Versican silencing in BeWo cells and its implication in gestational trophoblastic diseases
- 14 Downloads
Versican is a proteoglycan known to interact with cells to influence their ability to proliferate, differentiate, migrate, invade and assemble extracellular matrix, with all of these cell functions present during placentation. In the placenta, cytotrophoblast cells have the ability to differentiate into the syncytiotrophoblast, a mechanism that is greatly increased in gestational trophoblastic diseases (GTD). Nevertheless, the molecular signaling underlying the increased syncytiotrophoblast differentiation are still being unveiled and may result in novel therapeutic targets for GTD. Versican expression was investigated to establish its differential expression among GTD (partial moles, complete moles, invasive moles and choriocarcinoma) and the possible functional outcomes from versican gene silencing. Tissue samples had their versican expression evaluated using immunohistochemistry and RT-PCR. BeWo cells were employed for versican silencing with siRNA and the efficiency was confirmed by RT-PCR, immunofluorescence and flow cytometry. Cell death and forskolin-induced syncytialization were analyzed by a morphological analysis and human chorionic gonadotropin (hCG) production using immunofluorescence. Versican V0 and V1 isoforms were mainly expressed in the syncytiotrophoblast and they were the most expressed in benign rather than in malignant tumors. BeWo cells also expressed V0 and V1 isoforms, but only in cells undergoing syncytial fusion. After versican silencing, cell death was greatly increased, whereas spontaneous and forskolin-induced syncytialization decreased as well as hCG production. Versican is differentially expressed in GTD and is important for hydatidiform moles pathophysiology, protecting trophoblast cells from death and playing a role in their differentiation and functionality.
KeywordsHydatidiform moles Placenta Versican
Bcl-2 associated death promoter
Bovine serum albumin
Cyclic adenosine monophosphate
Methotrexate and actinomycin D with cyclophosphamide and vincristine
Etoposide and cisplatin with etoposide, methotrexate and dactinomycin
Extracellular signal-regulated kinases
Epithelioid trophoblastic tumor
Glyceraldehyde 3-phosphate dehydrogenase
Gestational trophoblastic disease
Gestational trophoblastic neoplasia
Human chorionic gonadotrophin
Placental site trophoblastic tumor
We are grateful to all the assistance provided by Prof. Dr. Emiliano de Oliveira Barreto and Prof. Dr. Salete Smaniotto from Federal University of Alagoas. We also thank all nurses and physicians from Federal University of Sao Paulo and Federal University of Alagoas for all the support on tissues and data collection. A special acknowledgement to Martin Knöfler, Ph.D. from the Medical University of Vienna for the donation of paraffin blocks of first trimester placentas. AUB is the guarantor of this work and, as such, had full access to all of the data in the study and takes full responsibility for the integrity of the data and the accuracy of the data analysis.
KSN Pires was responsible for siRNA Kit standardization and cell death analysis. SY Sun was responsible for clinical data and samples collection. CM Gonçalves and HGS Oliveira were responsible for RNA extraction, RT-PCR assay and helped in siRNA silencing. JC Santos and ALM Silva were responsible for versican immunohistochemistry. LPG Souza and RM Botelho were responsible for syncytial fusion and hCG expression analysis. KPT Pendelosky and S Daher helped with clinical data and samples collection. KSC Borbely was responsible for flow cytometry, statistical analysis and manuscript revision. AU Borbely was responsible for students and results supervision and manuscript composition.
This study has no funding and it was completely developed by participants own salary and material donations.
Compliance with ethical standards
Conflict of interest
All authors declare that they have no competing interests.
Ethics approval and consent to participate
Use of tissues was approved by the ethical committees from all involved institutions through the Brazilian unified system for human ethical committees, the Plataforma Brasil (protocol number: 43605515.9.0000.5013), requiring patients informed consent.
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
- Braga A, Uberti EMH, Fajardo MC, Viggiano M, Sun SY, Grillo BM, Padilha SL, Andrade JM, Souza CB, Madi JM, Maestá I, Silveira E (2014) Epidemiological report on the treatment of patients with gestational trophoblastic disease in 10 Brazilian referral centers. Results after 12 years since international FIGO 200 consensus. J Reprod Med 59:241–247PubMedGoogle Scholar
- Cattaruzza S, Schiappacassi M, Ljungberg-Rose A, Spessotto P, Perissinotto D, Mörgelin M, Mucignat MT, Colombatti A, Perris R (2002) Distribution of PG-M/versican variants in human tissues and de novo expression of isoform V3 upon endothelial cell activation, migration and neoangiogenesis in vitro. J Biol Chem 277:47626–47635CrossRefGoogle Scholar
- Fang L, Du WW, Yang X, Chen K, Ghanekar A, Levy G, Yang W, Yee AJ, Lu WY, Xuan JW, Gao Z, Xie F, He C, Deng Z, Yang BB (2013) Versican 3′-untranslated region (3′-UTR) functions as a ceRNA in inducing the development of hepatocellular carcinoma by regulating miRNA activity. FASEB J 7:907–919CrossRefGoogle Scholar
- Huppertz B, Kaufmann P, Kingdom J (2003a) Trophoblast turnover in health and disease. Fetal Maternal Med Rev 13:103–118Google Scholar
- Mitsui Y, Shiina H, Kato T, Maekawa S, Hashimoto Y, Shiina M, Imai-Sumida M, Kulkarni P, Dasgupta P, Wong RK, Hiraki M, Arichi N, Fukuhara S, Yamamura S, Majid S, Saini S, Deng G, Dahiya R, Nakajima K, Tanaka Y (2015) Versican promotes tumor progression, metastasis and predicts poor prognosis in renal carcinoma. Mol Cancer Res 15:884–895CrossRefGoogle Scholar
- Schmalfeldt M, Bandtlow CE, Dours-Zimmermann MT, Winterhalter KH, Zimmermann DR (2010) Brain derived versican V2 is a potent inhibitor of axonal growth. J Cell Sci 113:807–816Google Scholar
- Sun SY, Melamed A, Goldstein DP, Bernstein MR, Horowitz NS, Moron AF, Maestá I, Braga A, Berkowitz RS (2015) Changing presentation of complete hydatidiform mole at the New England Trophoblastic Disease Center over the past three decades: does early diagnosis alter risk for gestational trophoblastic neoplasia? Gynecol Oncol 138:46–49CrossRefGoogle Scholar
- Uberti EMH, Fajardo MC, Cunha AGV, Frota SS, Braga A, Ayub ACK (2015) Treatment of low-risk gestational trophoblastic neoplasia comparing biweekly eight-day methotrexate with folinic acid versus bolus-dose actinomycin-D, among Brazilian women. Rev Bras Ginecol Obstet 37:258–265CrossRefGoogle Scholar