PGRN通过mTOR信号通路促进宫颈癌细胞迁移与侵袭

发布时间：2018-03-21 11:30

本文选题：宫颈癌　切入点：颗粒蛋白前体　出处：《山东大学》2017年硕士论文　论文类型：学位论文

【摘要】：宫颈癌作为最常见的女性生殖系统恶性肿瘤之一,已成为15-44岁女性的第二杀手,仅次于乳腺癌。2012年全球新发宫颈癌超过50万例,其中85%以上发生在发展中国家,死亡病例超过26万例。大量证据证明高危型HPV感染是宫颈癌致癌机制中的关键因素,但仅有很少部分的HPV女性感染者发展为宫颈癌,提示其他一些因素促进了宫颈癌的进展。癌细胞恶性进展的主要特征包括:生长信号自给、生长抑制信号耐受、逃脱程序性死亡、无限的复制潜能、持续的血管生成、组织侵袭与转移。转移是肿瘤进展的终极且最凶险阶段,超过90%的癌症患者死于转移而非原发性肿瘤。宫颈癌的进展阶段性明显,包括正常宫颈、宫颈上皮内瘤变、原位癌、局部侵袭性癌和远处转移癌。早期宫颈癌(Ⅰa期-Ⅱa期)患者一般预后较好,但其中1/3死于转移和复发,晚期特别是有远处器官或淋巴结转移的宫颈癌(Ⅱb期-Ⅳ期)患者则难以达到根治目的。盆腔淋巴结转移是宫颈癌的重要扩散途径,有无淋巴结转移直接影响患者的预后。探讨宫颈癌的侵袭和转移的机制对增强疗效、防止复发和提高患者生存质量具有重大意义。癌细胞转移分为多个步骤,包括癌细胞从原位肿瘤脱离,侵入周围组织,内渗入血管或淋巴管,随血流或淋巴系统散播,最终外渗并在远端器官生长。每一步骤均需要独特的分子程序的参与,其中癌细胞黏附与迁移的调控和细胞骨架性质发挥重要的作用。此外,肿瘤转移的起始与癌细胞形态学改变,即上皮-间质转换(epithelial-mesenchymal transition,EMT)有关。颗粒蛋白前体(progranulin,PGRN)是一种新型多功能生长因子,具有促进细胞增殖、生存、迁移、抗炎等功能特点,参与多种重要的生理和疾病进程。PGRN最初被鉴定为癌细胞的生长因子,在许多肿瘤中高水平表达,与肿瘤的不良预后相关,并介导乳腺癌、卵巢癌、前列腺癌、膀胱癌和肝癌等的发生与发展。PGRN与癌细胞侵袭转移关系密切,可增强成纤维细胞的运动能力,驱动SW13和MCF-7细胞穿过基质胶滤膜,刺激乳腺癌细胞产生血管内皮生长因子和血管生成素,并可促进癌细胞基质金属蛋白酶的产生等,而近期的研究发现PGRN可通过增强EMT程序介导卵巢癌细胞的迁移与侵袭。我们的前期研究首次报道了 PGRN作为肿瘤微环境因素在宫颈癌中高表达,并促进宫颈癌细胞恶性增殖与转化。然而,PGRN在宫颈癌细胞的迁移与侵袭中的功能,以及PGRN对癌细胞迁移、侵袭关键机制的调控作用仍未见报道。目的:本论文旨在明确PGRN在宫颈癌细胞运动、迁移与侵袭中的功能;分析PGRN对宫颈癌细胞EMT和细胞骨架重排的影响,探索PGRN促进宫颈癌细胞迁移的分子机制。方法:利用重组人 PGRN(recombinant human PGRN,rhPGRN)处理宫颈癌细胞系(HeLa、SiHa)使用划痕修复实验、transwell迁移和侵袭实验,检测PGRN对宫颈癌细胞运动、迁移和侵袭能力的影响;免疫印迹法分析EMT相关蛋白谱表达的变化;比较正常宫颈组织和宫颈癌组织中PGRN和p-mTOR的蛋白水平表达变化及其相关性,检测mTOR下游蛋白的激活及PGRN调控mTOR的分子机制;采用F-actin免疫染色和激光共聚焦显微镜观察,表征宫颈癌细胞迁移相关细胞结构的改变;蛋白水平分析细胞骨架重排相关LIMK/cofilin信号途径的活化水平及FAK等信号分子的磷酸化,并分析PGRN对Rho小GTP酶活性的影响;利用FAK、Rho小GTP酶等的抑制剂,分析PGRN调控宫颈癌细胞迁移与侵袭对FAK和Rho小GTP酶的依赖性。结果:rhPGRN能够增强宫颈癌细胞运动、迁移和侵袭能力;PGRN对宫颈癌细胞EMT的影响较小,但可促进细胞骨架重排,表现为压力纤维增强、板状伪足和丝状伪足增多等;PGRN通过TNFR2激活mTOR信号通路,且在宫颈癌组织中与p-mTOR具有相关性,雷帕霉素抑制mTOR降低了宫颈癌细胞的迁移与侵袭;PGRN可激活细胞骨架重排相关的LIMK/cofilin信号途径,同时促进FAK的磷酸化水平增强;PGRN通过mTOR信号途径增强宫颈癌细胞中RhoA和Racl的活性,PGRN介导的宫颈癌细胞迁移和侵袭依赖于FAK、Racl和RhoA等的活化。结论:本论文证实了 PGRN具有促进宫颈癌细胞运动、迁移与侵袭的能力,首次发现PGRN主要通过mTOR信号途径促进细胞骨架重排能力调控癌细胞迁移,并深入分析了 FAK和mTOR活化的Rho小GTP酶介导的信号途径在其中的重要作用。这一研究发现了 PGRN促进宫颈癌侵袭转移,从而参与宫颈癌恶性进展的新机制,为控制宫颈癌侵袭转移提供了新的靶点;本研究首次提出PGRN对细胞骨架重排的调控作用,并将PGRN介导的FAK与Rho/LIMK/cofilin信号途径联系起来,为PGRN信号通路及生物学功能研究提供了新的视角。
[Abstract]:Cervical cancer is one of the most common malignant tumor of the female reproductive system, has become a 15-44 year old female second killer after breast cancer.2012 global new cervical cancer more than 500 thousand cases, of which more than 85% of deaths occur in developing countries, more than 260 thousand cases. A lot of evidence of high-risk HPV infection is the key factor for cervical cancer carcinogenesis the only small part of HPV infected women develop cervical cancer, suggesting that some other factors to promote the progression of cervical cancer. Key features include: the malignant progression of cancer cell growth inhibitory signal signal self-sufficiency, tolerance, evasion of programmed death, unlimited replicative potential, sustained angiogenesis, tissue invasion and transfer. Transfer is the ultimate and most dangerous tumor progression stage, more than 90% of cancer