Short tandem repeats genotyping of gestational choriocarcinoma - our experiences.

OBJECTIVE: This short communication demonstrates how short tandem repeat genotyping can identify the origin of gestational choriocarcinoma. MATERIALS AND METHODS: The origin of gestational choriocarcinoma in our three cases was determined using the short tandem repeats genotyping technique, which involved quantitative fluorescent PCR and fragmentation analysis. RESULTS: In Case 1 despite no medical history of molar pregnancy, DNA analysis indicated that the choriocarcinoma originated from a homozygous complete hydatidiform mole. We conclude, that the patient's complete abortion 10 years prior to the choriocarcinoma diagnosis was an undiagnosed complete hydatidiform mole. In Case 2 and Case 3 the clinically presumed origin of choriocarcinoma was confirmed. CONCLUSION: Determining the origin of choriocarcinoma is essential for clinical application, as it affects the FIGO scoring system for gestational trophoblastic neoplasia, which determines the patient's prognosis and treatment approach.

Insights into cAMP-dependent molecular mechanisms regulating expression and function of LGALS16 gene in choriocarcinoma JEG-3 cells.

The human choriocarcinoma cell line JEG-3 offers a valuable model to study galectin-16 gene (LGALS16) expression and functions in the context of placental cell differentiation and cancer cell biology. Recent evidence indicates that cAMP-mediated signaling pathways might be responsible for the upregulation of LGALS16; however, the underlying mechanisms are unknown. Here, we employed biochemical inhibitors of the cAMP cascade and CRISPR/Cas9 engineered cells to assess regulatory patterns and associations between cAMP-induced trophoblast differentiation and LGALS16 expression in JEG-3 cells. The expression of LGALS16 was significantly upregulated in parallel with human chorionic gonadotropin beta (CGB), a biomarker of syncytiotrophoblast differentiation, in response to 8-Br-cAMP. Inhibition of p38 MAPK and EPAC significantly altered LGALS16 expression during differentiation, while PKA inhibition failed to change LGALS16 and CGB3/5 expression in our cell model. The CRISPR/Cas9 LGALS16 knockout cell pool expressed a significantly lower amount of CGB3/5, a reduced level of CGB protein, and an unaltered cell growth rate in response to 8-Br-cAMP in comparison with wild-type JEG-3 cells. Collectively, these findings suggest that LGALS16 is required for the trophoblast-like differentiation of JEG-3 cells, and its expression is mediated through p38 MAPK and EPAC signaling pathway branches.

HIF-1α contributes to metastasis in choriocarcinoma by regulating DEC1 expression

Purpose To elucidate the underlying mechanism of HIF-1α in migration and invasion of choriocarcinoma. Methods Cell proliferation was determined by CCK-8 assay when cell invasion was detected by transwell assay. The protein expression was detected by western blotting, immunohistochemistry, and qPCR assay. Result HIF-1α was shown to be strongly expressed in both clinical tumour tissues and cell lines in choriocarcinoma. When HIF-1α was efficiently knocked down in JEG3 cells, the proliferation rate was reduced by approximately 50% and the number of cells that migrated through the transwell insert was greatly decreased. The cell invasion rate was also significantly reduced. Moreover, typical markers of epithelial–mesenchymal transition such as E-cadherin, were increased, while vimentin and α–SMA were decreased after HIF-1α knockdown. In contrast, overexpression of DEC1 reversed the effects of HIF-1α knockdown. Cell proliferation, migration, and invasion were partially recovered. The level of E-cadherin was decreased, while the level of vimentin and α–SMA was increased. In addition, the level of β-catenin and LEF1 was downregulated after HIF-1α knockdown. The expression of MMP2 and MMP9 also declined. However, overexpression of DEC1 after HIF-1α knockdown partially reversed the expression pattern of these molecules. Conclusion HIF-1α contributed to EMT and metastasis through activation of canonical β-catenin signalling in choriocarcinoma and this process was dependent on DEC1. This study provides a new mechanism of HIF-1α in choriocarcinoma and suggests that intervention with DEC1 might be a promising therapeutic choice for choriocarcinoma (AU)

NLRP7 Enhances Choriocarcinoma Cell Survival and Camouflage in an Inflammasome Independent Pathway.

