Sanjeev Raghuwanshi

Bio: Sanjeev Raghuwanshi is an academic researcher from University of Hyderabad. The author has contributed to research in topics: Megakaryocyte & Haematopoiesis. The author has an hindex of 6, co-authored 18 publications receiving 254 citations. Previous affiliations of Sanjeev Raghuwanshi include University of Delhi.

MicroRNA-9 promotes cell proliferation by regulating RUNX1 expression in human megakaryocyte development.

Abstract: To the editor: We read with interest the article by Tian et al,1 describing the role of MicroRNA9 (miR9) in the regulation of myeloidderived suppressor cell (MDSC) differentiation and function by targeting the runtrelated transcription factor 1 (Runx1). This finding has revealed posttranscriptional regulation of Runx1 by miR9. RUNX1, the master regulator of hematopoiesis,2 is known to regulate megakaryocyte (MK) polyploidization and cytoskeleton rearrangement in the process of MK maturation and proplatelet formation.3,4 Specifically, RUNX1 sequence is conserved for miR9 among vertebrates and miR9 was also identified with decreasing intensity of expression during human MK ontogeny,5 which shows the functional significance of miR9/RUNX1 axis during evolution. Thus, miR9 could be a potential therapeutic target in neonatal thrombocytopenia and other platelet disorders. Thrombocytopenia, the deficiency of platelets in the blood, is a major clinical problem encountered among several conditions and is common in all sick and preterm neonates admitted to NICU; the primary outcome of thrombocytopenia in infants is the incidence and severity of intraventricular haemorrhages (IVH), which is a leading cause of poor neurological outcome and mortality in sick neonates.6,7 It is believed that developmental differences between neonatal and adult MKs contribute to this vulnerability.8 Specifically, neonatal MK progenitors possess a high proliferative potential and give rise to MKs smaller and of low ploidy that generate fewer platelets compared with adult MKs. The regulatory mechanisms underlying these developmental differences are unknown, but we have revealed a critical role of small noncoding RNAs (miRNAs) in the regulation of MK development.9 Further, RUNX1 emerged as a putative target of miR9 by several bioinformatic databases such as TargetScan, miRbase and RNAhybrid. In our miRNA array study of neonatal vs adult MKs, four other RUNX1 targeting miRNAs were identified with higher levels of expression in neonatal MKs, although miR9 was the highest upregulated miRNA (Table 1, P < .05). HsamiR9 and RUNX1 mRNA duplex showed minimum free energy of −17.6 kcal/mole using bioinformatic tool RNAhybrid (Figure 1A). To test the role of miR9 in RUNX1 regulation in megakaryocytes, we cultured human CBand adult PBCD34+ cells (n = 3 for each group) in the presence of serum free medium with TPO (50 ng/mL), as previously described10; after 14 days of culture, >90% of cells were MKs (CD41+). The expression levels of hsamiR9 and RUNX1 mRNA were measured by qRTPCR (n = 3) and were normalized against internal control U6 and βactin, respectively.10,11 Initially, we confirmed the hsamiR9 expression levels and found that CB levels were significantly higher compared with PBderived MKs, and these differences were consistent through all the stages (0, 7, 11, 14 days) of megakaryocytopoiesis (n = 3, P = .02; Figure 1B), whereas its target RUNX1 mRNA was observed with significant lower expression in CBderived MKs compared with PBderived MKs (n = 3, P < .05; Figure 1C). RUNX1 protein levels were determined by western blot using antiRUNX1 (Santa Cruz, CA, USA) and antiβactin (Santa Cruz) antibodies. RUNX1 protein levels were quantified by densitometry using the ImageJ system and normalized with βactin. We further confirmed the developmental differences between CBderived MKs and PBderived MKs in RUNX1 protein expression (n = 3, P = .02; Figure 1D). To prove the functional significance of hsamiR9 in RUNX1 regulation, we transfected MEG01 (human megakaryoblastic leukaemia cell line) and Dami (human megakaryocytic leukaemia cell line) cells with either premiR9 or scrambled control (Cy3 labelled) using lipofectamine Hiperfect (Qiagen, Hilden, Germany). MEG01 and Dami cells, respectively, were maintained in DMEM and RPMI with 10% FBS. After 72 hours of transfection, we observed a significant increase in the miR9 levels (n = 3, P < .05; Figure 1E), along with high cell proliferation rate (n = 3, P < .05; Figure 1F) and reduced MK marker, CD61 expression (n = 3, P = .02; Figure 1G). Further, lower RUNX1 protein levels (n = 3, P < .001; Figure 1H) were noticed using western blot analysis. Over the past many years, multiple reports have identified the essential role of miRNAs in the regulation of lineageselective TFs.10 In vertebrates, recent studies evidenced the development stage and cellular context dependent role of miR9 on cell proliferation, migration and differentiation.12 In this study, we observed developmental differences in the expression levels of hsamiR9 in neonatal vs adult MKs (Figure 1B), and similar developmental differences were also observed in embryonic stem cell MKs vs adult MKs.5 Hypothetically, downregulated miRNAs during MK development and differentiation

