Regulation of Wnt signaling by non-coding RNAs during osteoblast differentiation.

Bone is a rigid, mineralized connective tissue that constitutes part of the skeleton in most vertebrate animals. Bone remodeling is a complex process that involves the coordination of ossification and bone resorption activities by osteoblasts and osteoclasts, respectively, resulting in maintaining bone mass. This process involves several growth factors/cytokines and hormones regulating the various signaling pathways. Wnt is one of the major molecular signaling pathways that positively regulate the osteogenic differentiation of mesenchymal stem cells. Dysregulation in the Wnt signaling leads to serious bone-related disorders like osteoporosis and osteosclerosis. Recently, several studies reported the critical role of non-coding RNAs (ncRNAs), including microRNAs, long ncRNAs, and circular RNAs, in the regulation of bone homeostasis via modulating the Wnt signaling cascade. This review summarizes the importance of such ncRNAs in mediating the Wnt cascade and its effect on osteoblast differentiation. Understanding the regulatory role of these ncRNAs would serve as a novel therapeutic strategy for treating bone-related disorders.

Role of p300, a histone acetyltransferase enzyme, in osteoblast differentiation.

Bone is a dynamic and tough connective tissue that undergoes constant remodeling throughout life. Bone-forming osteoblasts respond to various hormones, cytokines, and growth factors, and synthesize extracellular matrix components. Runx2 (Runt-related transcription factor 2), a bone transcription factor, is essential for ossification by stimulating the expression of osteoblast differentiation marker genes, including type I collagen, alkaline phosphatase, and osteocalcin. Coactivators, such as p300, CBP (CREB-binding protein), and PCAF (p300/CBP associated factor) tightly regulate osteoblast differentiation via Runx2. There is growing evidence indicating the role of p300, which possesses histone acetyltransferase (HAT) activity, in regulating histones and transcription factors such as Runx2 during osteoblast differentiation. In this review, we aim to delineate the role of p300 at the molecular level, emphasizing the importance of its HAT activity during osteoblast differentiation. Furthermore, this review intends to highlight the regulation of p300 at multiple levels, including post-translational and ncRNAs, that might exert an indirect influence on bone formation.

An osteoinductive effect of phytol on mouse mesenchymal stem cells (C3H10T1/2) towards osteoblasts.

In recent years, phytochemicals have been widely researched and utilized for the treatment of various medical conditions such as cancer, cardiovascular diseases, age-related problems and are also said to have bone regenerative effects. In this study, phytol (3,7,11,15-tetramethylhexadec-2-en-1-ol), an acyclic unsaturated diterpene alcohol and a secondary metabolite derived from aromatic plants was investigated for its effect on osteogenesis. Phytol was found to be nontoxic in mouse mesenchymal stem cells (C3H10T1/2). At the cellular level, phytol-treatment promoted osteoblast differentiation, as seen by the increased calcium deposits. At the molecular level, phytol-treatment stimulated the expression of Runx2 (a bone-related transcription factor) and other osteogenic marker genes. MicroRNAs (miRNAs) play an essential role in controlling bone metabolism by targeting genes at the post-transcriptional level. Upon phytol-treatment in C3H10T1/2 cells, mir-21a and Smad7 levels were increased and decreased, respectively. It was previously reported that mir-21a targets Smad7 (an antagonist of TGF-beta1 signaling) and thus, protects Runx2 from its degradation. Thus, based on our results, we suggest that phytol-treatment promoted osteoblast differentiation in C3H10T1/2 cells via Runx2 due to downregulation of Smad7 by mir-21a. Henceforth, phytol was identified to bolster osteoblast differentiation, which in turn may be used for bone regeneration.

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.

Runx2, a target gene for activating transcription factor-3 in human breast cancer cells.

