bims-mirbon 2021-11-21 papers

bims-mirbon

Biomed News

on MicroRNAs in bone

Issue of 2021–11–21
nineteen papers selected by
Japneet Kaur, Mayo Clinic

LINC02381, a sponge of miR-21, weakens osteogenic differentiation of hUC-MSCs through KLF12-mediated Wnt4 transcriptional repression.
miR-6807-5p Inhibited the Odontogenic Differentiation of Human Dental Pulp Stem Cells Through Directly Targeting METTL7A.
miR-544-3p mediates arthritis pain through regulation of FcγRI.
Human Bone Marrow Mesenchymal Stromal Cell-Derived Extracellular Vesicles Promote Proliferation of Degenerated Nucleus Pulposus Cells and the Synthesis of Extracellular Matrix Through the SOX4/Wnt/β-Catenin Axis.
Identification of Potential Osteoporosis miRNA Biomarkers Using Bioinformatics Approaches.
DNA methyltransferase 3 beta mediates the methylation of the microRNA-34a promoter and enhances chondrocyte viability in osteoarthritis.
Global miRNA expression of bone marrow mesenchymal stem/stromal cells derived from Fanconi anemia patients.
miR-23b mediates TNF-α-Inhibited Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Targeting Runx2.
Circulating miRNAs associated with bone mineral density in healthy adult baboons.
Sox9-Increased miR-322-5p Facilitates BMP2-Induced Chondrogenic Differentiation by Targeting Smad7 in Mesenchymal Stem Cells.
MiR-181a Targets RSPO2 and Regulates Bone Morphogenetic Protein - WNT Signaling Crosstalk During Chondrogenic Differentiation of Mesenchymal Stromal Cells.
Microarray analysis of circRNAs sequencing profile in exosomes derived from bone marrow mesenchymal stem cells in postmenopausal osteoporosis patients.
MicroRNA-497 inhibits inflammation in DSS-induced IBD model mice and lipopolysaccharide-induced RAW264.7 cells via Wnt/β-catenin pathway.
Screening of differentially expressed miRNAs during osteogenic/odontogenic differentiation of human dental pulp stem cells exposed to mechanical stress.
CircRNA-miRNA networks in regulating bone disease.
Integrative Analysis of the Expression of microRNA, Long Noncoding RNA, and mRNA in Osteoarthritis and Construction of a Competing Endogenous Network.
LncRNA HAGLR absorbing miR-214-3p promotes BMP2 expression and improves tibial fractures.
Role of microRNA Shuttled in Small Extracellular Vesicles Derived From Mesenchymal Stem/Stromal Cells for Osteoarticular Disease Treatment.
MicroRNA Variants and HLA-miRNA Interactions are Novel Rheumatoid Arthritis Susceptibility Factors.

J Bone Miner Metab. 2021 Nov 15.

LINC02381, a sponge of miR-21, weakens osteogenic differentiation of hUC-MSCs through KLF12-mediated Wnt4 transcriptional repression.

Gang Zhao, Wen-Dong Luo, Yong Yuan, Feng Lin, Li-Min Guo, Jing-Jing Ma, Han-Bo Chen, Huang Tang, Jun Shu.

   INTRODUCTION: Human umbilical cord blood-derived MSCs (hUC-MSCs) have the potential to differentiate into osteoblasts. This study investigated the function and potential mechanisms of a novel lncRNA LINC02381 in hUC-MSC osteogenic differentiation.
MATERIALS AND METHODS: hUC-MSCs were maintained in osteogenic differentiation medium. RT-qPCR assay was performed to assess LINC02381 expression. Alizarin Red S (ARS) and alkaline phosphatase (ALP) staining were performed to evaluate osteogenic differentiation. The interaction between miR-21 and LINC0238/KLF12 was determined by luciferase reporter and RNA immunoprecipitation (RIP) assays. Chromatin immunoprecipitation (ChIP) assay was used to confirm the transcriptional regulation of KLF12 on Wnt4 promoter. The nuclear translocation of β-catenin was evaluated using immunofluorescence. hUC-MSCs seeded on Bio-Oss Collagen scaffolds were transplanted into nude mice to assess in vivo osteogenesis. Bone formation was observed by H&E and Masson's trichrome staining. OSX and OPN levels were assessed by immunohistochemistry.
RESULTS: LINC02381 was up-regulated in the clinical samples of osteoporotic patients. However, LINC02381 expression was reduced during osteogenic differentiation of hUC-MSCs. Enforced expression of LINC02381 suppressed the osteogenic differentiation of hUC-MSCs. Mechanistically, LINC02381 sponged miR-21 to enhance KLF12 expression, which led to the inactivation of Wnt/β-catenin signaling pathway. Furthermore, miR-21 mimics or KLF12 silencing counteracted LINC02381-induced inhibition of osteogenic differentiation, whereas IWP-4 (an inhibitor of Wnt pathway) abolished this effect.
CONCLUSION: In summary, LINC02381 repressed osteogenic differentiation of hUS-MSCs through sponging miR-21 to enhance KLF12-mediated inactivation of Wnt/β-catenin pathway, indicating that LINC02381 might be a therapeutic target for osteoporosis.

Keywords:  KLF12; LINC02381; Osteoporosis; hUS-MSCs; miR-21

DOI:  https://doi.org/10.1007/s00774-021-01277-4
Front Cell Dev Biol. 2021 ;9 759192

miR-6807-5p Inhibited the Odontogenic Differentiation of Human Dental Pulp Stem Cells Through Directly Targeting METTL7A.