BACKGROUND: Gestational choriocarcinoma (GC) is a highly malignant trophoblastic tumor that often develops from a complete hydatidiform mole (HM). NLRP7 is the major gene responsible for recurrent HM and is involved in the innate immune response, inflammation and apoptosis. NLRP7 can function in an inflammasome-dependent or -independent pathway. Recently, we have demonstrated that NLRP7 is highly expressed in GC tumor cells and contributes to their tumorigenesis. However, the underlying mechanisms are still unknown. Here, we investigated the mechanism by which NLRP7 controls these processes in malignant (JEG-3) and non-tumor (HTR8/SVneo) trophoblastic cells. Cell survival, dedifferentiation, camouflage, and aggressiveness were compared between normal JEG-3 cells or knockdown for NLRP7, JEG-3 Sh NLRP7. In addition, HTR8/SVneo cells overexpressing NLRP7 were used to determine the impact of NLRP7 overexpression on non-tumor cells. NLRP7 involvement in tumor cell growth and tolerance was further characterized in vivo using the metastatic mouse model of GC. RESULTS: We demonstrate that NLRP7 (i) functions in an inflammasome-dependent and -independent manners in HTR8/SVneo and JEG-3 cells, respectively; (ii) differentially regulates the activity of NF-κB in tumor and non-tumor cells; (iii) increases malignant cell survival, dedifferentiation, and camouflage; and (iv) facilitates tumor cells colonization of the lungs in the preclinical model of GC. CONCLUSIONS: This study demonstrates for the first time the mechanism by which NLRP7, independently of its inflammasome machinery, contributes to GC growth and tumorigenesis. The clinical relevance of NLRP7 in this rare cancer highlights its potential therapeutic promise as a molecular target to treat resistant GC patients.

Ultra-High-Risk Gestational Choriocarcinoma of the Ovary Associated with Ectopic Pregnancy.

Gestational choriocarcinoma of the ovary is an exceptionally rare and highly aggressive tumor. Preoperative diagnosis of extrauterine choriocarcinoma is difficult due to nonspecific clinical presentation and its resemblance to ectopic pregnancy. Without molecular genetic analysis, it is not possible to reliably differentiate gestational from non-gestational choriocarcinoma. Here, we present a case of a 44-year-old woman who presented to our emergency department with complaints of pelvic pain, vaginal bleeding, and amenorrhea. Because of a recent history of conservatively managed ectopic pregnancy, the patient underwent emergency laparoscopy. Right-sided salpingo-oophorectomy was performed due to intraoperatively suspected ovarian ectopic pregnancy. Histopathology results revealed the diagnosis of ovarian choriocarcinoma of possible gestational origin. It was classified as FIGO stage IV and WHO ultra-high-risk, and she underwent multi-agent chemotherapy without major complications. She has remained in complete remission after a 12-month follow-up. Considering the rarity of this diagnosis, we conducted a literature review including all published cases of suspected gestational choriocarcinomas of the ovary. We conclude that due to the rarity of this entity, preoperative differentiating between ovarian ectopic pregnancy and ovarian choriocarcinoma is extremely challenging, and without molecular genetic analysis, it is not possible to identify the genetic origin of the tumor.

HIF-1α contributes to metastasis in choriocarcinoma by regulating DEC1 expression.