Activating transcription factor (ATF-3) is a stress response gene and is induced by transforming growth factor beta 1 (TGF-ß1) in breast cancer cells. In this study, we dissected the functional role of ATF-3 gene in vitro by knocking down its expression stably in human bone metastatic breast cancer cells (MDA-MB231). Knockdown of ATF-3 expression in these cells decreased cell number, altered cell cycle phase transition, and decreased mRNA expression of cell cycle genes. Knockdown of ATF-3 expression in MDA-MB231 cells also decreased cell migration, and the expression levels of invasive and metastatic genes such as MMP-13 and Runx2 were found to be decreased in these cells. Most importantly, ATF-3 was associated with Runx2 promoter in MDA-MB231 cells and knockdown of ATF-3 expression decreased its association with Runx2 promoter. Hence, our results suggested that ATF-3 plays a role in proliferation and invasion of bone metastatic breast cancer cells in vitro and we identified for the first time that Runx2 is a target gene of ATF-3 in MDA-MB231 cell line.

Nanohydroxyapatite-reinforced chitosan composite hydrogel for bone tissue repair in vitro and in vivo.

BACKGROUND: Bone loss during trauma, surgeries, and tumor resection often results in critical-sized bone defects that need to be filled with substitutionary materials. Complications associated with conventional grafting techniques have led to the development of bioactive tissue-engineered bone scaffolds. The potential application of hydrogels as three-dimensional (3D) matrices in tissue engineering has gained attention in recent years because of the superior sensitivity, injectability, and minimal invasive properties of hydrogels. Improvements in the bioactivity and mechanical strength of hydrogels can be achieved with the addition of ceramics. Based on the features required for bone regeneration, an injectable thermosensitive hydrogel containing zinc-doped chitosan/nanohydroxyapatite/beta-glycerophosphate (Zn-CS/nHAp/ß-GP) was prepared and characterized, and the effect of nHAp on the hydrogel was examined. METHODS: Hydrogels (Zn-CS/ß-GP, Zn-CS/nHAp/ß-GP) were prepared using the sol-gel method. Characterization was carried out by scanning electron microscopy (SEM), energy dispersive spectroscopy (EDX), Fourier transform infrared spectroscopy (FTIR), and X-ray diffraction (XRD) as well as swelling, protein adsorption, and exogenous biomineralization studies. Expression of osteoblast marker genes was determined by real-time reverse transcriptase polymerase chain reaction (RT-PCR) and western blot analyses. In vivo bone formation was studied using a rat bone defect model system. RESULTS: The hydrogels exhibited sol-gel transition at 37°C. The presence of nHAp in the Zn-CS/nHAp/ß-GP hydrogel enhanced swelling, protein adsorption, and exogenous biomineralization. The hydrogel was found to be non-toxic to mesenchymal stem cells. The addition of nHAp to the hydrogel also enhanced osteoblast differentiation under osteogenic conditions in vitro and accelerated bone formation in vivo as seen from the depositions of apatite and collagen. CONCLUSIONS: The synthesized injectable hydrogel (Zn-CS/nHAp/ß-GP) showed its potential toward bone formation at molecular and cellular levels in vitro and in vivo. The current findings demonstrate the importance of adding nHAp to the hydrogel, thereby accelerating potential clinical application toward bone regeneration.

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.

A potential function for MicroRNA-124 in normal and pathological bone conditions.

Cells produce short single-stranded non-coding RNAs (ncRNAs) called microRNAs (miRNAs), which actively regulate gene expression at the posttranscriptional level. Several miRNAs have been observed to exert significant impacts on bone health and bone-related disorders. One of these, miR-124, is observed in bone microenvironments and is conserved across species. It affects bone cell growth and differentiation by activating different transcription factors and signaling pathways. In-depth functional analyses of miR-124 have revealed several physiological and pathological roles exerted through interactions with other ncRNAs. Deciphering these RNA-mediated signaling networks and pathways is essential for understanding the potential impacts of dysregulated miRNA functions on bone biology. In this review, we aim to provide a comprehensive analysis of miR-124's involvement in bone physiology and pathology. We highlight the importance of miR-124 in controlling transcription factors and signaling pathways that promote bone growth. This review reveals therapeutic implications for the treatment of bone-related diseases.

