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Inhibition of γ/β Globin Gene Switching in CD 34+ Derived Erythroid Cells by BCL11A RNA Silencing

  • Seyyed Asadallah Taghavi
  • Kamran Mousavi HosseiniEmail author
  • Gholamhossein Tamaddon
  • Leila Kasraian
Original Article
  • 26 Downloads

Abstract

The induction of fetal haemoglobin (Hb F), due to the sustained clinical effects, is one of the most promising methods for the treatment of β hemoglobinopathies, such as thalassemia major and sickle cell disease (SCD). Inhibition of γ-globin gene silencing, possibly is a suitable strategy to induce HbF expression in these patients. In this study, the possibility of increasing HbF in the CD34+ derived erythroid cells was investigated by BCL11A inhibition using specific small-interfering RNAs (siRNAs). Human peripheral blood-derived hematopoietic stem cells were isolated and differentiated to erythroid cells. Erythroid maturation was investigated using cell morphology parameters and flow cytometry analysis of CD235a expression On day 20, siRNA complementary to BCL11A was transfected to differentiating cells via electroporation. BCL11A expression was evaluated through real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) and enzyme linked immunosorbant assay (ELISA). β actin was used as the reference gene to confirm the relative expression level of BCL11A gene mRNA. 48 hours after transfection, BCL11A siRNA significantly reduced BCL11A mRNA levels and consequently led to 2.0 fold decrease in corresponding protein. On the 28th day, haemoglobin electrophoresis results showed that Hb F levels in transfected erythroid cells increased 3.3-fold when compared with non transfected cells. In this study we showed that BCL11A inhibition in erythroid cells could increase fetal hemoglobin, and this strategy can be the basis for designing a γ globin expressing cellular system to increase Hb F in patients with thalassemia and SCD.

Keywords

Erythroid cells Gene silencing BCL11A SiRNA 

Notes

Acknowledgements

This article was extracted from a Ph.D. thesis and was financially supported by Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran. The authors declare no conflict of interest.

Author’s Contributions

S.A.T., K.M.H.; Contributed to conception and design. S.A.T., K.M.H., GH.T; Contributed to all experimental work and data. S.A.T., K.M.H., L.K.; Contributed to statistical analysis and interpretation of data. K.M.H., GH.T; were responsible for overall supervision. S.A.T.; Drafted the manuscript, which was revised by K.M.H. All authors read and approved the final manuscript.

Compliance with Ethical Standards

Conflict of interest

All authors of this article declare that they have no conflict of interest.

Human and Animal Rights

This article does not contain any studies with human participants performed by any of the authors.

Informed Consent

Informed consent was obtained from all individual participants included in the study.

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Copyright information

© Indian Society of Hematology and Blood Transfusion 2019

Authors and Affiliations

  1. 1.High Institute for Research and Education in Transfusion MedicineBlood Transfusion Research CenterTehranIran
  2. 2.Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical SciencesShiraz University of Medical SciencesShirazIran

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