【作者】 崔恒祥；

【导师】 刘明耀； 李大力；

【作者基本信息】 华东师范大学 ， 生物医学， 2011， 博士

【摘要】 G蛋白偶联受体(G protein-coupled receptor, GPCR)是细胞膜上具有七次跨膜结构的受体蛋白,也是最大的一类膜蛋白分子。人类GPCR共有800多个成员,约占全部基因组编码基因的3-4%。GPCR与配体作用后,激活G蛋白,生成细胞内信使物质(如cAMP, cGMP, N0, DAG, IP等),调节细胞增殖、分化、迁移、死亡,参与几乎所有生理活动的调控。由于数目众多并且功能广泛,GPCR及其下游信号在多种重大疾病中扮演关键的角色,而且位于细胞膜上容易被药物识别和发生作用,因此GPCR是令人瞩目的药物治疗靶点。一直以来,GPCR研究与人类健康、医药产业和经济发展直接相关,是生物医学基础、转化与临床研究以及创新药物研发的重大前沿领域。但在众多的GPCR中,很多成员的功能还不确切,并且有一半以上是配体未知的孤儿受体。黏附家族GPCR是数目第二多的GPCR家族,一般认为在发育中他们参与调节细胞与细胞、细胞与细胞外基质之间的相互作用,但是这些受体的功能大多未知。因此深入研究黏附GPCR的功能,具有非常重要的理论和现实意义。作为黏附家族GPCR成员,据文献报导,GPR126在蛋白质一级结构和功能上都非常保守。在斑马鱼和小鼠中,Gpr126促进施旺细胞(Schwann cell)细胞的成熟,影响外周神经系统髓鞘的形成。GPR126完全敲除,将致小鼠在胚胎期E10.5-E12.5期间死亡,并且约50%的胚胎内存在出血现象。另外有报道GPR126可能参与调节炎症,能被LPS在内皮细胞内诱导表达。上述结论提示,GPR126可能在血管发育中调节血管生成或血管新生。本研究从GPR126在内皮细胞的表达开始,深入对该受体在血管新生过程的作用进行了研究。同时发现GPR126在人类结肠癌细胞系和组织芯片的肿瘤组织中高表达,进而对其在结肠癌增殖中所起的作用进行了研究。研究主要分以下两个部分：一.GPR126在血管新生中的功能研究1.GPR126在血管内皮表达丰富利用拟胚体形成模型证实了GPR126在VEGFR2阳性细胞群中的表达显著高于阴性细胞；免疫组织化学法证明GPR126在肢芽、心脏、肾和肺组织的血管中特异表达；蛋白印迹杂交实验证明,HMEC-1和HUVEC细胞系中GPR126表达丰富；在HMEC-1细胞内,GPR126能被VEGF、IGF-1和FGF-2能促新生血管生长因子诱导；RT-PCR方法证明,在分选的斑马鱼内皮细胞中,Gpr126表达丰富；2.干扰GPR126在内皮细胞的表达,削弱了内皮细胞的血管新生活性。HMEC-1细胞内沉默表达,削弱了内皮细胞在三维基质胶中的出芽形成过程、抑制了内皮细胞的增殖、迁移和在基质胶上的成管能力；3.抑制GPR126表达抑制体内生理和病理血管新生。Matrigel Plug实验和视网膜造影实验证明,GPR126调节VEGF诱导的基质胶内血管新生和低氧诱导的视网膜血管新生。4.在斑马鱼胚胎中沉默Gpr126的表达导致血管新生缺陷。利用模式生物斑马鱼为研究材料,靶向Gpr126的反义吗啉代引起胚胎的体节间血管(ISVs)新生过程受到了严重的抑制；人源GPR126mRNA和GPR126的反义吗啉代寡核苷酸显微注射入斑马鱼胚胎,ISVs的血管新生过程部分到恢复；进一步研究证实,靶向Gpr126的反义吗啉代寡核苷酸主要影响节段动脉(segmental arteries)内皮细胞的数目,调节内皮细胞从背主动脉出芽以及顶端细胞(tip cell)的迁移和增殖。5.GPR126通过GATA2和STAT5调节VEGFR2的表达。在HMEC-1细胞中干扰GPR126的表达,抑制了VEGF诱导的ERK和FAK活性。通过对该信号通路的进一步研究发现,GPR126调节了VEGFR2的表达；通过RT-PCR法对已报导的调节血管新生、特别是调节VEGFR2表达的转录因子进行筛选,干扰GPR126在HMEC-1细胞的表达下调节了GATA2和STAT5的mRNA水平；蛋白质印迹也证明GPR126在内皮细胞的沉默表达也促使GATA2和STAT5蛋白水平的下调。GATA2和STAT5都能直接结合到VEGFR2的转录调节区,调节VEGFR2的表达；PKA激活剂forskolin增加了内皮细胞HMEC-1内CREB活性,并且以剂量依赖的方式诱导增加了STAT5和GATA2的表达；进一步发现STAT5和GATA2的启动调节区存在CREB的直接结合。总结起来,GPR126通过PKA-CREB调节STAT5和GATA2的表达,进而调节了VEGFR2的表达。6. STAT5和GATA2可回复因GPR126干扰而致的斑马鱼血管新生缺陷GPR126反义吗啉代联合注射STAT5mRNA或者和GATA2mRNA,部分恢复只注射GPR126反义吗啉代导致的ISVs血管新生缺陷；STAT5mRNA和GATA2mRNA同时与Gpr126反义吗啉代联合注射,将很大程度上恢复只注射GPR126反义吗啉代导致的ISVs血管新生缺陷；在HMEC-1细胞中,高表达STAT5或/和GATA2,都能部分恢复由于GPR126表达下调而受到削弱的内皮细胞成管能力,也恢复了由于GPR126表达下调而受到削弱的VEGF诱导的ERK和FAK的激活。二.GPR126在结肠癌增殖过程中的作用研究1.GPR126在结肠癌细胞中的表达RT-PCR和蛋白免疫印迹证明,GPR126在各结肠癌细胞中有比较高的表达。利用免疫组织化学法检测了结肠癌组织芯片中GPR126的表达,结果表明GPR126在恶性结肠癌组织中的表达显著高于同一病人的癌旁和正常组织的表达。2.体外实验证明GPR126调节结肠癌细胞的增殖GPR126在结肠癌细胞中的干扰表达,抑制了多种结肠癌细胞的增殖活性。3.GPR126在体内调节结肠癌细胞的生长荷瘤实验证明,干扰GPR126在HT-29, HCT116和Lovo细胞中的表达,抑制了这些细胞在裸鼠中形成的肿瘤的生长。4.GPR126可能通过调节mTOR途径调节结肠癌细胞生长利用高通量RNA测序方法在转录组水平研究GPR126调节结肠癌细胞系HT-29增殖的分子机理。更多还原

