| 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 LAMPS 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 ?-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/?-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,LAMPS 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. |
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