LAMP3介导上皮间质转化在绒癌侵袭迁移中的作用及机制研究

发布时间：2018-04-02 14:09

本文选题：EMT　切入点：绒癌　出处：《北京协和医学院》2017年博士论文

【摘要】：由于有效治疗措施如化疗的出现及人绒毛膜促性腺激素作为肿瘤标记物的广泛应用,妊娠滋养细胞肿瘤(Gestational trophoblastic neoplasia,GTN)成为有可能仅依靠化疗治愈的第一个妇科实体瘤,也是人类最早得以治愈的实体瘤之一。然而,仍然会有患者因为出现化疗耐药和多发转移而导致疾病进展。其中,转移性滋养细胞肿瘤主要经血行播散,转移发生早而且广泛,可同时出现原发灶和转移灶,而且很多患者原发灶消失,仅表现为转移灶症状,使疾病的诊断和治疗都变得更加困难。近年来学术界普遍认为,绒癌转移是多基因、多信号通路共同作用的结果,但具体机制尚不明确。因此,探讨绒癌侵袭转移的发生机制,寻找阻断或逆转绒癌侵袭的新靶点,对改善绒癌患者的预后具有重要的临床意义。溶酶体相关膜蛋白 3(1ysosomal associated membrane protein3,LAMP3)是一种高度糖基化的完整跨膜蛋白,主要位于细胞内的溶酶体膜上,其仅仅在少数几种细胞或者某些细胞的特定分化阶段表达。但是最近相关的体内体外试验均发现,LAMP3可能在肿瘤的侵袭转移中发挥着重要的作用,与肿瘤的预后不良密切相关。亦有研究发现LAMP3是一种新的低氧调节基因,参与低氧诱导的肿瘤的转移过程。文献报道低氧也可以诱导绒癌细胞发生EMT。LAMP3作为新的低氧调节基因是否在绒癌细胞的EMT中发挥作用需要进一步的研究。首先,本研究通过RT-PCR和免疫印迹法发现绒癌细胞JEG-3中LAMP3的mRNA和蛋白表达水平明显低于HTR-8/SVneo(P0.05),而HTR-8/SVneo被公认为具有更强的侵袭能力,提示LAMP3的表达水平与细胞侵袭能力呈正相关。同时,通过CCK-8、RT-PCR、免疫印迹法和体外侵袭迁移实验如Transwell、划痕实验等研究发现低氧诱导后绒癌细胞JEG-3中LAMP3的表达增加,细胞侵袭迁移能力增强。研究提示LAMP3的表达可能和JEG-3细胞的侵袭迁移能力呈正相关。其次,通过质粒转染和慢病毒转染技术分别构建过表达和敲减LAMP3的JEG-3细胞,并通过体外侵袭迁移实验如Transwell、划痕实验等检测不同细胞的侵袭迁移能力、并在过表达LAMP3后检测细胞对化疗药物的敏感性,另外通过RT-PCR和免疫印迹法检测不同细胞的EMT相关基因的表达情况。结果显示,过表达LAMP3可以通过在绒癌细胞JEG-3中介导EMT的激活促进其侵袭、迁移、耐药等表型;而对LAMP3敲减后,则会诱导相反的MET过程,使JEG-3细胞的侵袭迁移表型减弱。研究结果表明,LAMP3可以通过诱导EMT促进绒癌细胞JEG-3的侵袭迁移。最后,通过免疫印迹法分别对过表达和敲减LAMP3后以及分别应用小分子抑制剂XAV939和重组人Wnt-3a蛋白后的JEG-3细胞进行β-catenin和Wnt通路的相关靶基因蛋白表达的测定。结果表明,LAMP3的表达通过影响JEG-3细胞中Wnt/β-catenin通路的激活参与了对EMT的调控,这种调控作用可能是通过对EMT转录因子Twist和Snail的调节来实现的,同时也涉及MMP2及MMP9相关表达的改变。研究提示,Wnt信号通路参与了 LAMP3对JEG-3细胞EMT的调控。研究结果表明,LAMP3的表达可能和JEG-3细胞的侵袭迁移能力呈正相关,LAMP3可以通过诱导EMT促进绒癌细胞JEG-3的侵袭迁移,而Wnt信号通路参与了 LAMP3对JEG-3细胞EMT的调控;针对Wnt通路相关靶点设计的药物可能会在转移性绒癌的治疗中发挥重要作用。
[Abstract]:Because effective treatments such as chemotherapy and human chorionic gonadotropin used as tumor markers, gestational trophoblastic tumor (Gestational trophoblastic, neoplasia, GTN) have become impossible to rely on only the first chemotherapy cure gynecological tumor, is one of the earliest human solid tumors to cure. However, there will still be because of the emergence of resistance to chemotherapy and patients with multiple metastases and lead to disease progression. The metastatic trophoblastic tumor hematogenous spread, early metastasis can occur simultaneously and widely, the primary and metastatic cancer, and many patients with primary lesions disappeared, only showed metastatic symptoms, the diagnosis and treatment of diseases are it becomes more difficult. In recent years, academic circles generally believe that the transfer of choriocarcinoma is a multi gene, the interaction of multiple signaling pathways, but the specific mechanism is not clear. Therefore, to investigate the invasion of choriocarcinoma The occurrence mechanism of angiogenesis, to find a new target for preventing or reversing the invasion of choriocarcinoma, has important clinical significance for improving the prognosis of patients with choriocarcinoma. Lysosome associated membrane protein 3 (1ysosomal associated membrane protein3, LAMP3) is a highly glycosylated transmembrane protein lysosomal membrane integrity, mainly located inside the cell. The only expression in the differentiation stage specific a few cells or some cells. But in vivo and