Choriocarcinoma Tissues Research Articles

Hypermethylated RASAL1’s promotive role in chemoresistance and tumorigenesis of choriocarcinoma was regulated by TET2 but not DNMTs

BackgroundPatients with choriocarcinoma (CC) accompanying chemoresistance conventionally present a poor prognosis. Whether ras protein activator like-1 (RASAL1) functions as a tumor promoter or suppressor depends on tumor types. However, the role of RASAL1 in process of chemoresistance of CC and underlying molecular mechanism remain elusive.MethodsThe expression pattern of RASAL1 in CC cells and tissues was measured using Western blotting, immunohistochemistry and qRT-PCR. Cell viability and proliferative ability were assessed by MTT assay, Tunnel assay and flow cytometric analysis. Additionally, the stemness was evaluated by the colony formation and tumor sphere formation. Methotrexate (MTX) was applied to exam the chemosensitivity of CC cells.ResultsThe expression of RASAL1 was reduced both at the protein and mRNA levels in CC tissues and cells compared to hydatidiform mole (HM) and invasive mole (IM). Loss of RASAL1 was attributed to its promoter hypermethylation and could be restored by 5-Aza. Knock-down of RASAL1 promoted the viability, proliferative potential, stemness and EMT phenotype of JEG-3 cells. However, induced overexpression of RASAL1 by 5-Aza significantly prohibited cell proliferation and stemness potential of the JAR cell. Additionally, the xenograft model indicated that knockdown of RASAL1 led to a remarkable increase of tumor volume and weight in comparison with its counterpart. Moreover, the stimulatory activity brought by decrease of RASAL1 could be deprived by β-catenin inhibitor XAV 939, yet the suppressive activity resulted from its promoter demethylation could be rescued by β-catenin activator BML-284, indicating that function of RASAL1 depends on β-catenin. Besides, the co-immunoprecipitation assay confirmed the physical binding between RASAL1 and β-catenin. Further investigations showed hypermethylated RASAL1 was regulated by TET2 but not DNMTs.ConclusionTaken together, the present data elucidated that reduced RASAL1 through its promoter hypermethylation regulated by TET2 promoted the tumorigenicity and chemoresistance of CC via modulating β-catenin both in vitro and in vivo.

Metformin regulates autophagy via LGMN to inhibit choriocarcinoma

Choriocarcinoma has the problem of chemotherapy insensitivity and recurrence. Metformin may be a promising candidate to restrict choriocarcinoma progress because of its indirect and direct beneficial role on inhabitations of cancer cells without severe adverse side effects. In this study, metformin pressed the proliferation and invasion of choriocarcinoma JAR cells in vitro and the growth of the JAR subcutaneous xenografts in vivo. The high throughput sequencing and bioinformatics technology identified the low expression of legumain (LGMN) in lysosomal pathway caused by metformin, which was upregulated in human choriocarcinoma tissues compared with the early pregnancy tissues. As elevating metformin concentration and treatment time, the mRNA and protein expression of LGMN both depressed in two choriocarcinoma cell lines (JAR and JEG-3). LGMN was involved in metformin-mediated inhibition of cell proliferation and invasion. Furthermore, metformin induced autophagy via inhibiting LGMN through AKT/mTOR/LC3II signaling pathway of choriocarcinoma. Autophagy inhibitor could depress metformin-induced autophagy and improve cell proliferation and invasion ability dropped by metformin, while autophagy inducer could partially reverse the change of cell proliferation and invasion modulated by combination of metformin and LGMN overexpression. These results indicated that metformin inhibited cell proliferation and invasion ability by inducing autophagy in a LGMN-dependent manner so as to play a role in the treatment of choriocarcinoma.

METTL3 m6A-dependently promotes miR-21-5p maturation to accelerate choriocarcinoma progression via the HIF1AN-induced inactivation of the HIF1A/VEGF pathway.

