Versican silencing in BeWo cells and its implication in gestational trophoblastic diseases

  • Keyla S. N. Pires
  • Sue Y. Sun
  • Camilla M. Gonçalves
  • Jaqueline C. Santos
  • Liliane P. G. Tenorio
  • Rayane M. Botelho
  • Ana Lucia M. Silva
  • Hillary Gabriela S. Oliveira
  • Karen Priscila T. Pendeloski
  • Silvia Daher
  • Karen S. C. Borbely
  • Alexandre U. BorbelyEmail author
Original Paper


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.


Hydatidiform moles Placenta Versican 



Bcl-2 associated death promoter


Bovine serum albumin


Cyclic adenosine monophosphate


3,3′ diaminobenzidine


4′-6-Diamidine-2′-phenylindole dihydrochloride


Extracellular matrix


Methotrexate and actinomycin D with cyclophosphamide and vincristine


Etoposide and cisplatin with etoposide, methotrexate and dactinomycin


Extracellular signal-regulated kinases


Epithelioid trophoblastic tumor


Fluorescein isothiocyanate


Glyceraldehyde 3-phosphate dehydrogenase


Gestational trophoblastic disease


Gestational trophoblastic neoplasia


Human chorionic gonadotrophin


Phosphate-buffered saline


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.

Authors’ contributions

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.

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Supplementary material

418_2018_1739_MOESM1_ESM.doc (58 kb)
Supplementary material 1 (DOC 59 KB)
418_2018_1739_MOESM2_ESM.doc (47 kb)
Supplementary material 2 (DOC 47 KB)
418_2018_1739_MOESM3_ESM.tif (14.5 mb)
Supplementary material 3 (TIF 14810 KB)
418_2018_1739_MOESM4_ESM.docx (13 kb)
Supplementary material 4 (DOCX 14 KB)