in vitro experiments recently related were found, LAMP3 may play an important role in tumor invasion and metastasis of tumor, and the prognosis is closely related. Studies have found that LAMP3 is a new gene transfer process in hypoxic regulation. Hypoxia induced tumors. Hypoxia can induce the reported EMT.LAMP3 as the new hypoxia regulating gene can play a role in choriocarcinoma cell EMT in choriocarcinoma cells To further study. Firstly, the study by RT-PCR and Western blotting indicated the expression of mRNA and LAMP3 protein in choriocarcinoma cell JEG-3 was significantly lower than that of HTR-8/SVneo (P0.05), HTR-8/SVneo is recognized as the aggressive ability, suggesting that the expression of LAMP3 and cell invasion ability was positively correlated. At the same time, through the CCK-8, RT-PCR Western blotting, in vitro invasion and migration experiments such as Transwell, on the scratch test found increased LAMP3 expression in choriocarcinoma cell induced by JEG-3 after hypoxia in the cell invasion and migration ability. Studies suggest that expression of LAMP3 and JEG-3 cell invasion and migration ability were positively correlated. Secondly, through plasmid transfection and lentivirus transfection technology were constructed JEG-3 expression and knockdown of LAMP3, and the in vitro invasion and migration experiments such as Transwell, scratch test. The cell migration and invasion, And in the expression of sensitivity of cells to chemotherapeutic drugs LAMP3, the expression of EMT related genes RT-PCR and immunoblotting in different cells. The results showed that the migration of over expression of LAMP3 by activating JEG-3 mediated EMT in choriocarcinoma cells promote its invasion, drug resistance phenotype of knockdown of LAMP3; when will the MET process induced by the contrary, the invasion and migration of JEG-3 cell phenotype is weakened. The results show that the LAMP3 can promote the invasion and migration of EMT induced by JEG-3 choriocarcinoma cells. Finally, through Western blot respectively for overexpression and knockdown of LAMP3 and were applied to small molecule inhibitors of XAV939 and recombinant human Wnt-3a protein the JEG-3 cells to determine the expression of target beta -catenin and Wnt pathway protein. The results showed that the expression of LAMP3 in JEG-3 cells by affecting Wnt/ beta -catenin pathway activation. With the control of EMT, the regulation may be achieved through the regulation of EMT transcription factor Twist and Snail, also involving MMP2 and MMP9 related expression changes. Studies suggest that Wnt signaling pathway is involved in the regulation of LAMP3 on JEG-3 EMT cells. The results showed that expression of LAMP3 and JEG-3 cells may the invasion ability was positively related to LAMP3 can induce EMT to promote invasion of choriocarcinoma cells JEG-3, Wnt signaling pathway is involved in the regulation of LAMP3 on JEG-3 cell EMT; drug target design for Wnt pathway may play an important role in the treatment of metastatic choriocarcinoma.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.33

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