Gestational choriocarcinoma is a highly malignant neoplastic disease derived from pathological changes in trophoblastic cells. Recent evidences have shown that N6-methyladenosine (m6A) modifications play important role in modulating the development of multiple cancers, but the detailed mechanisms by which m6A-mediated choriocarcinoma progression have not been fully delineated. This study aimed to investigate the role of m6A in choriocarcinoma and reveal its underlying molecular mechanisms. The expression of METTL3, miR-21-5p and HIF1AN was detected using RT-qPCR in tissues and cells. The protein expression of METTL3, HIF1AN, HIF1A and VEGF were measured by western blot. The luciferase reporter assays and RNA immunoprecipitation (RIP) were used to verify the relationship between miR-21-5p and HIF1AN. The CCK-8, colony formation and transwell assays were used to detected cell proliferation and cell migration, respectively. Here, we demonstrated that the m6A methyltransferase-like 3 (METTL3) was aberrantly high-expressed in the clinical choriocarcinoma tissues and choriocarcinoma cell lines compared to the corresponding normal counterparts. The following functional experiments verified that silencing of METTL3 suppressed cell proliferation, migration, epithelial-mesenchymal transition (EMT) and tumorigenesis in vitro and in vivo to hamper the aggressiveness of choriocarcinoma. Next, the mechanical experiments confirmed that METTL3 promoted the maturation of miR-21-5p in an m6A-dependent manner, and elevated miR-21-5p subsequently degraded its downstream hypoxia-inducible factor asparagine hydroxylase (HIF1AN) by targeting its 3' untranslated regions (3'-UTR), resulting in the activation of the tumor-promoting HIF1A/VEGF pathway. Finally, the rescuing experiments verified that METTL3 ablation-induced inhibitory effects on the malignant phenotypes in choriocarcinoma were all abrogated by both miR-21-5p overexpression and HIF1AN downregulation. Collectively, this study firstly reported the involvement of the METTL3/m6A/miR-21-5p/HIF1AN signaling cascade in regulating the progression of choriocarcinoma, which provided novel biomarkers for the diagnosis and treatment of this disease.

Cell surface membrane lysosome-associated membrane glycoprotein 2 promotes cell adhesion via abundant N-glycans in choriocarcinoma.

Lysosome-associated membrane glycoprotein 2 (LAMP-2) is a target protein for glycosylation by N-acetylglucosaminyltransferase IV (GnT-IV), which catalyzes the formation of β1,4GlcNAc branches on the mannose core of N-glycans in choriocarcinoma cells. However, the role of LAMP-2, especially when it is expressed in the cell surface membrane of choriocarcinoma cells, has not been well investigated in the progression of choriocarcinoma. This study aimed to elucidate the function of the cell surface membrane LAMP-2 in the malignancy of choriocarcinoma. We evaluated the localization of LAMP-2 in some choriocarcinoma cell lines and clinical samples of choriocarcinoma, normal placenta, hydatidiform mole, and invasive mole by flow cytometry, immunocytochemistry, and immunohistochemistry. We performed functional experiments using the knockout or overexpression model of LAMP-2 in the presence or absence of galectins. LAMP-2 was observed in the cell surface membrane of some choriocarcinoma cell lines and tumor cells of choriocarcinoma tissue and trophoblasts of the placenta, hydatidiform mole, and invasive mole. Cell surface membrane LAMP-2 knockout decreased cell adhesion and invasion in choriocarcinoma cells. Conversely, cell surface membrane LAMP-2A overexpression increased cell adhesion and invasion. Experiments in the presence of galectins revealed that abundant N-glycans bound to the peptide core of the luminal side of the cell surface membrane LAMP-2 mediated cell adhesion of choriocarcinoma cells by interacting with galectins in the extracellular matrix (ECM). Cell surface membrane LAMP-2, which is glycosylated by GnT-IV, contributes to the malignancy of choriocarcinoma by promoting cell adhesion with the ECM via abundant N-glycans.

Immunoscreening of Alpha-Tocopherol in Breast, Prostate Cancers and in Gestational Choriocarcinoma Tissues

Background: Many literature reviews report vitamin E supplementation as a benefit chemopreventive and adjuvant therapy in breast and prostate cancers. We investigated in the present work, alpha-tocopherol (α-T) expression (the current active form of vitamin E) from tissues of Congolese patients neither smokers nor alcohol drinkers and without intake antioxidant vitamins supplement. Methods: α-T was analysed in one normal placenta of nine weeks of gestation and in nineteen cancerous tissues, including seven breast cancers, six prostate cancers, and six gestational choriocarcinomas. The study was performed by immunohistochemistry method after diagnosis confirmation by histological analysis. Results: α-T staining in membrane cells and collagen fibers presented a moderate expression in healthy sections of tissues (positive control), but the labelling was strong in breast, prostate adenocarcinomas, and in choriocarcinomas. Conclusion: Tumors immunohistochemistry of α-tocopherol in breast, prostate cancers and in choriocarcinoma show elevated immunostaining suggesting a probable oncogenic effect of the micronutrient.