Regulatory and therapeutic implications of competing endogenous RNA network in breast cancer progression and metastasis: A review.

Breast cancer (BC) is a global health concern, and development of diagnostic tools and targeted treatments for BC remains challenging. Therapeutic approaches for BC often involve a combination of surgery, radiation therapy, chemotherapy, targeted therapy, and hormone therapy. In recent years, there has been a growing interest in the role of noncoding RNAs (ncRNAs), including long ncRNAs (lncRNAs) and microRNAs (miRNAs), in BC and their therapeutic implications. Various biological processes such as cell proliferation, migration, and apoptosis rely on the activities of these ncRNAs, and their dysregulation has been implicated in BC progression. The regulatory function of the competitive endogenous RNA (ceRNA) network, which comprises lncRNAs, miRNAs, and mRNAs, has been the subject of extensive pathophysiological research. Most lncRNAs serve as molecular sponges for miRNAs and sequester their activities, thereby regulating the expression of target mRNAs and contributing to the promotion or inhibition of BC progression. This review summarizes recent findings on the role of ceRNA networks in BC progression, metastasis, and therapeutic resistance, and highlights the association of ceRNA networks with transcription factors and signaling pathways. Understanding the ceRNA network can lead to the discovery of biomarkers and targeted treatment methods to prevent the spread and metastasis of BC.

In vivo validation of the functional role of MicroRNA-4638-3p in breast cancer bone metastasis.

PURPOSE: Skeletal metastases are increasingly reported in metastatic triple-negative breast cancer (BC) patients. We previously reported that TGF-ß1 sustains activating transcription factor 3(ATF3) expression and is required for cell proliferation, invasion, and bone metastasis genes. Increasing studies suggest the critical regulatory function of microRNAs (miRNAs) in governing BC pathogenesis. TGF-ß1 downregulated the expression of miR-4638-3p, which targets ATF3 in human BC cells (MDA-MB-231). In the present study, we aimed to identify the functional role of miR-4638-3p in BC bone metastasis by the caudal artery injection of the MDA-MB-231 cells overexpressing mir-4638 in the mice. METHODS: MDA-MB-231 cells overexpressing miR-4638 were prepared by stable transfections. Reverse transcriptase quantitative PCR was carried out to determine the expression of endogenous miR-4638-3p and bone resorption marker genes. X-ray, micro-CT, and Hematoxylin & Eosin studies were used to determine osteolytic lesions, trabecular structure, bone mineral density, and micrometastasis of cells. RESULTS: The mice injected with MDA-MB-231 cells overexpressing miR-4638-3p decreased the expression of bone resorption marker genes, compared to MDA-MB-231 cells injection. Reduced osteolytic lesions and restored bone density by MDA-MB-231 cells overexpressing miR-4638-3p were observed. Similarly, the mice injected with MDA-MB-231 cells overexpressing miR-4638-3p showed a better microarchitecture of the trabecular network. A few abnormal cells seen in the femur of MDA-MB-231 cells-injected mice were not found in MDA-MB-231 cells overexpressing miR-4638. CONCLUSION: The identified functional role of ATF3 targeting miR-4638-3p in BC bone metastasis in vivo suggests its candidature as BC therapeutics in the future.

Chitosan nanocarriers for non-coding RNA therapeutics: A review.

Non-coding RNA (ncRNA)-based therapies entail delivering ncRNAs to cells to regulate gene expression and produce proteins that combat infections, cancer, neurological diseases, and bone abnormalities. Nevertheless, the therapeutic potential of these ncRNAs has been limited due to the difficulties in delivering them to specific cellular targets within the body. Chitosan (CS), a biocompatible cationic polymer, interacts with negatively charged RNA molecules to form stable complexes. It is a promising biomaterial to develop nanocarriers for ncRNA delivery, overcoming several disadvantages of traditional delivery systems. CS-based nanocarriers can protect ncRNAs from degradation and target-specific delivery by surface modifications and intracellular release profiles over an extended period. This review briefly summarizes the recent developments in CS nanocarriers' synthesis and design considerations and their applications in ncRNA therapeutics for treating various diseases. We also discuss the challenges and limitations of CS-based nanocarriers for ncRNA therapeutics and potential strategies for overcoming these challenges.