patients died of metastasis and primary tumor. Advanced stage cervical cancer significantly, including Normal cervix, cervical intraepithelial neoplasia and carcinoma in situ, local invasive carcinoma and metastasis carcinoma. Early cervical cancer (stage I a - II a period) the general prognosis is better, but the 1/3 died of recurrence and metastasis, especially in advanced distant organs or lymph node metastasis of cervical cancer (stage II B - IV) patients is difficult to achieve the purpose of cure. Pelvic lymph node metastasis is an important way to spread of cervical cancer, lymph node metastasis directly affect the prognosis of patients of cervical cancer. The invasion and metastasis mechanism to enhance the curative effect, prevent recurrence and is of great significance to improve the quality of life of patients. The metastasis of cancer cells is divided into multiple steps, including cancer cells from the tumor in situ, invade surrounding tissue, infiltrated the blood or lymphatic vessels, with blood or lymphatic system spread, eventually extravasation and growth in the distal organs. Each step requires the participation of unique molecular programs, including cancer The regulation of cytoskeleton and cell adhesion and migration properties play an important role. In addition, the initial change of tumor metastasis and tumor cell morphology, namely epithelial mesenchymal transition (epithelial-mesenchymal transition, EMT). Progranulin (progranulin, PGRN) is a multifunctional growth factor, can promote cell proliferation survival, migration, anti-inflammatory and other features, is involved in many important physiological and disease progression of.PGRN was originally identified as a growth factor in cancer cells, the expression level is high in many cancers, poor prognosis associated with tumor, and mediated breast cancer, ovarian cancer, prostate cancer, metastasis and invasion of bladder cancer and hepatocellular carcinoma the occurrence and development of.PGRN and cancer cells, can enhance the ability of motion of fiber cells, drive SW13 and MCF-7 cells through Matrigel membrane, stimulated breast cancer cells and vascular endothelial growth factor production Ang, and can promote cancer cell matrix metalloproteinase production, but recent studies have found that PGRN can be enhanced by the invasion and migration of ovarian cancer cells mediated by EMT program. Our previous study reports for the first time PGRN as the tumor microenvironment factors in cervical cancer and high expression, and promote malignant cervical cancer cells cell proliferation and transformation. However, PGRN on migration and invasion of cervical cancer cells and the function of PGRN on cancer cell migration, invasion and regulation of key mechanism has not been reported. Objective: the aim of this paper is to clear the PGRN movement in the cervical cancer cell migration and invasion in function; analysis on the effect of PGRN on cervical cancer cell line EMT and cytoskeletal rearrangement, to