Evaluation of the angiogenic properties of Brugia malayi asparaginyl-tRNA synthetase and its mutants: A study on the molecular target for antifilarial drug development.

Brugia malayi asparaginyl-tRNA synthetase (BmAsnRS) has been identified as an immunodominant antigen and a physiocrine that mimics Interleukin-8 (IL-8) to induce chemotaxis and angiogenesis in endothelial cells. Computational analyses have shown that the N-terminal region of BmAsnRS has a novel fold, a lysine rich ß-hairpin α-helix, (FLIRTKKDGKQIWE) which is similar to that present in IL-8 chemokine, CXCR1. This novel fold is involved in tRNA binding and is integral for the manifestation of the disease, lymphatic filariasis (LF). Drug discovery programmes carried out so far for LF have not been successful because of the target (BmAsnRS) resistance due to the disease-associated mutation. Mutations in AARS targets have been shown to correlate with several diseases. However, no disease-associated mutational studies have been carried out for LF. BmAsnRS has been an established target for LF. It was proposed, therefore, to study the effect of single point mutations in BmAsnRS so as to elucidate the molecular target. An understanding of the molecular consequences of mutations will provide insight into how resistance develops in addition to the identification of the likely resistance-conferring mutations. Three mutants were, therefore, generated by site-directed mutagenesis using CUPSAT server and their angiogenic properties evaluated. Cytometric analysis of the mutants on endothelial cell cycle was also carried out. CUPSAT prediction of protein stability upon point mutations reveal that two mutants generated are likely resistance-conferring mutations. All the three mutants show significant reduction in their angiogenic properties and reduction in the DNA content in the cells of S and G2/M phases thus showing altered function of the gene encoding the drug target. The resistance- conferring mutants, however, show angiogenic properties nearer to the wild type protein, BmAsnRS. Future work on designing newer drugs may take into consideration these drug resistance-conferring mutations.

Formulation and biological actions of nano-bioglass ceramic particles doped with Calcarea phosphorica for bone tissue engineering.

The improvisation of the treatment procedures for treating the various kind of bone defects such as, bone or dental trauma and for diseases such as osteoporosis, osteomyelitis etc., need the suitable and promising biomaterials with resemblance of bone components. Bioactive glass ceramic (BGC) has recently acquired great attention as the most promising biomaterials; hence it has been widely applied as a filler material for bone tissue regeneration. Because it elicts specific biological responses after implantation in addition more potential in formation of strong interface with both hard and soft tissues by dissolution of calcium and phosphate ions. Hence, the current focus in treating the bone defects by orchestrating the biomaterial in combination of alternative medicine such as homeopathic remedies with biomaterials to prevent the adverse effects at minimal concentrations. So the current study was focused on constructing the nano-bioglass ceramic particles (nBGC) doped with novel homeopathic remedy Calcarea phosphorica for dental and bone therapeutic implants. The nBGC particles were synthesized by sol-gel method and reinforced with commercially available Calcarea phosphorica. The synthesized particles were characterized by SEM, DLS, EDS, FT-IR, and XRD studies. The SEM and DLS were shown the size of the particles at nano scale, also the EDS, and FT-IR investigations indicated that the Calcarea phosphorica was integrated with nBGC particles and also the crystalline nature of particles was confirmed by XRD studies. Both nBGC and Calcarea phosphorica doped nBGC (CP-nBGC) were found to be non toxic to mouse mesenchymal stem cells at lower concentrations and also illustrated the better bone forming ability in vitro.

MicroRNA-590-5p Stabilizes Runx2 by Targeting Smad7 During Osteoblast Differentiation.