Abstract 2988: PLAC8 confers the chemoresistance of placental site trophoblastic tumor and choriocarcinoma to etoposide in an autophagy-dependent manner

Abstract Chemotherapy is commonly used to treat gestational trophoblastic neoplasia (GTN), but placental site trophoblastic tumor (PSTT) and a small proportion of choriocarcinoma demonstrate chemotherapy resistance, which becomes the major obstacle for the treatment. However, the underlying mechanism remains elusive. In the present study, we found that PLAC8 expression in PSTT tissues was higher than that in placental site intermediate trophoblast cells. In addition, PLAC8 was also highly expressed in human choriocarcinoma tissues compared with normal placental villi. Over-expression of PLAC8 inhibited p53 protein stability and induced the autophagic flux in HTR-8 and JAR cells. PLAC8-induced autophagic activity could be partially recovered by p53 re-expression. Further, PLAC8 over-expression conferred chemoresistance of trophoblast cells to etoposide, which can be effectively abrogated by autophagic inhibitors 3-methyladenine (3-MA) or chloroquine (CQ). In conclusion, over-expression of PLAC8 in PSTT and choriocarcinoma tissues may endow the trophoblast cells with enhanced chemoresistance via inducing autophagy activity. Therefore, PLAC8 may serve as a potential therapeutic target for the treatment of PSTT and choriocarcinoma. Citation Format: Xuan Feng, Huandi Yu, Xiang Tao, Yan Du, Jing Wu, Yinhua Yu, Congjian Xu, Hongbo Zhao. PLAC8 confers the chemoresistance of placental site trophoblastic tumor and choriocarcinoma to etoposide in an autophagy-dependent manner [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2988.

MEG3 is restored by schisandrin A and represses tumor growth in choriocarcinoma cells.

Schisandrin A (SchA) has been reported as a multidrug resistance-reversing agent; however, its antitumor effects have been rarely reported. Consequently, we attempted to explore whether SchA per se possesses an antitumor property in choriocarcinoma JEG-3 and BeWo cells and its potential mechanisms. JEG-3, BeWo, and HTR-8/SVneo cells were stimulated with SchA at different concentrations (10-100 μM), and cellular viability was evaluated with Cell Counting Kit-8. After stimulation with SchA, proliferation, apoptosis, migration, and invasion were detected by bromodeoxyuridine assay, Annexin V-fluorescein isothiocyanate/propidium iodide (Annexin V-FITC/PI) method, and a Transwell system, in JEG-3 cells transfected with short hairpin-RNA for maternally expressed 3. Western blot was performed to quantify protein. MEG3 was examined by a quantitative reverse transcription-polymerase chain reaction. MEG3 was downregulated in choriocarcinoma tissues. SchA diminished cellular viability, decreased proliferative activity, inhibited migratory and invasive behaviors, and repressed phosphorylation of regulators of phosphatidylinositol 3 kinase/protein kinase B/nuclear factor κB (PI3K/AKT/NF-κB) signaling cascade in gestational choriocarcinoma cells. MEG3 was upregulated by SchA in JEG-3 and BeWo cells. SchA exhibited little suppressive effects in JEG-3 cells lacking MEG3. Besides, the phosphorylation of transducers was evoked in MEG3-silenced JEG-3 cells despite stimulation with SchA. SchA administration repressed the growth of JEG-3 and BeWo cells by upregulating MEG3. Besides, SchA blocked PI3K/AKT/NF-κB signal cascade by elevating MEG3.

Production of an anti-angiogenic factor sFLT1 is suppressed via promoter hypermethylation of FLT1 gene in choriocarcinoma cells