PURPOSE: To elucidate the underlying mechanism of HIF-1α in migration and invasion of choriocarcinoma. METHODS: Cell proliferation was determined by CCK-8 assay when cell invasion was detected by transwell assay. The protein expression was detected by western blotting, immunohistochemistry, and qPCR assay. RESULT: HIF-1α was shown to be strongly expressed in both clinical tumour tissues and cell lines in choriocarcinoma. When HIF-1α was efficiently knocked down in JEG3 cells, the proliferation rate was reduced by approximately 50% and the number of cells that migrated through the transwell insert was greatly decreased. The cell invasion rate was also significantly reduced. Moreover, typical markers of epithelial-mesenchymal transition such as E-cadherin, were increased, while vimentin and α-SMA were decreased after HIF-1α knockdown. In contrast, overexpression of DEC1 reversed the effects of HIF-1α knockdown. Cell proliferation, migration, and invasion were partially recovered. The level of E-cadherin was decreased, while the level of vimentin and α-SMA was increased. In addition, the level of ß-catenin and LEF1 was downregulated after HIF-1α knockdown. The expression of MMP2 and MMP9 also declined. However, overexpression of DEC1 after HIF-1α knockdown partially reversed the expression pattern of these molecules. CONCLUSION: HIF-1α contributed to EMT and metastasis through activation of canonical ß-catenin signalling in choriocarcinoma and this process was dependent on DEC1. This study provides a new mechanism of HIF-1α in choriocarcinoma and suggests that intervention with DEC1 might be a promising therapeutic choice for choriocarcinoma.

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.

Molecular Evidence for Epithelial Origin of Mixed Ovarian Epithelial-Germ Cell Neoplasms: Report of 2 Cases and Review of Literature.

Ovarian germ cell tumors (GCT) account for 2% to 3% of malignant ovarian neoplasms in Western countries and typically occur within the first 2 decades. When presenting later in life, GCTs may be associated with epithelial malignancies. In these circumstances, it has been theorized that these tumors may originate from a somatic, rather than germ cell origin, especially in the postmenopausal setting; however, the true derivation is not fully understood. Our database was searched for primary ovarian GCTs associated with a malignant epithelial component in patients above 35 yr of age, from 2006 to 2021. Two cases were identified and in each case, slides were reviewed and targeted next-generation sequencing was utilized to identify and compare gene mutation variants in morphologically distinct components. Patient A is a 58-yr-old, with choriocarcinoma and minor component of mucinous adenocarcinoma, and patient B is a 43-yr-old, with yolk sac tumor and minor component of endometrioid adenocarcinoma. The morphologically distinct areas in each case showed disparate staining patterns; however, next-generation sequencing demonstrated identical mutation variants within both the germ cell and epithelial components. Variants in CDKN2A , PIK3CA , PIK3R1 , and TP53 were present in patient A's tumor, while patient B's tumor showed CTNNB1 , PIK3R1 , and 2 PTEN variants. These mutational patterns are similar to those seen in pure epithelial counterparts, suggesting somatic derivation of the germ cell component. These rare tumors portend a poor prognosis and understanding their origin has clinical and therapeutic implications.

Uterine choriocarcinoma arising from serous carcinoma in a postmenopausal woman: an analysis of next-generation sequencing and PD-L1 immunochemistry.

BACKGROUND: Uterine somatic choriocarcinoma is a rare, clinically aggressive malignant tumor. They frequently concur with other cancer. However, the molecular pathogenesis between somatic choriocarcinoma and the concurrent carcinoma has rarely been addressed to date. CASE PRESENTATION: We report a 68-years old Chinese woman with a uterine choriocarcinoma arising from serous carcinoma. The patient underwent radical surgery including total abdominal hysterectomy with bilateral salpingo-oophorectomy, omentectomy and pelvic lymph node resection. She received 10 courses of post-operative chemotherapy. She died of disease 13 months after her surgery. Microscopically, the tumor showed a biphasic pattern of choriocarcinoma and serous carcinoma. The choriocarcinomatous component showed a combination of cytotrophoblast, intermediate trophoblast and syncytiotrophoblast with hemorrhage and necrosis. The component of serous carcinoma was characterized by solid sheets of small cells with marked nuclear atypia and occasional glandular and papillary formation. PD-L1 was exclusively expressed in the choriocarcinomatous component. Next-generation sequencing revealed that the genetic abnormalities were overlapping between the two components.