Mesenchymal stem cells (MSCs) are multipotent cells and their differentiation into the osteoblastic lineage is strictly controlled by several regulators, including microRNAs (miRNAs). Runx2 is a bone transcription factor required for osteoblast differentiation. Here, we used in silico analysis to identify a number of miRNAs that putatively target Runx2 and its co-factors to mediate both positive and negative regulation of osteoblast differentiation. Among these miRNAs, miR-590-5p was selected and its expression was found to be increased during osteoblast differentiation. When mouse MSCs (mMSCs) were transiently transfected with a miR-590-5p mimic, we detected an increase in both calcium deposition and the mRNA expression of osteoblast differentiation marker genes such as alkaline phosphatase (ALP) and type I collagen genes. Smad7 was found to be among the putative target genes of miR-590-5p and its mRNA and protein expression decreased after miR-590-5p mimic transfection in human osteoblast-like cells (MG63). Our analysis indicated that Runx2 was not a putative target of miR-590-5p. However, Runx2 protein, but not mRNA expression, increased after miR-590-5p mimic transfection in MG63 cells. Runx2 protein expression was increased with knockdown of Smad7 expression by Smad7 siRNA in these cells. We further identified that the 3'-untranslated region of Smad7 was directly targeted by miR-590-5p; this was done using the luciferase reporter gene system. It is known that Smad7 inhibits osteoblast differentiation via Smurf2-mediated Runx2 degradation. Hence, based on our results, we suggest that miR-590-5p promotes osteoblast differentiation by indirectly protecting and stabilizing the Runx2 protein by targeting Smad7 gene expression. J. Cell. Physiol. 232: 371-380, 2017. © 2016 Wiley Periodicals, Inc.

A Combinatorial effect of carboxymethyl cellulose based scaffold and microRNA-15b on osteoblast differentiation.

The present study was aimed to synthesize and characterize a bio-composite scaffold containing carboxymethyl cellulose (CMC), zinc doped nano-hydroxyapatite (Zn-nHAp) and ascorbic acid (AC) for bone tissue engineering applications. The fabricated bio-composite scaffold was characterized by SEM, FT-IR and XRD analyses. The ability of scaffold along with a bioactive molecule, microRNA-15b (miR-15b) for osteo-differentiation at cellular and molecular levels was determined using mouse mesenchymal stem cells (mMSCs). miR-15b acts as posttranscriptional gene regulator and regulates osteoblast differentiation. The scaffold and miR-15b were able to promote osteoblast differentiation; when these treatments were combined together on mMSCs, there was an additive effect on promotion of osteoblast differentiation. Thus, it appears that the combination of CMC/Zn-nHAp/AC scaffold with miR-15b would provide more efficient strategy for treating bone related defects and bone regeneration.

Role of Mesoporous Wollastonite (Calcium Silicate) in Mesenchymal Stem Cell Proliferation and Osteoblast Differentiation: A Cellular and Molecular Study.

Wollastonite (calcium silicate) has been widely used in bone tissue engineering, but its mechanism of action on the regulation of mesenchymal stem cell proliferation and differentiation to osteoblasts still remains unclear. The current study utilized an inexpensive source of rice straw ash to synthesize wollastonite with mesoporous architecture. Mesoporous-wollastonite (m-WS) particles were characterized by transmission electron microscopy (TEM), N2 adsorption-desorption isotherms, and Fourier transform infrared (FT-IR) spectroscopy. These particles were found to be biocompatible with mouse mesenchymal stem cells (C3H10T1/2) and significantly stimulated cell proliferation by promoting the entry of the cell population from the G0/G1 phase into the S and G2/M phases via the upregulated expression of the cyclin B1 and cyclin E genes. Under osteogenic conditions, m-WS particles promoted osteoblast differentiation as indicated by calcium deposits and upregulated mRNA expression of osteoblast differentiation marker genes determined by real-time RT-PCR, depicting the osteoconductive nature of these particles. Runx2, a bone-specific transcription factor responsible for the expression of osteoblast differentiation marker genes, was upregulated in C3H10T1/2 cells. The expression of Runx2 co-regulators like Sirt-1, a positive regulator, and HDAC-4, a negative regulator, were upregulated and downregulated, respectively, by m-WS particles in these cells. Thus, this study provides a detailed insight into the effect of m-WS particles on mesenchymal stem cells at the molecular and cellular levels for in vitro bone formation.

Regulation of proliferation and apoptosis in human osteoblastic cells by microRNA-15b.