BackgroundSoluble Fms-like tyrosine kinase-1 (sFLT1) as an anti-angiogenic factor is abundantly expressed in placental trophoblasts. Choriocarcinoma, a malignant tumor derived from trophoblasts, is known to be highly angiogenic and metastatic. However, the molecular mechanism underlying angiogenesis in choriocarcinoma pathogenesis remains unclear. We aimed to investigate the mRNA expression and DNA methylation status of the FLT1 gene in human choriocarcinoma cells and trophoblast cells.MethodsqRT-PCR, Western blotting and ELISA were conducted to evaluate the mRNA and protein expression levels of sFLT1. 5-aza-2′-deoxycytidine (5azadC) treatment and bisulfite sequencing were used to study the FLT1 gene promoter methylation. The effect of sFLT1 on choriocarcinoma growth and angiogenesis was evaluated in a xenograft mouse model.ResultsExpression of the FLT1 gene was strongly suppressed in choriocarcinoma cell lines compared with that in the primary trophoblasts. Treatment of choriocarcinoma cell lines with 5azadC, a DNA methyltransferase inhibitor, markedly increased in mRNA expression of three FLT1 splice variants and secretion of sFLT1 proteins. Bisulfite sequencing revealed that the CpG hypermethylation was observed at the FLT1 promoter region in choriocarcinoma cell lines and a human primary choriocarcinoma tissue but not in human trophoblast cells. Interestingly, in 5azadC-treated choriocarcinoma cell lines, sFLT1 mRNA expression and sFLT1 production were further elevated by hypoxic stimulation. Finally, as expected, sFLT1-expressing choriocarcinoma cells implanted into nude mice showed significantly slower tumor growth and reduced microvessel formation compared with GFP-expressing control choriocarcinoma cells.ConclusionsInhibition of sFLT1 production by FLT1 silencing occurs via the hypermethylation of its promoter in choriocarcinoma cells. The stable expression of sFLT1 in choriocarcinoma cells resulted in the suppression of tumor growth and tumor vascularization in vivo. We suggest that the FLT1 gene may be a cell-type-specific tumor suppressor in choriocarcinoma cells.

Quantitative proteomic analysis identifies novel regulators of methotrexate resistance in choriocarcinoma

ObjectiveAlthough methotrexate (MTX) is commonly used for the treatment of choriocarcinoma, chemoresistance to MTX may occur in a considerable fraction of patients. Further understanding on the mechanisms of MTX resistance would help to develop more effective therapy for choriocarcinoma. MethodsQuantitative proteomic approach involving TMT labeling and LC-MS/MS was used to identify MTX resistance-related proteomic profiles in choriocarcinoma cell models. Pathway and process enrichment analysis were conducted to identify MTX resistance-related biological processes/molecular pathways. CCK-8 viability assay, clonogenic survival assay, and BrdU incorporation analysis were used to examine the chemosensitivity to MTX in choriocarcinoma cells. ResultsIn total, 5704 protein groups were identified, among which 4997 proteins were quantified. Bioinformatic analysis revealed that multiple biological processes/molecular pathways might be associated with MTX resistance in JEG3/JEG3/MTX cell systems. DPP4 and METTL7A were selected for further investigation. Increased expression of DPP4 or METTL7A was observed in MTX-resistant cancer cell lines and choriocarcinoma tissues. Knockdown of DPP4 or METTL7A significantly decreased cell viability, impaired clonogenesis, and increased apoptosis after MTX treatment in JEG3/MTX and JAR/MTX cells; while over-expression of DPP4 or METTL7A promoted cell viability and reduced apoptosis following exposure to MTX in JEG3, JAR and BEWO cells. Further, DPP4 and METTL7A differentially activated prosurvival signaling pathways including PI3K/AKT, ERK1/2 and STAT3, and attenuated the accumulation of reactive oxygen species (ROS) in choriocarcinoma cell lines. ConclusionsDPP4 and METTL7A might promote MTX resistance through activating pro-survival signaling pathways and attenuating the accumulation of ROS in choriocarcinoma cells. Targeting DPP4 and METTL7A might be useful to sensitize choriocarcinoma cells to MTX-based chemotherapy.

CD105 overexpression mediates drug-resistance in choriocarcinoma cells through BMP9/Smad pathway.