【Abstract】 G protein-coupled receptors (GPCRs), also known as seven-transmembrane domain receptors (7TM receptors) comprise the largest protein family of transmembrane proteins. There are nearly800different human GPCR genes, about4%of the entire protein-coding genome, being predicted from genome sequence analysis. When ligands binding and activating these receptors, they transducer intracellular signal molecules, such as cAMP, cGMP, NO, DAG and IP, which modulating cell proliferation and differentiation, cell migration and death, thus participating in nearly all physiological processes. Due to broad spectrum of physiological functions and structural advantages, GPCRs are the most successful targets for moden medicine.Functional investigation of GPCR, a frontier field for medicinal chemistry, clinical research and drug discovery, has been shown to be important for pharmaceutical industry and human health. However, many members of the GPCRs have not been well characterized, and even more than half of them are still orphans, meaning their endogenous ligands are still unknown. As the second largest family, adhesion GPCRs are commonly considered to be involved in interaction of cell to cell, or cell to ECM, but the precise physiological roles of most of the adhesion GPCRs are to be identified. GPR126, as a member of adhesion GPCRs, is identified to be required for Schwann cell maturation in zebrafish and mouse during peripheral nerve development. GPR126knock-out mice were observed a sharp loss of viability occurs from10.5dpf to12.5dpf, accompanying with intra-embryonic hemorrhage in about50%(24/49) of embryos. And GPR126was initially isolated from HUVEC cultures that had been challenged with lipopolysaccharides or thrombin. All these indicate that GPR126might be implicated in endothelial cell function and in vascular development including processes of vasculogenesis and angiogenesis. In this thesis, we started the investigation of GPR126from its expression in endothelial cells, and subsequently revealed the role of GPR126in vascular angiogenesis and colon cancer progression.I. Role of GPR126in angiogenesis 1. Gpr126is highly enriched in endothelial cells and induced by pro-angiogenic factors in HMEC-1cellsUsing model of embryoid bodies (EB) formation and subsequent differentiation of embryonic stem cells, we identified Gpr126mRNA expression was highly enriched in Flkl-positive cells at day4of EB formation. And immunohistochemistry staining revealed that GPR126positive cells were specifically detected in the endothelial cells of vessels of embryonic limb bud, lung, heart and kidney. Western blot showed that GPR126expresses in HUVEC and HMEC-1cells. Meanwhile, VEGF, IGF-1and FGF-2increased GPR126expression in HMEC-1cells. And also RT-PCR assay revealed GPR126mRNA is highly enriched in Flk+endothelial cells sorted from zebrafish.2. Knockdown of GPR126impaired endothelial sprout formation in three-dimensional collagen, and also inhibited endothelial activity of proliferation, migration and tube formation.3. Inhibition of GPR126expression impairs physiological and pathological angiogenesis. Matrigel Plug assay and oxygen-induced retinopathy assay demonstrated that GPR126regulated VEGF induced angiogenesis in matrigel and ischemia-induced retinal neovascularization.4. Silencing of GPR126causes angiogensis defects during embryogenesis. Knockdown of Gpr126in zebrafish resulted to defects of intersegmental vessel formation, while