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.

MicroRNAs mediated interaction of tumor microenvironment cells with breast cancer cells during bone metastasis.

Breast cancer (BC) bone metastasis is primarily osteolytic and has limited therapeutic options. Metastasized BC cells prime the secondary environment in bone by forming a tumor niche, which favors their homing and colonization. The tumor microenvironment (TME) is primarily generated by the cancer cells. Bone TME is an intricate network of multiple cells, including altered bone, tumor, stromal, and immune cells. Recent findings highlight the significance of small non-coding microRNAs (miRNAs) in influencing TME during tumor metastasis. MiRNAs from TME-resident cells facilitate the interaction between the tumor and its microenvironment, thereby regulating the biological processes of tumors. These miRNAs can serve as oncogenes or tumor suppressors. Hence, both miRNA inhibitors and mimics are extensively utilized in pre-clinical trials for modulating the phenotypes of tumor cells and associated stromal cells. This review briefly summarizes the recent developments on the functional role of miRNAs secreted directly or indirectly from the TME-resident cells in facilitating tumor growth, progression, and metastasis. This information would be beneficial in developing novel targeted therapies for BC.

Chitosan-coated and thymol-loaded polymeric semi-interpenetrating hydrogels: An effective platform for bioactive molecule delivery and bone regeneration in vivo.

Thymol (2-isopropyl-5-methylphenol; Thy) is a monoterpene phenolic phytocompound with medicinal properties; however, its impact on osteogenesis is yet to be thoroughly investigated. Its distribution is often hampered because of its intricate hydrophobic structure, which reduces its bioavailability. In this study, we synthesized a drug delivery vehicle using semi-interpenetrating polymer network (SIPN) hydrogels containing sodium alginate and poly(2-ethyl-2-oxazoline) (SA/Pox) loaded with Thy at varying concentrations (100, 150, and 200 µM). Subsequently, they were coated with chitosan (CS) to increase bioactivity and for sustained and prolonged release of Thy. Thy-loaded CS-coated SIPN hydrogels (SA/Pox/CS-Thy) were developed using ionic gelation and polyelectrolyte-complexation techniques. The addition of CS to hydrogels enhanced their physicochemical and material properties. These hydrogels were cytofriendly toward mouse mesenchymal stem cells (mMSCs). When mMSCs were cultured on hydrogels, Thy stimulated osteoblastic differentiation, as evidenced by calcium deposits at the cellular level. The expression of RUNX2, a key bone transcriptional factor, and other differentiation biomarkers was significantly enhanced in mMSCs cultured on SA/Pox/CS-Thy hydrogels. Notably, Thy in the SA/Pox/CS hydrogels significantly activated the TGF-ß/BMP signaling pathway, which is involved in osteogenesis. A rat tibial bone defect model system revealed that the incorporation of Thy into SA/Pox/CS hydrogels augmented bone regeneration. Thus, sustained and prolonged release of Thy from the SA/Pox/CS hydrogels promoted osteoblast differentiation in vitro and bone formation in vivo. These findings shed light on the effect of Thy bioavailability in fostering osteoblast differentiation and its prospective application in bone rejuvenation.

Circ_CUX1/miR-130b-5p/p300 axis for parathyroid hormone-stimulation of Runx2 activity in rat osteoblasts: A combined bioinformatic and experimental approach.