Background: CD105 (endoglin, ENG) is a membranous protein that is overexpressed in tumor-associated endothelial cells and some actual tumor cells and is associated with poor prognosis. However, the association between CD105 and response to chemoresistance in choriocarcinoma cells has not been clearly defined. The present study aimed to investigate the effects of targeting CD105 in drug-resistant choriocarcinoma.Methods: CD105 expression was evaluated in drug-resistant and parental choriocarcinoma cells by qRT-PCR, western blotting, and immunofluorescence. CD105 overexpressing and knockdown cells were established by lentiviral transfection. CCK8, transwell, and flow cytometric assays were used to measure changes in drug-sensitivity, invasion, migration, and apoptosis. Drug-sensitivity and Smad1/5/8, Smad2, and Smad3 expression were also detected after BMP9 treatment. Immunohistochemical staining for CD105 and BMP9 was performed on choriocarcinoma tissues and the relationships between clinical and pathological characteristics were analysed.Results: Data demonstrated that CD105 overexpression could decrease drug sensitivity, promote invasion and migration, and inhibit apoptosis in choriocarcinoma cells, and this protein was confirmed to mediate drug resistance through the BMP9/Smad pathway. Further experiments showed that the expression of CD105 and BMP9 was consistent in choriocarcinoma tissues and significantly associated with disease recurrence.Conclusions: This study provides evidence suggesting that CD105 is critical for the development of drug-resistance in choriocarcinoma and might serve as a therapeutic target for reversing chemoresistance in choriocarcinoma patients.

β-catenin/LIN28B promotes the proliferation of human choriocarcinoma cells via Let-7a repression.

Choriocarcinoma is a rare and malignant trophoblastic tumor. However, the molecular mechanisms by which choriocarcinoma is regulated remain unknown. In the present study, we first elucidated that LIN28B was highly expressed in human choriocarcinoma tissues and choriocarcinoma cell lines. Our data further demonstrated that knockdown of LIN28B by small interfering RNA caused an increase in Let-7a expression in JAR cells. In addition, silencing of LIN28B inhibited IGF2BP1 expression and suppressed cell proliferation capacity, both of which can be markedly restored by Let-7a inhibitor. In contrast, LIN28B over-expression-improved cell proliferation was inhibited by Let-7a mimic. Knockdown of β-catenin resulted in reduced expression of LIN28B and increased expression of Let-7a. Knockdown of β-catenin also caused a decrease in cell proliferation, which can be recovered by re-expression of LIN28B or by Let-7a inhibitor. Collectively, our data indicate that β-catenin/LIN28B/Let-7a pathway may be crucial for the regulation of cell proliferation in human choriocarcinoma cells.

Abstract 540: β-Catenin/LIN28B promotes cell proliferation of human trophoblast cells via let7a repression

Abstract LIN28 is a highly conserved RNA-binding proteins, have been implicated in key biological functions such as development, glucose metabolism and pluripotency. LIN28 overexpression induces the reprogramming of pluripotent stem cells (iPSCs) and supports the proliferative and metabolic capacities of iPSCs. Therefore LIN28 is crucial for the regulation of the cell fate and human embryonic development. In addition, LIN28B is frequently up-regulated in a variety of cancers and correlates with poor prognosis and cancer recurrence, indicating the crucial role of LIN28B in tumorigenesis. Aberrantly expression of LIN28B is responsible for the decrease in the let-7 microRNA family observed in many cancers and Let-7 is frequently down-regulated in numerous different tumors and lower levels of let-7 correlates with poor prognosis. Therefore, LIN28/Let7 may be crucial for the regulation of cell cycle and cell proliferation. In our present study, We first detected the expression of LIN28B in normal placental villi and choriocarcinoma tissue. Our data showed LIN28B was highly expressed in human choriocarcinoma tissues and most LIN28B staining was localized in the nucleus. Comparatively, LIN28B staining was faint in normal placental villi. Our data then demonstrated that LIN28B was highly expressed in human choriocarcinoma cell lines JEG-3 and JAR. However, LIN28B expression was undetectable in HTR-8/Svneo normal trophoblast cells. Our data then demonstrated that LIN28B knockdown by small interfering RNA (siRNA) caused an increase in Let-7a expression. Further, silencing of LIN28 inhibited IGF2BP1 expression and suppressed cell proliferation capacity, which can be markedly restored by Le7a siRNA. These findings reveal that LIN28 may promote IGF2BP1 expression and cell proliferation by Let-7a repression. Moreover, β-catenin knockdown resulted in reduced LIN28 expression and Let-7a up-regulation. β-catenin is the major mediator of canonical Wnt signaling pathway, and activation of canonical Wnt signaling has been reported to play a critical role in the proliferation, invasion, and differentiation of various tumors. Collectively, our present data indicated that β-catenin/LIN28B/Let7a pathway may be crucial for cell proliferation regulation in human choriocarcinoma cells. Citation Format: Jing Wu, Baoxin Luan, Huandi Yu, Yinhua Yu, Congjian Xu, Hongbo Zhao. β-Catenin/LIN28B promotes cell proliferation of human trophoblast cells via let7a repression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 540.