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

BACKGROUND: 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. OBJECTIVES: This study aimed to investigate the role of m6A in choriocarcinoma and reveal its underlying molecular mechanisms. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

LRSAM1 E3 Ubiquitin Ligase Promotes Choriocarcinoma Progression and Metastasis via p53/p21 Signaling Impediment.

Objective: The E3 ubiquitin ligase LRSAM1 (LRSAM1) was involved in many cancers, but whether it exerts anti- or protumor efficacies on choriocarcinoma cellular structures remains unknown. We wanted to explore the efficacies of aberrant LRSAM1 expression on human choriocarcinoma cellular structures and the underlying mechanisms. Methods: LRSAM1 mRNA expressions in choriocarcinoma lines of cells JEG-3 and JAR cellular structures, as well as HTR8/sev8 human trophoblastic cell line cellular structures, were assessed using assay analysis of quantitative real-time polymerase chain reactions. We compared cell proliferation, migratory flow, invasive force, adhesion, and apoptotic process between cellular structures infected with si-LRSAM1 plasmids versus negative controls using CCK-8, clone formation, Transwell, adhesion, and flow cytometry assays. Protein expressions of LRSAM1, E-cadherin, and N-cadherin (indicators of epithelial-mesenchymal transformation) and p53/p21 pathway components were quantitated using a Western blot assay. The morphology of tumor lesions was observed in xenografted nude mice using immunohistochemistry (IHC) analyses. Results: LRSAM1 was markedly overexpressed within JEG-3 and JAR choriocarcinoma cellular structures compared to HTR8/sev8 trophoblast cellular structures. Compared to si-NC, LRSAM1 knockdown robustly restricted cell proliferating, migratory flow, invasive force, and adhesion and fueled apoptotic cell process in JEG-3 as well as JAR cellular structures and suppressed tumor growth, as evidenced by the reduction in tumor volume and weight in naked mice inoculated with transfected cellular structures. Compared to si-negative control (si-NC), si-LRSAM1 significantly decreased Ki67 (a proliferating indicator) and N-cadherin expressions but reduced E-cadherin expression in JEG-3 and JAR cellular structures. Blocking the p53/p21 pathway by pifithrin-a (a p53 restrictor) successfully reversed the anti-inhibitory effect of LRSAM1 depletion, resulting in enhanced proliferating and metastasis in JEG-3 and JAR cellular structures. Conclusion: LRSAM1 exerts tumorigenic roles in choriocarcinoma. Via the activating of the p53/p21 pathway of signaling and impediment of choriocarcinoma cell proliferating, migratory flow, and invasive force, LRSAM1 knockdown slows the course of the disease. For choriocarcinoma diagnosis and treatment, it serves as a new therapeutic target.

CBR3-AS1 Accelerates the Malignant Proliferation of Gestational Choriocarcinoma Cells by Stabilizing SETD4.

Background: Gestational choriocarcinoma (GC) is a rare malignant gestational trophoblastic tumor. Long noncoding RNA (lncRNA) CBR3 antisense RNA 1 (CBR3-AS1) has been reported to serve as a critical oncogene and facilitate tumor progression. Besides, we found that CBR3-AS1 is implicated in GC progression. Materials and Methods: Gene and protein expression was detected via quantitative reverse transcription PCR (RT-qPCR) and western blot analyses, respectively. CCK-8 assay and colony formation assay were performed to assess cell proliferative abilities while flow cytometry analysis was applied for cell cycle and apoptosis. To analyze the specific mechanism among CBR3-AS1, SET domain containing 4 (SETD4), and polypyrimidine tract binding protein 1 (PTBP1), RNA binding protein immunoprecipitation (RIP), RNA pulldown, and mRNA stability assays were conducted. Results: CBR3-AS1 was markedly upregulated in GC cells, and its downregulation suppressed cell proliferation, induced cell cycle arrest, but promoted cell apoptosis in GC. SETD4 was determined as the downstream mRNA of CBR3-AS1 and positively regulated by CBR3-AS1 in GC cells. Furthermore, CBR3-AS1 could interact with its RNA binding protein (RBP) PTBP1, thereby stabilizing SETD4 mRNA. Rescue assays verified that CBR3-AS1 facilitates GC cell malignant proliferation via SETD4. Conclusion: CBR3-AS1 accelerates the malignant proliferation of GC cells via stabilizing SETD4.