Epigenetic Mechanisms: Role in Hematopoietic Stem Cell Lineage Commitment and Differentiation.

Abstract: Major breakthroughs in the last several decades have contributed to our knowledge of the genetic regulation in development. Although epigenetics is not a new concept, unfortunately, the role of epigenetics has not come to fruition in the past. But the field of epigenetics has exploded within the past decade. Now, growing evidences show a complex network of epigenetic regulation in development. The epigenetic makeup of a cell, tissue or individual is much more complex than their genetic complement. Epigenetic modifications are more important for normal development by maintaining the gene expression pattern in tissue- and context-specific manner. Deregulation of epigenetic mechanism can lead to altered gene expression and its function, which result in altered tissue specific function of cells and malignant transformation. Epigenetic modifications directly shape Hematopoietic Stem Cell (HSC) developmental cascades, including their maintenance of self-renewal and multilineage potential, lineage commitment, and aging. Hence, there is a growing admiration for epigenetic players and their regulatory function in haematopoiesis. Epigenetic mechanisms underlying these modifications in mammalian genome are still not completely understood. This review mainly explains 3 key epigenetics mechanisms including DNA methylation, histone modifications and non-coding RNAs inference in hematopoietic lineage commitment and differentiation.

MicroRNA function in megakaryocytes

Abstract: Megakaryocytes (MKs), the largest cells in the bone marrow, are generated from hematopoietic stem cells (HSCs) in a sequential process called megakaryocytopoiesis in which HSCs undergo MK-progenitor (MP) commitment and maturation to terminally differentiated MK. Megakaryocytopoiesis is controlled by a complex network of bone marrow niche factors. Traditionally, the studies on megakaryocytopoiesis were focused on different cytokines, growth factors and transcription factors as the regulators of megakaryocytopoiesis. Over the past two decades many research groups have uncovered the key role of microRNAs (miRNAs) in megakaryocytopoiesis. miRNAs are a class of small length non-coding RNAs which play key regulatory role in cellular processes such as proliferation, differentiation and development and are also known to be involved in disease development. This review summarizes the current state of knowledge of miRNAs which have changed expression during megakaryocytopoiesis, also focuses on miRNAs which are differentially regulated during developmental maturation of MKs. Further, we aimed to discuss potential mechanisms of miRNAs-mediated regulation underlying megakaryocytopoiesis and developmental maturation of MKs.

MicroRNAs: Target Recognition and Regulatory Functions

Abstract: MicroRNAs (miRNAs) are endogenous ∼23 nt RNAs that play important gene-regulatory roles in animals and plants by pairing to the mRNAs of protein-coding genes to direct their posttranscriptional repression. This review outlines the current understanding of miRNA target recognition in animals and discusses the widespread impact of miRNAs on both the expression and evolution of protein-coding genes.

The role of m6A modification in the biological functions and diseases.

Abstract: N6-methyladenosine (m6A) is the most prevalent, abundant and conserved internal cotranscriptional modification in eukaryotic RNAs, especially within higher eukaryotic cells. m6A modification is modified by the m6A methyltransferases, or writers, such as METTL3/14/16, RBM15/15B, ZC3H3, VIRMA, CBLL1, WTAP, and KIAA1429, and, removed by the demethylases, or erasers, including FTO and ALKBH5. It is recognized by m6A-binding proteins YTHDF1/2/3, YTHDC1/2 IGF2BP1/2/3 and HNRNPA2B1, also known as “readers”. Recent studies have shown that m6A RNA modification plays essential role in both physiological and pathological conditions, especially in the initiation and progression of different types of human cancers. In this review, we discuss how m6A RNA methylation influences both the physiological and pathological progressions of hematopoietic, central nervous and reproductive systems. We will mainly focus on recent progress in identifying the biological functions and the underlying molecular mechanisms of m6A RNA methylation, its regulators and downstream target genes, during cancer progression in above systems. We propose that m6A RNA methylation process offer potential targets for cancer therapy in the future.