Selvaraj Vimalraj

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.

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.

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‐15bs 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. J. Cell. Physiol. 229: 1236–1244, 2014.

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.

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.

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.

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.

MicroRNAs: Impaired vasculogenesis in metal induced teratogenicity

Certain metals have been known for their toxic effects on embryos and fetal development. The vasculature in early pregnancy is extremely dynamic and plays an important role in organogenesis. Nascent blood vessels in early embryonic life are considered to be a primary and delicate target for many teratogens since the nascent blood islands follow a tightly controlled program to form vascular plexus around and inside the embryo for resourcing optimal ingredients for its development. The state of the distribution of toxic metals, their transport mechanisms and the molecular events by which they notch extra-embryonic and embryonic vasculatures are illustrated. In addition, pharmacological aspects of toxic metal induced teratogenicity have also been portrayed. The work reviewed state of the current knowledge of specific role of microRNAs (miRNAs) that are differentially expressed in response to toxic metals, and how they interfere with the vasculogenesis that manifests into embryonic anomalies.

Mixed-ligand copper(II) complex of quercetin regulate osteogenesis and angiogenesis

Copper(II) complex of quercetin Cu+Q, mixed ligand complexes, quercetin-Cu(II)-phenanthroline [Cu+Q(PHt)] and quercetin-Cu(II)-neocuproine [Cu+Q(Neo)] have been synthesized and characterized. From the FT-IR spectroscopic studies, it was evident that C-ring of quercetin is involved in the metal chelation in all the three copper complexes. C-ring chelation was further proven by UV-Visible spectra and the presence of Cu(II) from EPR spectroscopic investigations. These complexes were found to have osteogenic and angiogenic properties, observed through in vitro osteoblast differentiation and chick embryo angiogenesis assay. In osteoblast differentiation, quercetin-Cu(II) complexes treatment increased calcium deposition and alkaline phosphatase activity (ALP) activity at the cellular level and stimulated Runx2 mRNA and protein, ALP mRNA and type 1 collagen mRNA expression at the molecular level. Among the complexes, Q+Cu(PHt) showed more effects on osteoblast differentiation when compared to that of other two copper complexes. Additionally, Q+Cu(Neo) showed more effect compared to Q+Cu. Furthermore, the effect of these complexes on osteoblast differentiation was confirmed by the expression of osteoblast specific microRNA, pre-mir-15b. The chick embryo angiogenesis assay showed that angiogenic parameters such as blood vessel length, size and junctions were stimulated by these complexes. Thus, the present study demonstrated that quercetin copper(II) complexes exhibit as a pharmacological agent for the orthopedic application.

Nitric oxide signaling regulates tumor-induced intussusceptive-like angiogenesis

Existing animal models for screening tumor angiogenic process have various setbacks that necessitate further investigations. In this study, we developed an ex-ovo egg yolk angiogenesis model to screen the angiogenic potency of tumor cells (HeLa and SiHa cell lines). The egg yolk angiogenesis assay was applied to study the nitric oxide (NO) influence on switching from sprouting angiogenesis (SA) to intussusceptive angiogenesis (IA) under tumor microenvironment. Morphological analysis and SA-like or IA-like markers expression were determined during the development of chicken chorioallantoic membrane (CAM) from day 5 to 13. Expression of Notch1, Notch2, EphrinB2, and Tie2 were considered as SA-like while TEM8, CALD1, CXCR4 and HOMX1 were followed as IA-like markers. The HeLa and SiHa cell lines embedded CAM showed an increase in micro and macro blood vessels and vascular size, junction and length which are the pivotal morphological parameters of angiogenesis. Further, the study revealed that HeLa is more aggressive than SiHa in inducing tumor angiogenesis. To determine the NO signaling implication in tumor milieu, NO donor (Spermine NONOate (SPNO)), NOS inhibitor (L-nitro-L-arginine-methyl ester (L-NAME) and VEGF inhibitor (Avastin) were administrated to chick embryo vascular bed with and without HeLa cells. The results demonstrated that HeLa cells promote IA through NO signaling, VEGF and eNOS and it was documented by angiogenic morphological parameters and SA-like or IA-like markers expression. Therefore, our study claims that ex-ovo egg yolk angiogenesis model could be used to study tumor angiogenesis and NO plays a key role in switching of IA under tumor microenvironment.