Extracellular vesicle-mediated delivery of miR-127-3p inhibits the proliferation and invasion of choriocarcinoma cells by targeting ITGA6.

BACKGROUND: Choriocarcinoma (CC) is a highly aggressive malignant tumor that mostly occurs in women of childbearing age. Chemotherapy is the main treatment for CC, but it has side effects and causes drug resistance, which can lead to treatment failure. Extracellular vesicles (EVs) that deliver microRNAs (miRNAs) have emerged as a novel and promising therapeutic tool for inhibiting tumor progression and metastasis. This research aimed to study the effects of miR-127-3p-enriched EVs (EV-miR-127-3p) on CC and underlying mechanisms. METHODS: Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and western blotting were performed to determine the miR-127-3p and integrin subunit alpha-6 (ITGA6) expression levels. The interaction between miR-127-3p and ITGA6 was confirmed by a dual-luciferase reporter assay. Human umbilical cord mesenchymal stem cells (hUCMSCs) were identified using flow cytometry and multilineage differentiation. Uptake of labeled EVs was demonstrated using immunofluorescence staining and flow cytometry assays. EV-miR-127-3p were isolated from the culture medium of hUCMSCs and co-cultured with JEG-3 or JAR cells to evaluate their effects on cell proliferation, invasion, migration, and apoptosis, using the cell counting kit-8, Transwell, and flow cytometry assays. Epithelial-mesenchymal transition (EMT) and the transforming growth factor (TGF)-ß1/Smad pathway were investigated using qRT-PCR and western blotting. RESULTS: The expression of miR-127-3p was downregulated, while that of ITGA6 was upregulated in CC cell lines. ITGA6 was identified as a target gene of miR-127-3p. EV-miR-127-3p could inhibit the proliferation, invasion, migration, and promote the apoptosis of CC cells. We observed that EV-miR-127-3p suppressed EMT of CC cells by targeting ITGA6. In addition, the knockdown of ITGA6 inhibited the TGF-ß1/Smad pathway and reversed the EMT-promoting effect. CONCLUSION: These results indicate that EV-miR-127-3p from hUCMSCs exhibits anti-tumor effects by targeting ITGA6, which may be used as a novel therapeutic strategy for CC treatment.

Clinicopathologic and Molecular Characteristics of and Diagnostic Dilemmas in Invasive Breast Carcinoma with Choriocarcinomatous Pattern apropos a New Case: A Literature Review with New Findings.

BACKGROUND: Invasive breast carcinoma with a choriocarcinomatous pattern (IBC-CP) is extremely rare, and its molecular basis is yet unclear. The choriocarcinomatous pattern is characterized by the biphasic arrangement of multinucleated syncytiotrophoblast-like cells around clusters of monotypic tumor cells in a hemorrhagic background, along with ß-human chorionic gonadotropin (ß-hCG) expression. The differentiation of IBC-CP from metastatic choriocarcinoma of the breast (MC-B) is difficult due to the histologic similarity. METHODS: Based on a literature review and our own case, the clinicopathologic differences between IBC-CP patients (n = 17) and MC-B patients (n = 8) were analyzed. Moreover, in our case of IBC-CP, next-generation sequencing (NGS) comparative analysis was conducted for both choriocarcinomatous and invasive breast carcinoma (IBC) components. RESULTS: Compared to the MC-B patients, the IBC-CP patients were older (p < 0.001) and less frequently had past histories of gestational trophoblastic disease/pregnancy/abortion (p = 0.001) and distant metastases (p = 0.005). Our case, a 49-year-old female patient, presented with masses in the right breast and axilla. Following neoadjuvant chemotherapy, a radical mastectomy found an 8.5-cm-sized tumor. Microscopically, multinucleated syncytiotrophoblast-like cells were observed around mononuclear tumor cells with hemorrhage and necrosis. Some tumor cells showed ß-hCG immunopositivity, which was compatible with IBC-CP. NGS results showed a missense mutation in exon 5 of the TP53 gene in both the choriocarcinomatous and IBC components. Meanwhile, copy number loss in the PTEN gene was only identified in the choriocarcinomatous components. CONCLUSION: The present IBC-CP case is triple-negative breast cancer with TP53 mutation. The PTEN gene may be associated with choriocarcinomatous differentiation. Obtaining a medical history is mandatory to exclude metastatic lesions.

