Dataset Information

MiR-539-3p impairs osteogenesis by suppressing Wnt interaction with LRP-6 co-receptor and subsequent inhibition of Akap-3 signaling pathway.

ABSTRACT: X-linked hypophosphatemia (XLH), an inheritable form of rickets is caused due to mutation in Phex gene. Several factors are linked to the disease's aetiology, including non-coding RNA molecules (miRNAs), which are key post-transcriptional regulators of gene expression and play a significant role in osteoblast functions. MicroRNAs sequence analysis showed differentially regulated miRNAs in phex silenced osteoblast cells. In this article, we report miR-539-3p, an unidentified novel miRNA, in the functional regulation of osteoblast. MiR-539-3p overexpression impaired osteoblast differentiation. Target prediction algorithm and experimental confirmation by luciferase 3' UTR reporter assay identified LRP-6 as a direct target of miR-539-3p. Over expression of miR-539-3p in osteoblasts down regulated Wnt/beta catenin signaling components and deteriorated trabecular microarchitecture leading to decreased bone formation in ovariectomized (Ovx) mice. Additionally, biochemical bone resorption markers like CTx and Trap-5b were elevated in serum samples of mimic treated group, while, reverse effect was observed in anti-miR treated animals along with increased bone formation marker P1NP. Moreover, transcriptome analysis with miR-539-3p identified a novel uncharacterized Akap-3 gene in osteoblast cells, knock down of which resulted in downregulation of osteoblast differentiation markers at both transcriptional and translational level. Overall, our study for the first time reported the role of miR-539-3p in osteoblast functions and its downstream Akap-3 signalling in regulation of osteoblastogenesis.

SUBMITTER: Tripathi A

PROVIDER: S-EPMC9577939 | biostudies-literature | 2022

REPOSITORIES: biostudies-literature

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MiR-539-3p impairs osteogenesis by suppressing Wnt interaction with LRP-6 co-receptor and subsequent inhibition of Akap-3 signaling pathway.

Tripathi Alok A John Aijaz A AA Kumar Deepak D Kaushal Saurabh Kumar SK Singh Devendra Pratap DP Husain Nazim N Sarkar Jayanta J Singh Divya D

Frontiers in endocrinology 20220929

X-linked hypophosphatemia (XLH), an inheritable form of rickets is caused due to mutation in Phex gene. Several factors are linked to the disease's aetiology, including non-coding RNA molecules (miRNAs), which are key post-transcriptional regulators of gene expression and play a significant role in osteoblast functions. MicroRNAs sequence analysis showed differentially regulated miRNAs in phex silenced osteoblast cells. In this article, we report miR-539-3p, an unidentified novel miRNA, in the f ...[more]

PMID: 36267566

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Project description:Osteoporosis is a systemic metabolic bone disease during which bone mass decreases and bone quality is reduced. Maintaining the bone formation capacity of osteoblasts is crucial for the treatment of osteoporosis. In the present study, bioinformatics analysis was performed on online microarray expression profiles to identify miRNA(s) related to osteoblast proliferation and bone marrow‑derived mesenchymal stem cell (BMSC) osteogenic differentiation. The specific effects of candidate miRNAs on cell proliferation, osteogenic differentiation and Wnt signaling‑related factors were examined. As regards the downstream mechanisms, online tools were employed to predict the downstream targets of candidate miRNAs and the predicted miRNA‑mRNA binding was verified. Finally, the dynamic effects of miRNAs and mRNAs were examined. The results revealed that miR‑483‑3p expression was decreased in bone tissue samples from patients with osteoporosis. In miR‑483‑3p‑overexpressing human osteoblasts, cell viability, DNA synthesis capacity and osteogenesis were promoted, and the protein levels of Wnt1, β‑catenin and cyclin D1 were increased. However, the protein receptor activator of nuclear factor kappa‑Β ligand (RANKL)/osteoprotegerin (OPG) ratio and cell apoptotic rate were decreased. The Wnt signaling, antagonist Dikkopf 2 (DKK2), was targeted and negatively regulated by miR‑483‑3p. DKK2 knockdown exerted similar effects as miR‑483‑3p overexpression, while DKK2 overexpression inhibited cell viability, DNA synthesis capacity and osteogenesis. DKK2 overexpression also decreased the Wnt1, β‑catenin, and cyclin D1 protein levels, whereas it promoted the the RANKL/OPG ratio and the apoptosis of human osteoblasts. DKK2 overexpression reversed the functions of miR‑483‑3p overexpression. On the whole, the findings of the present study demonstrate that the miR‑483‑3p/DKK2 axis modulates the bone formation process by affecting osteoblast proliferation, pre‑osteoblast differentiation into mature osteoblasts and new bone matrix formation.