MicroRNAs (miRNAs) play an important role in proliferation and differentiation of osteoblasts. We recently reported that miR-15b acts as a positive regulator of osteoblast differentiation, whereas its functional role in osteoblastic proliferation remains not known. In this study, we found that there was increased proliferation of human osteoblastic cells (MG63) when they were transiently transfected with miR-15b inhibitor. A significant level of cell population was found to be decreased at G0/G1 phase, and increased at S and G2/M phase by miR-15b inhibitor treatment. Cyclin E1 was found to be one of the putative target genes of miR-15b, and miR-15b mimic and miR-15b inhibitor treatments in cells decreased and increased cyclin E1 expression, respectively. We further identified that the cyclin E1 3'UTR is directly targeted by miR-15b using the luciferase reporter gene system. No significant effect was found on apoptosis of MG63 cells with miR-15b inhibitor. Thus, these findings provide new insights in understanding the role of miR-15b expression as negative regulator of osteoblast proliferation.

Runx2: Structure, function, and phosphorylation in osteoblast differentiation.

Runx2 is a master transcription factor for osteogenesis. The most important phenomenon that makes this protein a master regulator for osteogenesis is its structural integrity. In response to various stimuli, the domains in Runx2 interact with several proteins and regulate a number of cellular events via posttranslational modifications. Hence, in this review we summarized the structural integrity of Runx2 and its posttranslational modifications, especially the phosphorylation responsible for either stimulation or inhibition of its regulatory role in osteogenesis.

A feedback expression of microRNA-590 and activating transcription factor-3 in human breast cancer cells.

MicroRNAs (miRNAs) are small non coding RNA molecules (∼ 23 nt) that are capable of regulating several physiological and pathological processes by targeting mRNAs post transcriptionally, and miRNAs are also known to be regulated by their own target gene(s) in a feedback manner. In this study, we analysed the expression of miRNAs (pre-mir-93, pre-mir-20b, pre-mir-520 c, pre-mir-143, pre-mir-154 and pre-mir-590) by body map, an in silico method and by qRT-PCR in MDA-MB231 (highly invasive and metastatic in nature), and MCF-7 (poor invasive and metastatic in nature) cells. These miRNAs were down regulated in MDA-MB231 cells, and among these, miR-590 was found to putatively target activating transcription factor-3 (ATF-3), a stress response gene. ATF-3 expression level was significantly increased in MDA-MB231 cells and inhibition of ATF-3 expression in these cells increased the expression of pre-mir-590. Thus, these results suggest that there is a negative feedback expression of pre-mir-590 and its putative target gene, ATF-3 in human breast cancer cells.

MicroRNAs expression and their regulatory networks during mesenchymal stem cells differentiation toward osteoblasts.

MicroRNAs (miRNAs) are small endogenous noncoding RNAs which regulate mRNAs post-transcriptionally. In this study, a selective number of miRNAs was investigated for their expression and intracellular regulatory networks involved in differentiation of human mesenchymal stem cells (hMSCs) toward osteoblasts. The expression of miR-424, miR-106a, miR-148a, let-7i and miR-99a miRNAs was found to be specific in hMSCs; whereas expression of miR-15b, miR-24, miR-130b, miR-30c, and miR-130a miRNAs was found to be specific in differentiated osteoblasts. A bioinformatics approach identified that the MAPK pathway was mostly targeted by hMSCs specific miRNAs; whereas JAK-STAT, p53, Focal adhesion, gap junction, ubiquitin mediated proteolysis pathways were targeted by osteblastic specific miRNAs. Altering expression of osteoblast specific miRNA (miR-15b) promoted adipogenesis and myogenesis lineages. Thus, we suggest that miRNAs' regulatory networks and their target genes might provide an insight of their role during differentiation of hMSCs toward osteoblasts, and alteration in the expression of miRNAs would be a valuable approach for controlling osteoblast differentiation.

A positive role of microRNA-15b on regulation of osteoblast differentiation.