Aberrantly Expressed SALL4 Promotes Cell Proliferation via β-Catenin/c-Myc Pathway in Human Choriocarcinoma Cells.

Sal-like protein 4 (SALL4) has been proved to play a pivotal role in the development and progression of various cancers. Previous studies showed that SALL4 was highly expressed in human choriocarcinoma tissues. However, the role of SALL4 in the biological behavior of human choriocarcinoma cells remains largely unknown. In this study, we first elucidated that SALL4 was highly expressed in human choriocarcinoma cell line JEG-3 and JAR. Sal-like protein 4 knockdown by small interfering RNA (siRNA) decreased c-Myc expression, whereas SALL4 overexpression by transfection of human pLenti-SALL4 construct promoted c-Myc expression. Further data showed that SALL4 overexpression improved cell proliferation of JEG-3 cells, which can be abrogated by c-Myc siRNA. Moreover, our data showed that SALL4 interact with β-catenin and SALL4 overexpression promoted the localization of β-catenin in the nucleus and β-catenin siRNA abrogated SALL4-induced c-Myc expression in JEG-3 cells. These data indicate that aberrantly expressed SALL4 in human choriocarcinoma cells may promote cell proliferation via β-catenin/c-Myc pathway, indicating that SALL4 may be potential treatment targets of human choriocarcinoma.

Secreted Frizzled Related Protein 2 Modulates Epithelial–Mesenchymal Transition and Stemness via Wnt/β-Catenin Signaling in Choriocarcinoma

Background/Aims: Choriocarcinoma (CC) is a highly aggressive gestational trophoblastic neoplasia; however, the underlying molecular mechanisms of its invasiveness and metastasis remain poorly understood. Human secreted frizzled-related protein 2 (SFRP2) could function as a tumor promoter or suppressor in different tumors, yet the role it plays in CC’s invasion and metastasis is thoroughly unclear. The current study was aimed to explore the function and underlying mechanism of SFRP2 in CC. Methods: The expression of SFRP2 in CC tissues was examined via immunohistochemistry. The methylation level and expression of SFRP2 in CC cell lines, JEG-3 and JAR were examined via bisulfite sequencing PCR (BSP), western blotting and quantitative RT-PCR. The biological role of increasing expressed SFRP2 through its promoter demethylation with 5-Aza-2’-deoxycytidine (5-Aza) was examined by a series of in vitro functional studies. Furthermore, lentivirus transfection technology was adopted to investigate the biological roles of SFRP2 knockdown in JEG-3 and JAR cells in vitro and in vivo. Moreover, its downstream signaling pathway was investigated. Results: SFRP2 was downregulated in CC tissues, and its expression was inversely related to its promoter hypermethylation frequency in JEG-3 and JAR cells. Increased SFRP2 through its promoter demethylation inhibited cell migration, invasion and colony formation in JEG-3 and JAR cells, whereas decreased SFRP2 reversed the epithelial-mesenchymal transition (EMT) process and stemness in JEG-3 and JAR cells both in vitro and vivo. Mechanistically, SFRP2 regulated the EMT and stemness of CC cell lines via canonical Wnt/β-catenin signaling, validated by the usage of a Wnt activator and inhibitor. Conclusion: The current study indicates that downregulated SFRP2 has potent tumor-promotive effects in CC through the modulation of cancer stemness and the EMT phenotype via activation of Wnt/β-catenin signaling in vitro and in vivo.

Hypermethylation and loss of retinoic acid receptor responder 1 expression in human choriocarcinoma