Project description:BackgroundOsteoporosis (OP) is a progressive metabolic bone disease characterized by impaired bone microarchitecture, decreased bone strength, and dysregulated bone remodeling, leading to an increased risk of fractures. Among osteoporotic fractures, osteoporotic vertebral compression fractures (OVCF) are the most common and can significantly impact patients' quality of life. Growing evidence suggests that microRNAs (miRNAs) play a crucial role in bone homeostasis by regulating osteoblast differentiation, bone metabolism, and remodeling processes. Notably, miR-147b-3p has been found to be downregulated in OVCF; however, its specific role in osteogenic regulation remains largely unknown. Therefore, further investigation is warranted to elucidate the function and underlying mechanism of miR-147b-3p in osteogenic differentiation.MethodsThe GSE93883 and GSE74209 datasets were retrieved from the Gene Expression Omnibus (GEO) database to investigate specific microRNAs involved in the regulation of osteogenesis. Differential expression of miR-147b-3p and NDUFA4 was assessed between healthy controls and patients with osteoporotic vertebral compression fractures (OVCF) using real-time quantitative PCR.To modulate the expression levels of miR-147b-3p in MC3T3-E1 cells, both the miR-147b-3p mimic and inhibitor were utilized. Cell viability was evaluated via the CCK-8 assay to assess the impact of miR-147b-3p on MC3T3-E1 cell proliferation. Real-time PCR and Western blot analysis were conducted to quantify the expression levels of osteogenic markers across different experimental groups. Alizarin red staining (ARS) was employed to examine the effect of miR-147b-3p on the mineralization capacity of MC3T3-E1 cells. In vivo experiments were performed to evaluate the functional role of miR-147b-3p. Bioinformatics databases were used to predict the potential target gene of miR-147b-3p (NDUFA4), and the predictions were validated by a dual luciferase reporter gene assay.To investigate the regulatory role of the miR-147b-3p/NDUFA4 axis in osteogenic differentiation, MC3T3-E1 cells were transfected with the NDUFA4 overexpression plasmid and miR-147b-3p mimic. Western blot analysis was performed to assess the phosphorylation levels of PI3K and AKT, in order to explore whether the miR-147b-3p/NDUFA4 axis regulates osteogenic differentiation through the PI3K/AKT signaling pathway.ResultsOur results indicated a significant downregulation of miR-147b-3p and a concurrent upregulation of NDUFA4 in patients with osteoporotic vertebral compression fractures (OVCF). A luciferase reporter assay confirmed that NDUFA4 is a direct target gene of miR-147b-3p.To examine the functional role of miR-147b-3p, both in vitro and in vivo experiments were conducted.The experimental findings revealed that the miR-147b-3p mimic significantly enhanced cell viability, increased protein expressions of Alkaline Phosphatase (ALP) and Runt-related Transcription Factor 2 (RUNX2), and promoted mineralization as evidenced by Alizarin Red S staining. Conversely, treatment with the miR-147b-3p inhibitor or overexpression plasmid for NDUFA4 (pNDUFA4) produced opposite effects.Furthermore, the miR-147b-3p/NDUFA4 axis was found to regulate the PI3K/AKT signaling pathway.The miR-147b-3p mimic significantly increased the phosphorylation levels of PI3K (p-PI3K) and AKT (p-AKT), whereas pNDUFA4 led to their reduction.ConclusionsThis study demonstrated that miR-147b-3p plays a crucial role in promoting osteogenic differentiation in osteoporotic vertebral compression fractures (OVCF) by targeting NDUFA4 and modulating the PI3K/AKT signaling pathway. These findings provide new insights into the molecular mechanisms underlying the progression of osteoporotic vertebral fractures.

Project description:Human endogenous retroviruses (HERVs) are remnants of ancestral germline infections by exogenous retroviruses. Human endogenous retroviruses W family envelope gene (HERV-W env, also called ERVWE1), located on chromosome 7q21-22, encodes an envelope glycoprotein from the HERV-W family. Mounting evidence suggests that aberrant expression of ERVWE1 involves the etiology of schizophrenia. Moreover, the genetic and morphological studies indicate that dendritic spine deficits may contribute to the onset of schizophrenia. Here, we reported that ERVWE1 changed the density and morphology of the dendritic spine through inhibiting Wingless-type (Wnt)/c-Jun N-terminal kinases (JNK) non-canonical pathway via miR-141-3p in schizophrenia. In this paper, we found elevated levels of miR-141-3p and a significant positive correlation with ERVWE1 in schizophrenia. Moreover, serum Wnt5a and actin-related protein 2 (Arp2) levels decreased and demonstrated a significant negative correlation with ERVWE1 in schizophrenia. In vitro experiments disclosed that ERVWE1 up-regulated miR-141-3p expression by interacting with transcription factor (TF) Yin Yang 1 (YY1). YY1 modulated miR-141-3p expression by binding to its promoter. The luciferase assay revealed that YY1 enhanced the promoter activity of miR-141-3p. Using the miRNA target prediction databases and luciferase reporter assays, we demonstrated that miR-141-3p targeted Wnt5a at its 3' untranslated region (3' UTR). Furthermore, ERVWE1 suppressed the expression of Arp2 through non-canonical pathway, Wnt5a/JNK signaling pathway. In addition, ERVWE1 inhibited Wnt5a/JNK/Arp2 signal pathway through miR-141-3p. Finally, functional assays showed that ERVWE1 induced the abnormalities in hippocampal neuron morphology and spine density through inhibiting Wnt/JNK non-canonical pathway via miR-141-3p in schizophrenia. Our findings indicated that miR-141-3p, Wnt5a, and Arp2 might be potential clinical blood-based biomarkers or therapeutic targets for schizophrenia. Our work also provided new insight into the role of ERVWE1 in schizophrenia pathogenesis.

Dataset Information

MiR-539-3p impairs osteogenesis by suppressing Wnt interaction with LRP-6 co-receptor and subsequent inhibition of Akap-3 signaling pathway.

Publications

MiR-539-3p impairs osteogenesis by suppressing Wnt interaction with LRP-6 co-receptor and subsequent inhibition of Akap-3 signaling pathway.

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