Osteoblast differentiation is tightly regulated by several factors including microRNAs (miRNAs). In this paper, we report that pre-mir-15b is highly expressed in differentiated osteoblasts. The functional role of miR-15b in osteoblast differentiation was determined using miR-15b mimic/inhibitor and the expression of osteoblast differentiation marker genes such as alkaline phosphatase (ALP), type I collagen genes was decreased by miR-15b inhibitor. Runx2, a bone specific transcription factor is generally required for expression of osteoblast differentiation marker genes and in response to miR-15b inhibitor treatment, Runx2 mRNA expression was not changed; whereas its protein expression was decreased. Even though Smurf1 (SMAD specific E3 ubiquitin protein ligase 1), HDAC4 (histone deacetylase 4), Smad7, and Crim1 were found to be few of miR-15b's putative target genes, there was increased expression of only Smurf1 gene at mRNA and protein levels by miR-15b inhibitor. miR-15b mimic treatment significantly increased and decreased expressions of Runx2 and Smurf1 proteins, respectively. We further identified that the Smurf1 3'UTR is directly targeted by miR-15b using the luciferase reporter gene system. This is well documented that Smurf1 interacts with Runx2 and degrades it by proteasomal pathway. Hence, based on our results we suggest that miR-15b promotes osteoblast differentiation by indirectly protecting Runx2 protein from Smurf1 mediated degradation. Thus, this study identified that miR-15b can act as a positive regulator for osteoblast differentiation.

Regulation of breast cancer and bone metastasis by microRNAs.

Breast cancer progression including bone metastasis is a complex process involving numerous changes in gene expression and function. MicroRNAs (miRNAs) are small endogenous noncoding RNAs that regulate gene expression by targeting protein-coding mRNAs posttranscriptionally, often affecting a number of gene targets simultaneously. Alteration in expression of miRNAs is common in human breast cancer, possessing with either oncogenic or tumor suppressive activity. The expression and the functional role of several miRNAs (miR-206, miR-31, miR-27a/b, miR-21, miR-92a, miR-205, miR-125a/b, miR-10b, miR-155, miR-146a/b, miR-335, miR-204, miR-211, miR-7, miR-22, miR-126, and miR-17) in breast cancer has been identified. In this review we summarize the experimentally validated targets of up- and downregulated miRNAs and their regulation in breast cancer and bone metastasis for diagnostic and therapeutic purposes.

Expression of microRNA-30c and its target genes in human osteoblastic cells by nano-bioglass ceramic-treatment.

Osteoblast differentiation is tightly regulated by post transcriptional regulators such as microRNAs (miRNAs). Several bioactive materials including nano-bioglass ceramic particles (nBGC) influence differentiation of the osteoblasts, but the molecular mechanisms of nBGC-stimulation of osteoblast differentiation via miRNAs are not yet determined. In this study, we identified that nBGC-treatment stimulated miR-30c expression in human osteoblastic cells (MG63). The bioinformatics tools identified its regulatory network, molecular function, biological processes and its target genes involved in negative regulation of osteoblast differentiation. TGIF2 and HDAC4 were found to be its putative target genes and their expression was down regulated by nBGC-treatment in MG63 cells. Thus, this study advances our understanding of nBGC action on bone cells and supports utilization of nBGC in bone tissue engineering.

MicroRNAs: Synthesis, Gene Regulation and Osteoblast Differentiation.

The central dogma of transfer of genetic information from DNA to protein via mRNA is now challenged by small fragment of non coding RNAs typically 19-25 nucleotides in length namely microRNAs (miRNAs). miRNAs regulate expression of the protein coding genes by interfering in their mRNAs and, thus, act as key regulators of diverge cellular activities. Osteoblast differentiation, a key step in skeletal development involves activation of several signalling pathways including TGFb, BMP, Wnt and transcription factors, which are tightly regulated by miRNAs. In this review, we provide information on recent developments of the synthesis and gene regulation of miRNAs as well as the potential nature of miRNAs that regulate mesenchymal stem cell towards osteoblast differentiation for therapeutic purpose.