In silico analysis of miRNAs in hydroxyurea induced foetal haemoglobin in sickle cell anaemia patients Smruti Sudha Biswal1, Anand Bodade2 1National Institute of Technology, Rourkela; 2NIIH, ICMR, Mumbai Methods and Materials
Abstract Sickle cell disease is a hereditary disease caused due to a change in 6 th position of beta globin chain from glutamic acid to valine. Various therapies are underway for the treatment, but hydroxyurea remains to be at the most sought form of treatment. However the exact molecular mechanisms of the miRNAs in this process remains elusive. Here in this study we have tried to elucidate the differentially expressed miRNAs and their plausible role in induction of foetal haemoglobin.
Introduction 1. Sickle cell anaemia is caused due to substitution of valine to glutamic acid in the 6th position of the beta globin 2.
Hydroxyurea (HU) is an oral, S-phase, cytotoxic, anti-metabolic and anti-neoplastic drug. It induces HbF in the sickle cell anaemic patients and is known to reduce the episodes of pain in the same 3. However, there the role of miRNAs in HUinduced HbF production in SCD patients is still unknown. 4. Here in this study we attempted to elucidate the role of top 50 differentially expressed miRNAs from the analysis of microarray data obtained through online databases. Their molecular mechanisms have been identified through functional annotation and KEGG pathway enrichment analysis
Aims and objectives Identifying and In silico analysis of miRNAs in hydroxyurea induced foetal haemoglobin in SCD patients
1. 2. 3. 4. 5.
Collection of dataset from Array express database (GSE32035 and GSE11060) Identification of DE expressed miRNAs Finding out targets of the identified DEmiRNAs Gene set enrichment analysis of the gene targets Pathway enrichment analysis of the identified gene targets
Fig 3: Heat maps of normalised data
Fig 1: Box plots of normalized data of GSE32025 and GSE11060
GSE32025
GSE11060
TYPE OF STUDY
Observational
Observational
OBSERVATIONAL
Cohort
Cohort
TIME PERSPECTIVE
Prospective
Prospective
ACTUAL
260 Participants
260 Participants
3
2
Fig 2: Volcano plots of the normalised data
hsa-miR-223*
Pathways in cancer
hsa-miR-126
Chronic myeloid leukemia
hsa-miR-30a*
MAPK signaling pathway
kshv-miR-K12-1
Neurotrophin signaling pathway
hsa-miR-342-3p
Pancreatic cancer
Table 2: DE miRNAs and the pathways enriched
Discussion
MODEL
ENROLLMENT NO OF COHORTS
Table 1: Details of study
Results 1. Out of the 50 DE miRNAs chosen from the lot of 2000 produced by the software, 32 DE miRNAs were identified to be downregulated while the remaining ones were upregulated. 2. A total of around 29000 genes were identified as targets for the DE miRNAs. Upon identification of targets, the gene set enrichment analysis was performed which identified around 125 enriched biological functions were identified. 3. Similarly through the pathway enrichment analysis, B cell receptor signalling pathway, T cell receptor signalling pathway, B-cell receptor signalling pathway, Maturity onset diabetes of the young (MODY), endocytosis, apoptosis, MAPK Signalling pathway, Axon guidance, ErB Signalling pathway, renal cell carcinoma, pancreatic cancer and endocytosis pathways were identified.
1. Through this study, we were able to identify different kinds of miRNAs like miR-29b-2, miR-552, miR-765, kshv-miR-K12-1 which were not only downregulated but also involved in different kinds of biological functions and pathways. 2. These miRNAs might be plausibly involved in the HbF induction process following HU 3. This information can be exploited to find out novel therapeutic targets for the treatment of sickle cell anaemia using the recently developed genome editing and gene therapy tools
Future Directions With this background we plan to conduct similar analysis in Indian SCD patient’s cohort. This will help in understanding the mechanism of action of HU in SCD patients via miRNAs. This will help in optimizing HU therapy in Indian cohort. This will definitely prove a better therapeutic approach in managing Sickle cell disease which is still a public health challenge in front of our country
References 1.Mnika K, Mazandu GK, Jonas M, et al. Hydroxyurea-Induced miRNA Expression in Sickle Cell Disease Patients in Africa. Front Gene 2019;10:509. 2.Gift D Pule, ShaheenMowla, Nicolas Novitzky, Charles S Wiysonge&AmbroiseWonkam (2015): A systematic review of known mechanisms of hydroxyureainduced fetalhemoglobin for treatment of sickle cell disease, Expert Review of Hematology
Acknowledgements
CSIR SRTP 2020