞生长减慢、凋亡增加、侵袭和迁移能力下降，泛素羧基末端水解酶UCH-L3表达升高，推测UCH-L3可能受HIF-1负反馈调节。UCH-L3表达载体转染细胞又能降低HIF-1a mRNA表达，可见HIF-1和UCH-L3之间存在相互影响；本项目拟构建UCH-L3表达载体和RNA干扰体分别转染细胞，检测细胞内泛素、UCH-L3、HIF-1a和与HIF-1凋亡、侵袭和转移相关蛋白表达以及信号通路活化；流式和Hoechst染色检测细胞凋亡；划痕和穿膜实验检测迁移侵袭；细胞计数和MTT检测细胞生长能力，建立UCH-L3过表达或低表达裸鼠移植瘤和转移瘤模型验证体外发现；体内对比人乳腺癌组织，探讨UCH-L3对HIF-1a表达的影响，寻找抑制HIF-1a表达的新途经

缺氧、基因改变和生长因子及其受体的过度表达和信号通路的活化是肿瘤发生和转移等恶性演进的重要因素，低氧诱导因子（HIF-1）受其诱导而活化，保障了恶性肿瘤在缺氧时保持生存、凋亡抑制、侵袭乃至远处转移能力的主要原因。三阴乳腺癌细胞MDA-MB-231呈现HIF-1a的高表达和高侵袭转移能力。前期研究发现干扰HIF-1a，MDA-MB-231 乳腺癌细胞生长减慢、凋亡增加、侵袭和迁移能力下降。HIF-1的活化取决于HIF-1a的表达，HIF-1a主要通过泛素蛋白酶体系降解。UCH-L3是去泛素化酶的一种，属于羧基末端水解酶家族成员。对泛素化过程起着调控作用。越来越多的证据表明UCH-L1和UCH-L3的异常表达与肿瘤的生长和演进有关。本项目蛋白质组学研究显示干扰MDA-MB-231细胞的HIF-1a蛋白，泛素羧基末端水解酶UCH-L3表达升高。然而UCH-L3表达载体转染细胞又能降低HIF-1a 蛋白表达，可见HIF-1和UCH-L3之间存在相互影响。免疫共沉淀实验表明HIF-1a和泛素结合，并且UCH-L3过表达细胞内游离的HIF-1a和泛素化的HIF-1a均明显低于UCH-L3低表达细胞。蛋白酶体抑制剂未能显示增加HIF-1a蛋白的作用，推测UCH-L3促进了HIF-1的降解可能不依赖于蛋白酶体。高表达的UCH-L3削弱MDA-MB-231细胞增殖、侵袭、迁移和克隆形成能力，促进其凋亡，裸鼠移植瘤生长变慢。siＲＮＡ干扰UCH-L3,一定程度上增加HIF-1a的表达，反向验证了UCH-L3与HIF-1之间的相互负反馈调节作用。然而，UCH-L3高表达却增加MCF-7细胞的HIF-1a蛋白，并增强其生长、迁移、侵袭和克隆形成能力。说明UCH-L3对乳腺癌细胞生物学行为的影响取决于乳腺癌细胞系本身的特征，依赖于HIF-1a的表达。构建UCH-L3高表达的措施可能为三阴乳腺癌的治疗提高新的思路和靶点。