BackgroundHuman placental development resembles tumorigenesis, due to the invasive and fusogenic potential of trophoblasts. However, these features are tightly controlled in trophoblasts. Disturbance of this spatial and temporal regulation is thought to contribute to the rare formation of choriocarcinomas. Promoter hypermethylation and loss of the tumor suppressor Retinoic acid receptor responder 1 (RARRES1) were shown to contribute to cancer progression. Our study investigated the epigenetic and transcriptional regulation of RARRES1 in healthy human placenta in comparison to choriocarcinoma cell lines and cases.MethodsThree choriocarcinoma cell lines (Jeg-3, JAR and BeWo) were treated with three different retinoic acid derivates (Am580, Tazarotene and all-trans retinoic acid) and 5-aza-2′-deoxycytidine. We analyzed RARRES1 promoter methylation by pyrosequencing and performed realtime-PCR quantification to determine RARRES1 expression in placental tissue and trophoblastic cell lines. Additionally, RARRES1 was stained in healthy placentas and in biopsies of choriocarcinoma cases (n = 10) as well as the first trimester trophoblast cell line Swan71 by immunofluorescence and immunohistochemistry.ResultsIn the choriocarcinoma cell lines, RARRES1 expression could not be induced by sole retinoic acid treatment. Stimulation with 5-aza-2′-deoxycytidine significantly induced RARRES1 expression, which then could be further increased with Am580, Tazarotene and all-trans retinoic acid. In comparison to healthy placenta, choriocarcinoma cell lines showed a hypermethylation of the RARRES1 promoter, which correlated with a reduced RARRES1 expression. In concordance, RARRES1 protein expression was lost in choriocarcinoma tissue. Additionally, in the trophoblastic cell line Swan71, we found a significant induction of RARRES1 expression with increased cell density, during mitosis and in syncytial knots.ConclusionsOur findings showed that RARRES1 expression is absent in choriocarcinoma due to promoter methylation. Based on our analysis, we hypothesize that RARRES1 might exert tumor suppressive functions in multiple cellular processes (e.g. cell cycle regulation, adhesion, invasion and apoptosis).

Long Noncoding RNA LINC00261 Suppresses Cell Proliferation and Invasion and Promotes Cell Apoptosis in Human Choriocarcinoma.

Choriocarcinoma is one of the gestational trophoblastic neoplasias (GTNs) that originate in the chorionic villi and the extravillous trophoblast. Long noncoding RNAs (lncRNAs) are a type of non-protein-coding RNAs that have recently been implicated in human tumorigenesis. The present study investigated the role of the lncRNA LINC00261 in cell proliferation, metastasis, and apoptosis in choriocarcinoma cell lines. The transcription level of LINC00261 was significantly lower in choriocarcinoma tissues and in choriocarcinoma cell lines. Overexpression of LINC00261 caused a decrease in cell proliferation and arrested the cell cycle at the G0/G1 phase. Furthermore, overexpression of LINC00261 inhibited cell migration and invasion. Meanwhile, it promoted cell apoptosis and the relative activities of caspase 3 and caspase 9 in choriocarcinoma JEG-3 and JAR cells. These data suggested that LINC00261 promotes cell proliferation and metastasis in choriocarcinoma. Our data might provide novel insight into the early diagnosis and treatment of choriocarcinoma in clinics.

Glycan profiling of gestational choriocarcinoma using a lectin microarray

Glycosylation is an important post-translational modification, in which attachment of glycans to proteins has effects on biological functions and carcinogenesis. Analysis of human chorionic gonadotropin, a glycoprotein hormone produced by placental trophoblasts and trophoblastic tumors, has contributed to the diagnosis and treatment of trophoblastic disease, resulting in reduced incidence and mortality. However, alterations of the glycan structure itself in choriocarcinoma have not been characterized. We established a new choriocarcinoma cell line, induced choriocarcinoma cell-1 (iC3-1), which mimics the clinical pathohistology invivo, to examine the tumorigenesis and pathogenesis of choriocarcinoma. In this study, the alterations of glycan structures in the development of choriocarcinoma were examined by performance of comprehensive glycan profiling in clinical samples and in iC3-1 cells using a conventional microarray and the recently introduced lectin microarray. Microarray comparison showed significant upregulation of several characteristic glycogenes in the iC3-1 cells as compared to the parental HTR8/SVneo cells. The lectin array showed increased α-2-6-sialic acid, Galβ1-4GlcNAc, GlcNAcβ1-3GalNAc, and decreased α-1-6 core fucose, high mannose, GalNacβ1-4Gal, GALNAc (Tnantigen) and Galβ1-3Gal in choriocarcinoma tissue compared to normal villi. This is the first report of a lectin array analysis in choriocarcinoma and provides useful information for understanding of the disease.

