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 protein 3，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（P＜0.05），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。

Due to precise measurement of human chorionic gonadotropin (hCG) levels, effective combination chemotherapy and other viable procedures such as surgeries, gestational trophoblastic neoplasia (GTN) is considered to be one of the most curable human malignancies as well as the first gynecological solid tumor. However，there are still disease progressions because of chemoresistance and multiple metastases. The metastatic choriocarcinoma can disseminated widely through blood stream at very early stage. Primary and metastatic lesions could be detected simultaneously and many of them show metastatic symptoms only which makes it more difficult to diagnose and manage. It is acknowledged that metastasis involved plenty of genes and signal pathways. Exploring mechanisms of metastatic choriocarcinoma as well as finding new target for treatment are necessary to improve the prognosis of choriocarcinoma patients.

    Lysosomal associated membrane protein 3 (LAMP3) is a kind of transmembrane proteins containing highly glycosylated domain, mainly located in cellular lysosomal membrane in certain differentiated stages of some cells. Recent studies have shown that LAMP3 may be involved in the invasion and metastasis of tumors and related to the adverse prognosis. It is also a new hypoxic regulated gene. Studies revealed that hypoxia is a inducer of EMT in choriocarcinoma cells. However, the role of LAMP3 and its relationship with EMT in choriocarcinoma cells still remains largely unknown.

    Firstly, we found that expression of LAMP3 in JEG-3 was related to invasion and migration abilities. RT-PCR and western blot were used to assess mRNA and protein levels of LAMP3 in JEG-3 and HTR-8/SVneo and results show that expression of LAMP3 in JEG-3 were much lower than HTR-8/SVneo. HTR-8/SVneo was considered to be the positive control of invasion and migration ability of trophoblastic cells. Results show that expression level of LAMP3 was positively related to the invasion ability of JEG-3. After hypoxia induction expression of LAMP3, invasion and migration abilities of JEG-3 were increased according to the results of CCK-8, RT-PCR, western blot, transwell test and scratch test. Evidences suggest that alteration of LAMP3 expression could significantly change the invasion and migration phenotype of JEG-3.

    Secondly, we constructed LAMP3 overexpression and knockdown vectors through plasmid and lentivirus transfections separately. Invasion and migration abilities of different cells were tested by transwell and scratch test. Sensitivity to chemotherapy drugs was evaluated by CCK-8 and RT-PCR and western blot were used to assess mRNA and protein levels of EMT-related genes in different cells. Results show that after transfection of LAMP3 overexpression plasmid in JEG-3, EMT was activated as well as the invasion, migration and chemoresistance phenotypes. On the other side, knowdown of LAMP3 could induce MET and attenuate invasion and migration phenotypes in JEG-3. Evidences show that LAMP3 can mediate invasion and migration of JEG-3 through modulation of EMT.

    Finally, western blot was used to detect the expression of β-catenin and target genes of Wnt pathway in LAMP3 overexpression and knockdown cells. The small molecule inhibitor of Wnt pathway XAV939 and human recombinant Wnt-3a protein were added to LAMP3 overexpression and knockdown JEG-3 cells. Results show that expression of LAMP3 could modulate EMT through activation of Wnt/β-catenin pathway and EMT transcription factor Twist, Snail and MMP2, MMP9 may be involved. Evidences suggest that Wnt signalling participate the modulation of LAMP3-mediated EMT in JEG-3 cells.

    In conclusions, alteration of LAMP3 expression could significantly change the invasion and migration phenotype of JEG-3, LAMP3 can mediate invasion and migration of JEG-3 through modulation of EMT and Wnt signalling participate the modulation of LAMP3-mediated EMT in JEG-3 cells which suggest therapies targeting Wnt signal may aid in the reversal and treatment of metastatic choriocarcinma.