ATR and CDK4/6 inhibition target the growth of methotrexate-resistant choriocarcinoma.

Low-risk gestational trophoblastic neoplasia including choriocarcinoma is often effectively treated with Methotrexate (MTX) as a first line therapy. However, MTX resistance (MTX-R) occurs in at least ≈33% of cases. This can sometimes be salvaged with actinomycin-D but often requires more toxic combination chemotherapy. Moreover, additional therapy may be needed and, for high-risk patients, 5% still die from the multidrug-resistant disease. Consequently, new treatments that are less toxic and could reverse MTX-R are needed. Here, we compared the proteome/phosphoproteome of MTX-resistant and sensitive choriocarcinoma cells using quantitative mass-spectrometry to identify therapeutically actionable molecular changes associated with MTX-R. Bioinformatics analysis of the proteomic data identified cell cycle and DNA damage repair as major pathways associated with MTX-R. MTX-R choriocarcinoma cells undergo cell cycle delay in G1 phase that enables them to repair DNA damage more efficiently through non-homologous end joining in an ATR-dependent manner. Increased expression of cyclin-dependent kinase 4 (CDK4) and loss of p16Ink4a in resistant cells suggested that CDK4 inhibition may be a strategy to treat MTX-R choriocarcinoma. Indeed, inhibition of CDK4/6 using genetic silencing or the clinically relevant inhibitor, Palbociclib, induced growth inhibition both in vitro and in an orthotopic in vivo mouse model. Finally, targeting the ATR pathway, genetically or pharmacologically, re-sensitised resistant cells to MTX in vitro and potently prevented the growth of MTX-R tumours in vivo. In short, we identified two novel therapeutic strategies to tackle MTX-R choriocarcinoma that could rapidly be translated into the clinic.

ZBED1 Regulates Genes Important for Multiple Biological Processes of the Placenta.

The transcription factor ZBED1 is highly expressed in trophoblast cells, but its functions in the processes of trophoblast and placental biology remain elusive. Here, we characterized the role of ZBED1 in trophoblast cell differentiation using an in vitro BeWo cell model. We demonstrate that ZBED1 is enhanced in its expression early after forskolin-induced differentiation of BeWo cells and regulates many of the genes that are differentially expressed as an effect of forskolin treatment. Specifically, genes encoding markers for the differentiation of cytotrophoblast into syncytiotrophoblast and factors essential for trophoblast cell fusion and invasion were negatively regulated by ZBED1, indicating that ZBED1 might be important for maintaining a steady pool of cytotrophoblast cells. In addition, ZBED1 affected genes involved in the regulation of trophoblast cell survival and apoptosis, in agreement with the observed increase in apoptosis upon knockdown of ZBED1 in forskolin-treated BeWo cells. In addition, genes implicated in the differentiation, recruitment, and function of innate immune cells by the placenta were affected by ZBED1, further suggesting a role for this protein in the regulation of maternal immune tolerance. In conclusion, our study implicates ZBED1 in major biological processes of placental biology.

Lectin galactoside-binding soluble 3 binding protein mediates methotrexate resistance in choriocarcinoma cell lines.