  1. 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
  2. Braga A, Maestá I, Rocha Soares R, Elias KM, Custódio Domingues MA, Barbisan LF, Berkowitz RS (2016) Apoptotic index for prediction of postmolar gestational trophoblastic neoplasia. Am J Obstet Gynecol 215:336.e1-336.e12CrossRefGoogle Scholar
  3. Bukong TN, Maurice SB, Chahal B, Schaeffer DF, Winwood PJ (2016) Versican: a novel modulator of hepatic fibrosis. Lab Invest 96:361–374CrossRefGoogle Scholar
  4. Carthy JM, Meredith AJ, Boroomand S, Abraham T, Luo Z, Knight D, McManus BM (2015) Versican V1 overexpression induces a myofibroblast-like phenotype in cultured fibroblasts. PLoS One 10:e0133056CrossRefGoogle Scholar
  5. Castellucci M, Scheper M, Scheffen I, Celona A, Kaufmann P (1990) The development of the human placental villous tree. Anat Embryol 181:117–128CrossRefGoogle Scholar
  6. 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
  7. Chan CC, Lao TT, Cheung AN (1999) Apoptotic and proliferative activities in first trimester placentae. Placenta 20:223–227CrossRefGoogle Scholar
  8. Corps AN, Robinson AH, Movin T, Costa ML, Ireland DC, Hazleman BL, Riley GP (2014) Versican splice variant messenger RNA expression in normal human Achilles tendon and tendinopathies. Rheumatology 43:969–972CrossRefGoogle Scholar
  9. Crocker IP, Tansinda DM, Jones CJ, Baker PN (2004) The influence of oxygen and tumor necrosis factor-alpha on the cellular kinetics of term placental villous explants in culture. J Histochem Cytochem 52:749–757CrossRefGoogle Scholar
  10. Cummings BS, Wills LP, Schnellmann RG (2012) Measurement of cell death in Mammalian cells. Curr Protoc Pharmacol 56(1):12.8.1–12.8.24CrossRefGoogle Scholar
  11. Desjardins M, Xie J, Gurler H, Muralidhar GG, Sacks JD, Burdette JE, Barbolina MW (2014) Versican regulates metastasis of epithelial ovarian carcinoma cells and spheroids. J Ovarian Res 7:67–70CrossRefGoogle Scholar
  12. Du WW, Yang W, Yee AJ (2013) Roles of versican in cancer biology- tumorigenesis, progression and metastasis. Histol Histopathol 28:701–713PubMedGoogle Scholar
  13. Evanko SP, Tammi MI, Tammi RH, Wight TN (2007) Hyaluronan-dependent pericellular matrix. Adv Drug Deliv Rev 59:1351–1365CrossRefGoogle Scholar
  14. 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
  15. Hui P, Buza N, Murphy KM, Ronnett BM (2017) Hydatidiform moles: genetic basis and precision diagnosis. Ann Rev Pathol Mech Dis 12:449–485CrossRefGoogle Scholar
  16. Huppertz B, Kaufmann P, Kingdom J (2003a) Trophoblast turnover in health and disease. Fetal Maternal Med Rev 13:103–118Google Scholar
  17. Huppertz B, Kingdom J, Caniggia I, Desoye G, Black S, Korr H, Kaufmann P (2003b) Hypoxia favours necrotic versus apoptotic shedding of placental syncytiotrophoblast into the maternal circulation. Placenta 24:181–190CrossRefGoogle Scholar
  18. Jeffers MD, Richmond JA, Smith R (1996) Trophoblast proliferation rate does not predict progression to persistent gestational trophoblastic disease in complete hydatidiform mole. Int J Gynecol Pathol 15:34–38CrossRefGoogle Scholar
  19. Kato HD, Terao Y, Ogawa M, Matsuda T, Arima T, Kato K, Yong Z, Wake N (2002) Growth-associated gene expression profiles by microarray analysis of trophoblast of molar pregnancies and normal villi. Int J Gynecol Pathol 21:255–260CrossRefGoogle Scholar
  20. Kischel P, Walttregny D, Dumont B, Turtoi A, Greffe Y, Kirsch S, De Pauw E, Castronovo E (2010) Versican overexpression in human breast cancer lesions: known and new isoforms for stromal tumor targeting. Int J Cancer 126:640–650CrossRefGoogle Scholar
  21. Lemire JM, Merrilees MJ, Braun KR, Wight TN (2002) Overexpression of the V3 variant of versican alters arterial smooth muscle cell adhesion, migration and proliferation in vitro. J Cell Physiol 190:38–45CrossRefGoogle Scholar
  22. Li F, Li S, Cheng T (2014) TGF-beta1 promotes osteosarcoma cell migration and invasion through the miR-143-versican pathway. Cell Physiol Biochem 34:2169–2179CrossRefGoogle Scholar
  23. Lurain JR (2011) Gestational trophoblastic disease II: classification and management of gestational trophoblastic neoplasia. Am J Obstet Gynecol 204:11–18CrossRefGoogle Scholar
  24. 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
  25. Ngan HY, Seckl MJ, Berkowitz RS, Xiang Y, Golfier F, Sekharan PK, Lurain JR (2015) Update on the diagnosis and management of gestational trophoblastic disease. Int J Gynaecol Obstet 131:S123–S126CrossRefGoogle Scholar
  26. Oktem G, Sercan O, Guven U, Uslu R, Uysal A, Goksel G, Ayla S, Bilir A (2014) Cancer stem cell differentiation: TGFbeta1 and versican may trigger molecules for the organization of tumor spheroids. Oncol Rep 32:641–649CrossRefGoogle Scholar
  27. 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
  28. Seckl MJ, Sebire NJ, Fisher RA, Golfier F, Massuger L, Sessa C, ESMO Guidelines Working Group (2013) Gestational trophoblastic disease: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 24:39–50CrossRefGoogle Scholar
  29. Sheng W, Wang G, Wang Y, Liang J, Wen J, Zheng PC, Wu Y, Lee Y, Slingerland J, Dumont D, Yang BB (2005) The roles of versican V1 and V2 isoforms in cell proliferation and apoptosis. Mol Biol Cell 16:1330–1340CrossRefGoogle Scholar
  30. 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
  31. 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
  32. Werfel J, Krause S, Bischof AG, Mannix RJ, Tobin H, Bar-Yam Y, Bellin RM, Ingber DE (2013) How changes in extracellular matrix mechanics and gene expression variability might combine to drive cancer progression. PLoS One 8:e76122CrossRefGoogle Scholar
  33. Wice B, Menton D, Geuze H, Schwartz AL (1990) Modulators of cyclic AMP metabolism induce syncytiotrophoblast formation in vitro. Exp Cell Res 186:306–316CrossRefGoogle Scholar
  34. Wight TN, Kinsella MG, Evanko SP, Potter-Perigo S, Merrilees J (2014) Versican and the regulation of cell phenotype in disease. Biochem Biophys Acta 1840:2441–2451CrossRefGoogle Scholar
  35. Wolfberg AJ, Berkowitz RS, Goldstein DP, Feltmate C, Lieberman E (2005) Postevacuation hCG levels and risk of gestational trophoblastic neoplasia in women with complete molar pregnancy. Obstet Gynecol 106:548–552CrossRefGoogle Scholar
  36. Wu Y, Chen L, Zheng PS, Yang BB (2002) Beta 1-integrin-mediated glioma cell adhesion and free radical-induced apoptosis are regulated by binding to a C-terminal domain of PG-M/versican. J Biol Chem 277:12294–12301CrossRefGoogle Scholar
  37. Wu Y, Sheng W, Chen L, Dong H, Lee V, Lu F, Wong CS, Lu WY, Yang BB (2004) Versican V1 isoform induces neuronal differentiation and promotes neurite outgrowth. Mol Biol Cell 15:2093–2104CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  • Keyla S. N. Pires
    • 1
  • Sue Y. Sun
    • 2
  • Camilla M. Gonçalves
    • 1
  • Jaqueline C. Santos
    • 1
  • Liliane P. G. Tenorio
    • 1
  • Rayane M. Botelho
    • 1
  • Ana Lucia M. Silva
    • 1
  • Hillary Gabriela S. Oliveira
    • 1
  • Karen Priscila T. Pendeloski
    • 2
  • Silvia Daher
    • 2
  • Karen S. C. Borbely
    • 1
  • Alexandre U. Borbely
    • 1
    Email author
  1. 1.Cell Biology Laboratory, Institute of Health and Biological SciencesFederal University of AlagoasMaceioBrazil
  2. 2.Department of ObstetricsUniversidade Federal de Sao Paulo-UNIFESP, Escola Paulista de MediinaSao PauloBrazil

Personalised recommendations