explore the molecular mechanism of PGRN promoting cervical cancer cell migration. Methods: using recombinant human PGRN (recombinant human PGRN, rhPGRN) treatment of cervical cancer cell lines (HeLa, SiHa) using scratch repair After experiment, the invasion and migration of experimental Transwell, detection of PGRN on cervical cancer cells, effects of migration and invasion; analysis of the changes of the expression of EMT related protein immunoblotting; protein levels of PGRN and p-mTOR in normal cervical tissue and cervical cancer tissue expression changes and correlation detection, molecular mechanism of activation and regulation of PGRN mTOR mTOR downstream proteins; using the microscope F-actin staining and confocal laser, related to the change of cell structure characterization of cervical cancer cell migration; protein level analysis and activation level of FAK signaling molecules cytoskeleton related LIMK/ cofilin signaling pathway, phosphorylation, and analysis the influence of PGRN on Rho GTP activity; the use of FAK, GTP enzyme inhibitor Rho small, analysis of PGRN expression of cervical cancer cell migration and invasion depends on FAK and Rho GTP enzyme. Results: rhPGRN can enhance the cervical Cancer cell motility, migration and invasion; PGRN has little effect on cervical cancer EMT cells, but could promote the cytoskeleton, as pressure reinforced, lamellipodia and filopodia increased; PGRN through TNFR2 and mTOR signal pathway in cervical cancer tissues and the correlation with p-mTOR, mTOR reduced the inhibitory effect of rapamycin the migration and invasion of cervical cancer cells; PGRN activation of LIMK/cofilin signaling pathway related to cytoskeleton rearrangement, while promoting the phosphorylation level of FAK increased; PGRN enhanced RhoA and Racl activity in cervical cancer cells through the mTOR signaling pathway, PGRN mediated migration and invasion of cervical cancer cells depends on the activation of Racl and RhoA FAK. Etc.. Conclusion: This study confirmed that PGRN can promote the movement of cervical cancer cell migration and invasion ability, for the first time that PGRN mainly through the mTOR signaling pathway promotes cell skeleton rearrangement regulating ability Control of cancer cell migration, and in-depth analysis of the FAK signaling pathway and activation of mTOR Rho GTP enzyme mediated in the important role. This study found that PGRN promotes the invasion and metastasis of cervical cancer, a new mechanism to participate in the malignant progression of cervical cancer, for the control of cervical cancer invasion and metastasis will provide a new target; this is the first study of the role of PGRN in regulation of cytoskeletal rearrangement, and the link between FAK and Rho/LIMK/cofilin signaling pathway mediated by PGRN, provides a new perspective for the study of PGRN signaling pathway and biological function.

【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R737.33

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