Choriocarcinoma is one of the most aggressive gestational trophoblastic neoplasias (GTN). Methotrexate (MTX) resistance is the main cause of treatment failure in choriocarcinoma. However, the mechanism of MTX resistance in choriocarcinoma is poorly known. This study aims to explore the function of Lectin galactoside-binding soluble 3 binding protein (LGALS3BP) in MTX-resistance in choriocarcinoma cells. Gradual dose escalation of MTX was used to establish MTX-resistant choriocarcinoma cells (JAR-MTX and JEG3-MTX cell lines). RNA-sequencing was used to explore the differentially expressed genes. Plasmids or SiRNA transfection was used to regulate the expression of LGALS3BP. ELISA was used to detect the concentrations of LGALS3BP in the serum of MTX-sensitive and MTX-resistant patients. qRT-PCR, Western blot, and CCK-8 assay were used to determine the effects of LGALS3BP on MTX-resistance in JAR and JEG3 cells. The results showed the relative resistance index (RI) of MTX is 791.50 and 1040.04 in JAR-MTX and JEG3-MTX, respectively. LGALS3BP was up-regulated in MTX-resistant cells compared to original cells in both RNA and protein level. The concentrations of LGALS3BP were higher in the sera of MTX-resistant patients than in MTX-sensitive patients. Knocking down LGALS3BP can reverse the MTX-resistance in JAR-MTX and JEG3-MTX cells. In summary, we preliminarily established two MTX-resistant cells, and performed RNA-sequencing, and found LGALS3BP may play important role in MTX-resistance. Our work not only provides a research tool (MTX-resistant cells) for other researchers, but gives some hint on how MTX resistance is regulated.

Ropivacaine Retards the Viability, Migration, and Invasion of Choriocarcinoma Cells by Regulating the Long Noncoding RNA OGFRP1/MicroRNA-4731-5p/HIF3A Axis.

Choriocarcinoma is an aggressive gestational trophoblastic neoplasm. This study attempted to explore the biological functions and underlying mechanisms by which ropivacaine restrains the progression of choriocarcinoma. The expression of long noncoding RNA OGFRP1, microRNA-4731-5p (miR-4731-5p), and HIF3A in choriocarcinoma cells was assessed by qRT-PCR. Choriocarcinoma cells treated with ropivacaine at the concentration of 100, 500, and 1000 µM were cultured for 24, 48, and 72 h, respectively. Choriocarcinoma cell viability was evaluated by MTT assay. Transwell assay was conducted to examine choriocarcinoma cell migration and invasion. Additionally, the target relationship between OGFRP1 and miR-4731-5p or between miR-4731-5p and HIF3A was predicted by bioinformatics analysis and confirmed by dual-luciferase reporter assays. OGFRP1 and HIF3A expression were enhanced in choriocarcinoma cells, while miR-4731-5p expression was inhibited. Treatment with ropivacaine impeded choriocarcinoma cell viability, migration, and invasion. Choriocarcinoma cells treated with 1000 µM ropivacaine for 48 h were selected for subsequent experiments. OGFRP1 elevation or miR-4731-5p deficiency mitigated the reduction effect of ropivacaine on tumorigenesis of choriocarcinoma cells. Besides, miR-4731-5p was predicted as the potential OGFRP1 target by StarBase and LncBase, and HIF3A was predicted as the potential miR-4731-5p target by StarBase and TargetScan. Dual-luciferase reporter assays determined that miR-4731-5p was a target of OGFRP1 and HIF3A was a target of miR-4731-5p. Feedback experiments declared that miR-4731-5p elevation or HIF3A suppression reversed the promoting effect of OGFRP1 overexpression on the malignant behaviors of ropivacaine-treated choriocarcinoma cells. Ropivacaine constrained choriocarcinoma cell viability, migration, and invasion through modulating the OGFRP1/miR-4731-5p/HIF3A axis. Our study may provide a novel strategy for choriocarcinoma prevention and treatment.