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

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Abstract

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.

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Abbreviations

Bad:

Bcl-2 associated death promoter

BSA:

Bovine serum albumin

cAMP:

Cyclic adenosine monophosphate

DAB:

3,3′ diaminobenzidine

DAPi:

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

ECM:

Extracellular matrix

EMA-CO:

Methotrexate and actinomycin D with cyclophosphamide and vincristine

EP-EMA:

Etoposide and cisplatin with etoposide, methotrexate and dactinomycin

ERK:

Extracellular signal-regulated kinases

ETT:

Epithelioid trophoblastic tumor

FITC:

Fluorescein isothiocyanate

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

GTD:

Gestational trophoblastic disease

GTN:

Gestational trophoblastic neoplasia

hCG:

Human chorionic gonadotrophin

PBS:

Phosphate-buffered saline

PSTT:

Placental site trophoblastic tumor

TRITC:

Tetramethylrhodamine

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Acknowledgements

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.

Funding

This study has no funding and it was completely developed by participants own salary and material donations.

Author information

Authors and Affiliations

  1. Cell Biology Laboratory, Institute of Health and Biological Sciences, Federal University of Alagoas, Campus A.C. Simões. Av. Lourival Melo Mota, s/n, 57072-970, Maceio, Alagoas, Brazil

    Keyla S. N. Pires, Camilla M. Gonçalves, Jaqueline C. Santos, Liliane P. G. Tenorio, Rayane M. Botelho, Ana Lucia M. Silva, Hillary Gabriela S. Oliveira, Karen S. C. Borbely & Alexandre U. Borbely

  2. Department of Obstetrics, Universidade Federal de Sao Paulo-UNIFESP, Escola Paulista de Mediina, Sao Paulo, Brazil

    Sue Y. Sun, Karen Priscila T. Pendeloski & Silvia Daher

Authors
  1. Keyla S. N. Pires
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  2. Sue Y. Sun
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  3. Camilla M. Gonçalves
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  4. Jaqueline C. Santos
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  5. Liliane P. G. Tenorio
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  6. Rayane M. Botelho
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  7. Ana Lucia M. Silva
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  8. Hillary Gabriela S. Oliveira
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  9. Karen Priscila T. Pendeloski
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  10. Silvia Daher
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  11. Karen S. C. Borbely
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  12. Alexandre U. Borbely
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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.

Corresponding author

Correspondence to Alexandre U. Borbely.

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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.

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Pires, K.S.N., Sun, S.Y., Gonçalves, C.M. et al. Versican silencing in BeWo cells and its implication in gestational trophoblastic diseases. Histochem Cell Biol 151, 305–313 (2019). https://doi.org/10.1007/s00418-018-1739-9

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