Parathyroid hormone (PTH) regulates the expression of bone remodeling genes by enhancing the activity of Runx2 in osteoblasts. p300, a histone acetyltransferase, acetylated Runx2 to activate the expression of its target genes. PTH stimulated the expression of p300 in rat osteoblastic cells. Increasing studies suggested the potential of non-coding RNAs (ncRNAs), such as microRNAs (miRNAs) and circular RNAs (circRNAs), in regulating gene expression under both physiological and pathological conditions. In this study, we hypothesized that PTH regulates Runx2 activity via ncRNAs-mediated p300 expression in rat osteoblastic cells. Bioinformatics and experimental approaches identified PTH-upregulation of miR-130b-5p and circ_CUX1 that putatively target p300 and miR-130b-5p, respectively. An antisense-mediated knockdown of circ_CUX1 was performed to determine the sponging activity of circ_CUX1. Knockdown of circ_CUX1 promoted miR-130b-5p activity and reduced p300 expression, resulting in decreased Runx2 acetylation in rat osteoblastic cells. Further, bioinformatics analysis identified the possible signaling pathways that regulate Runx2 activity and osteoblast differentiation via circ_CUX1/miR-130b-5p/p300 axis. The predicted circ_CUX1/miR-130b-5p/p300 axis might pave the way for better diagnostic and therapeutic approaches for bone-related diseases.

Synthesis, characterization, and antimicrobial activity of nano-hydroxyapatite-zinc for bone tissue engineering applications.

The bone implants used in tissue repair are susceptible to infections caused by staphylococci, specifically Staphylococcus aureus. Hence, the development of better biological materials that provide antimicrobial activity in bone tissue engineering is required. The nanoparticles of hydroxyapatite (nHAp) and nHAp dopped with Zn (nHAp-Zn) were prepared by the wet chemical method and the ion exchange method, respectively. They were characterized using SEM, AFM, FTIR and XRD. The antibacterial activity of nHAp and nHAp-Zn was determined with Gram-negative and Gram-positive bacterial strains. The results indicated that nHAp alone was acting as an inert matrix and when substituted with Zn, it showed better antibacterial activity. The nHAp-Zn was found to be non-toxic to osteoprogenitor cells. Thus, due to the antimicrobial property of nHAp-Zn nanoparticles, we suggest that they would have potential applications towards bone tissue engineering.

Orsellinic acid-loaded chitosan nanoparticles in gelatin/nanohydroxyapatite scaffolds for bone formation in vitro.

AIM: Orsellinic acid (2,4-Dimethoxy-6-methylbenzoic acid) (OA) is a hydrophobic polyphenolic compound with therapeutic potential, but its impact on actuating osteogenesis remains unknown. The bioavailability of OA is hampered by its hydrophobic nature. This study aimed to fabricate nano-drug delivery system-based scaffolds for OA and test its potential for osteogenesis in vitro. MATERIALS AND METHODS: OA was loaded into chitosan nanoparticles (nCS + OA) using the ionic gelation technique at different concentrations. nCS + OA were incorporated onto the scaffolds containing gelatin (Gel) and nanohydroxyapatite (nHAp) by the lyophilization method. Biocomposite scaffolds were examined for their physicochemical and material characteristic properties. The effect of OA in the scaffolds for osteoblast differentiation was determined by alizarin red and von Kossa staining at the cellular level and by reverse transcriptase-qPCR and western blot analysis at the molecular level. KEY FINDINGS: The scaffolds showed excellent physiochemical and material characteristics and remained cyto-friendly to mouse mesenchymal stem cells (mMSCs, C3H10T1/2). The release of OA from Gel/nHAp/nCS scaffolds enhanced the differentiation of mMSCs towards osteoblasts, as observed through cellular and molecular studies. Moreover, the osteogenic potential of OA was mediated by the activation of FAK and ERK signaling pathways through integrins. SIGNIFICANCE: The inclusion of OA into Gel/nHAp/nCS biocomposite scaffolds at 80 µM concentration promoted osteoblast differentiation via cell adhesion mediated signaling, compared with that shown by Gel/nHAp/nCS alone. Overall, this study identified the potential therapeutic OA containing Gel/nHAp/nCS scaffolds, accelerating its potential for clinical application towards bone regeneration.