coinjecting antisense morpholinos targeting Gpr126(Gpr126MO) with human GPR126mRNA in the embryos restored intersegmental vessel formation. Further investigation showed that antisense morpholinos targeting Gpr126dramatically reduced cell numbers of segmental arteries. The phenotypes of zebrafish Gpr126knockdown showed that the formation of dorsal aorta and the subsequent ECs sprouting from dorsal aorta to the horizontal myoseptum were obviously affected, and the subsequent endothelial tip cell migration and proliferation were severely inhibited.5. GPR126regulated VEGFR2expression via STAT5and GATA2.In GPR126deficient HMEC-1cells, VEGF induced ERK and FAK activities were downregulated. Further GPR126was found to regulate VEGFR2transcription. By RT-PCR and western blot methods, we identified both mRNA and protein of STAT5and GATA2were down-regulated. While both GATA2and STAT5were demonstrated to directly bind to the promoter of VEGFR2and regulate expression of VEGFR2in HMEC-1cells. Forskolin, a PKA agonist, increased CREB activity and upregulated GATA2and STAT5expression in dose dependent way in HMEC-1cells. Furthermore, chip assays revealed that there contain direct binding in promoters of GATA2and STAT5respectively.Together, GPR126regulates GATA2and STAT5expression through PKA-CREB pathway, through which further modulates VEGFR2expression in HMEC-1cells.6. STAT5and GATA2restored the angiogenic activity of endothelial cells attenuated by silencing of GPR126in vivo and in vitro. Coinjection of GATA2mRNA or STAT5mRNA with Gpr126MO partially restored ISVs growth which was disrupted by Gpr126MO alone. When STAT5and GATA2mRNA together were introduced with Gpr126MO at the same time, the impaired ISV morphology was greatly rescued than individual injection of STAT5mRNA or GATA2mRNA. And forced expression of STAT5and/or GATA2in GPR126deficit HMEC-1cells restored the accumulated length of tubes significantly compared to that of GPR126knockdown group in tube formation assay, and also regained VEGF induced activation of ERK and FAK which were attenuated by GPR126knockdown.II. Role of GPR126in Colorectal Cancer Proliferation1. GPR126overexpressed in colorectal cancer cellRT-PCR and Western blot experiment showed that GPR126mRNA and protein level was high. Taking advantage of immunohistochemistry staining, we confirmed that GPR126expression in the malignant tissues from colon cancer is significantly higher than that in tissues beside the malignant ones from colon cancer.2. Knockdown of GPR126in vitro inhibited proliferation of colorectal cancer cells.3. Knockdown of GPR126inhibited tumor growth in vivo. Using xenograft nude mice, we demonstrated that silence of GPR126in HT-29, HCT116and Lovo cells inhibited tumor growth.4. GPR126might regulate proliferation of colorectal cancer cells via mTOR pathway. RNA sequencing technology was used to investigate the mechanism of GPR126modulating proliferation of colorectal cancer cells. The result showed that knockdown of GRR126in HT-29cells decreased mTOR protein.更多还原