IMP1 promotes choriocarcinoma cell migration and invasion through the novel effectors RSK2 and PPME1

ObjectiveOncofetal protein insulin-like growth factor II mRNA-binding protein 1 (IMP1) regulates cellular proliferation and migration. Expression of IMP1 is limited to a few adult human tissues. However, it commonly expresses in a variety of cancers. Our objective was to study the regulatory mechanism of IMP1 on the cellular functions of choriocarcinoma (CC) JAR cells. MethodsIMP1 protein levels were measured in CC tissues via immunohistochemistry. Specific siRNAs were used to down-regulate gene expressions. The abilities of migration and invasion were estimated by wound-healing and Matrigel chamber assays. The profile of IMP1-binding genes was investigated with an Agilent microarray. RT-qPCR, RNA immunoprecipitation, and IMP1 rescue experiments were performed to confirm the association between IMP1 and its binding genes. Gene expression was further analyzed by using RT-PCR and Western blotting. ResultsStrong IMP1 expressions were frequently detected in CC tissues. Knockdown of IMP1 expression in JAR cells inhibited cell migration and invasion, but did not affect cellular proliferation and morphology. Microarray and RNA-immunoprecipitation results revealed several candidate genes regulated by IMP1. Among them, ribosomal protein S6 kinase (RSK2) and protein phosphatase methylesterase 1 (PPME1) were confirmed to be down-regulated in IMP1-depleted JAR cells. Re-expression of IMP1 into the cells restored the expressions of RSK2 and PPME1. Furthermore, the depletion of RSK2 or PPME1 decreased the migration and invasion of JAR cells. ConclusionOur results suggest that IMP1 plays an essential role in the regulation of migration and invasion of human CC cells, possibly through the novel effectors RSK2 and PPME1.

Down-Regulation of Poly(rC)-Binding Protein 1 Correlates With the Malignant Transformation of Hydatidiform Moles

To analyze the expression patterns of poly(rC)-binding protein 1 (PCBP1) in complete hydatidiform moles (HMs) and to determine the predictive value of PCBP1 during postmolar follow-up after uterine evacuation. The PCBP1 protein expression profile was investigated in 10 complete moles that remained benign, 10 complete moles that underwent malignant transformation, and 10 choriocarcinoma tissues using Western blot analysis. The PCBP1 protein expression patterns in complete HM samples gathered from 69 patients were also detected by immunohistochemical analysis. The association of PCBP1 protein expression with the progression in HMs was subsequently assessed. The expression of PCBP1 was significantly lower in malignant-transformed moles than benign moles. The PCBP1 expression level was negatively associated with malignant transformation and serum human chorionic gonadotropin levels. Logistic regression analysis indicated that complete moles with high PCBP1 expression levels had a significantly lower risk of progression to gestational trophoblastic tumors (odds ratio, 0.22; 95% confidence interval, 0.07-0.67). These observations suggest that PCBP1 may be important in the pathogenesis of gestational trophoblastic tumors. In addition to the β-fraction of human chorionic gonadotropin, decreased expression of PCBP1 protein may be a strong predictor of the malignant transformation of complete moles.

LOW DOSES OF HIGH FREQUENCY LOW MAGNITUDE LOADING INCREASE MODULUS & MAINTAIN GAG IN CULTURED TENDONS

Objective: The purpose of this study was to evaluate the association of telomerase activity in complete hydatidiform moles with subsequent development of persistent gestational trophoblastic tumor. Study Design: By means of the standard telomerase repeat assay, we examined telomerase activity in 4 normal placentas, 31 complete hydatidiform moles (16 cases of uneventful regression, according to serum levels of β-human chorionic gonadotropin, after evacuation and 15 cases in which persistent gestational trophoblastic disease developed after evacuation), 7 invasive moles, and 5 choriocarcinoma tissue samples. Results: Telomerase activity was detected in 13 of 15 (86.7%) complete hydatidiform moles in patients who eventually underwent chemotherapy for the treatment of persistent gestational trophoblastic tumors. All 9 patients with metastatic disease (International Federation of Gynecology and Obstetrics stage III) had telomerase activity in the initial molar tissue sample. In contrast, telomerase activity was evident in only 3 of 16 (12.5%) complete hydatidiform moles from patients with spontaneous remission after evacuation (P < .05). Telomerase activity was detected in all 7 invasive moles and all 5 choriocarcinoma tissue samples but was not detected in normal placentas. Conclusion: The presence of telomerase activity in complete hydatidiform moles is associated with the development of persistent gestational trophoblastic tumors, such as invasive moles and choriocarcinoma. (Am J Obstet Gynecol 1999;180:328-33.)