A regulatory role of circRNA-miRNA-mRNA network in osteoblast differentiation.

Osteoblast differentiation is an important process in skeletal development and bone remodelling. Serious bone diseases occur from any delay, defect, or imbalance in osteoblastic differentiation. Non-coding RNAs (ncRNAs) play a regulatory role in controlling the expression of proteins under physiological and pathological conditions via inhibiting mRNA translation or degrading mRNA. Circular RNAs (circRNAs) and microRNAs (miRNAs) are the long and small ncRNAs, respectively, which have been reported to regulate the expression of osteoblast marker genes directly or indirectly. Also, recent studies identified the regulatory mechanisms involving the crosstalk among circRNAs, miRNAs, and mRNAs during osteoblast differentiation. Understanding these regulatory mechanisms behind osteoblastic differentiation would help to diagnose or treat bone and bone-related disorders. Hence, the current review comprehensively discussed the regulatory relationship of circRNAs, miRNAs and mRNAs, and their functional role as circRNA-miRNA-mRNA axis in osteoblast differentiation.

Epigenetic modifications of histones during osteoblast differentiation.

In bone biology, epigenetics plays a key role in mesenchymal stem cells' (MSCs) commitment towards osteoblasts. It involves gene regulatory mechanisms governed by chromatin modulators. Predominant epigenetic mechanisms for efficient osteogenic differentiation include DNA methylation, histone modifications, and non-coding RNAs. Among these mechanisms, histone modifications critically contribute to altering chromatin configuration. Histone based epigenetic mechanisms are an essential mediator of gene expression during osteoblast differentiation as it directs the bivalency of the genome. Investigating the importance of histone modifications in osteogenesis may lead to the development of epigenetic-based remedies for genetic disorders of bone. Hence, in this review, we have highlighted the importance of epigenetic modifications such as post-translational modifications of histones, including methylation, acetylation, phosphorylation, ubiquitination, and their role in the activation or suppression of gene expression during osteoblast differentiation. Further, we have emphasized the future advancements in the field of epigenetics towards orthopaedical therapeutics.

Parathyroid Hormone-regulation of Runx2 by MiR-290 for Matrix Metalloproteinase-13 Expression in Rat Osteoblastic Cells.

BACKGROUND: The dynamic changes that bone undergoes during the ensemble of remodeling are administered by vital factors like Runx2 (a bone transcription factor) and matrix metalloproteinases (MMPs). AIMS: Parathyroid hormone (PTH), an FDA approved drug for bone-related ailments, was seen to stimulate MMP-13 expression via Runx2 to ultimately aid in the bone remodeling process. MicroRNAs (miRNAs) have been shown to play a major role in controlling bone metabolism, and the use of miRNAs has recently become promising therapeutic avenues for the treatment of many diseases, including bone disorders. Thus, in this study, we attempted to investigate and evaluate the expression of MMP-13 via a miRNA profile targeting Runx2 under PTH-regulation in rat osteoblastic cells. METHODS: PTH stimulated the expression of MMP-13 mRNA significantly at 4 h in rat osteoblastic cells (UMR106-01). Runx2 was required for PTH-stimulation of MMP-13 expression, in silico scrutiny generated 14 unique miRNAs targeting Runx2, and among these miRNAs, miR-290 was significantly downregulated by PTH-treatment in UMR106- 01 cells and in rat primary osteoblasts. RESULTS: Overexpression of miR-290 decreased the expression of Runx2, the binding of Runx2 at the MMP-13 promoter, and the expression of MMP-13 mRNA in PTH-treated UMR106-01 cells. A dual luciferase reporter assay identified the direct targeting of Runx2 mRNA by miR-290 in these cells. CONCLUSION: Our findings indicate that the PTH-responsive miR-290 regulated Runx2- mediated MMP-13 expression in rat osteoblastic cells, suggesting miR-290 as a molecular marker or target in bone and bone-related diseases.