Ning Wang, Xiao Han, Haoqing Yang, Dengsheng Xia, Zhipeng Fan.

  Background: Tooth tissue regeneration mediated by mesenchymal stem cells (MSCs) has become the most ideal treatment. Although the known regulatory mechanism and some achievements have been discovered, directional differentiation cannot effectively induce regeneration of tooth tissue. In this study, we intended to explore the function and mechanism of miR-6807-5p and its target gene METTL7A in odontogenic differentiation. Methods: In this study, human dental pulp stem cells (DPSCs) were used. Alkaline phosphatase (ALP), Alizarin red staining (ARS), and calcium ion quantification were used to detect the odontogenic differentiation of miR-6807-5p and METTL7A. Real-time RT-PCR, western blot, dual-luciferase reporter assay, and pull-down assay with biotinylated miRNA were used to confirm that METTL7A was the downstream gene of miR-6807-5p. Protein mass spectrometry and co-immunoprecipitation (Co-IP) were used to detect that SNRNP200 was the co-binding protein of METTL7A. Results: After mineralized induction, the odontogenic differentiation was enhanced in the miR-6807-5p-knockdown group and weakened in the miR-6807-5p-overexpressed group compared with the control group. METTL7A was the downstream target of miR-6807-5p. After mineralized induction, the odontogenic differentiation was weakened in the METTL7A-knockdown group and enhanced in the METTL7A-overexpressed group compared with the control group. SNRNP200 was the co-binding protein of METTL7A. The knockdown of SNRNP200 inhibited the odontogenic differentiation of DPSCs. Conclusion: This study verified that miR-6807-5p inhibited the odontogenic differentiation of DPSCs. The binding site of miR-6807-5p was the 3'UTR region of METTL7A, which was silenced by miR-6807-5p. METTL7A promoted the odontogenic differentiation of DPSCs. SNRNP200, a co-binding protein of METTL7A, promoted the odontogenic differentiation of DPSCs.

Keywords:  METTL7A; SNRNP200; human dental pulp stem cells (DPSCs); miR-6807-5p; mineralized induction; odontogenic differentiation

DOI:  https://doi.org/10.3389/fcell.2021.759192
Pain. 2021 Nov 12.

miR-544-3p mediates arthritis pain through regulation of FcγRI.

Yan Liu, Sang-Min Jeon, Michael J Caterina, Lintao Qu.

ABSTRACT: Chronic joint pain is a major symptom in rheumatoid arthritis (RA) and its adequate treatment represents an unmet medical need. Non-coding microRNAs (miRNAs) have been implicated in the pathogenesis of RA as negative regulators of specific target mRNAs. Yet, their significance in RA pain is still not well defined. We and other groups recently identified neuronally expressed FcγRI as a key driver of arthritis pain in mouse RA models. Thus, we tested the hypothesis that miRNAs that target and regulate neuronal FcγRI attenuate RA pain. Here, we show that miR-544-3p was robustly downregulated whereas FcγRI was significantly upregulated in the dorsal root ganglion (DRG) in mouse RA models. Intrathecal injection of miR-544-3p mimic attenuated established mechanical and heat hyperalgesia partly through the downregulation of FcγRI in the DRG in a mouse model of collagen II-induced arthritis (CIA). Moreover, this effect was likely mediated, at least in part, by FcγRI since miR-544-3p mimic downregulated Fcgr1 mRNA expression in the DRG during arthritis and genetic deletion of Fcgr1 produced similar antihyperalgesic effects in the CIA model. This notion was further supported by a dual luciferase assay showing that miR-544-3p directly targeted Fcgr1 3'UTR. In naïve mice, miR-544-3p mediated acute joint pain hypersensitivity induced by IgG immune complex through the regulation of FcγRI. These findings suggest that miR-544-3p causally participates in the maintenance of arthritis pain by targeting neuronal FcγRI, and thus define miR-544-3p as a new potential therapeutic target for treating RA pain.

DOI: https://doi.org/10.1097/j.pain.0000000000002531
Front Physiol. 2021 ;12 723220

Human Bone Marrow Mesenchymal Stromal Cell-Derived Extracellular Vesicles Promote Proliferation of Degenerated Nucleus Pulposus Cells and the Synthesis of Extracellular Matrix Through the SOX4/Wnt/β-Catenin Axis.

Haoyu Wang, Fei Li, Wenrui Ban, Jing Zhang, Guiqi Zhang.

  Objective: Intervertebral disk degeneration (IDD) is a major cause of pain in the back, neck, and radiculus. Mesenchymal stem cells (MSCs)-derived extracellular vesicles (EVs) are therapeutic in musculoskeletal degenerative diseases such as IDD. This study explored the effect and functional mechanism of human bone MSCs (hBMSCs)-derived EVs in proliferation and apoptosis of degenerated nucleus pulposus cells (DNPCs) and extracellular matrix (ECM) synthesis. Methods: Extracellular vesicles were isolated from hBMSCs and identified. DNPCs were induced by TNF-α. EVs were incubated with DNPCs for 24h. Internalization of EVs by DNPCs, DNPCs proliferation, apoptosis, and expressions of ECM synthetic genes, degrading genes and miR-129-5p were assessed. Downstream target genes of miR-129-5p were predicted. Target relation between miR-129-5p and SRY-box transcription factor 4 (SOX4) was verified. DNPCs proliferation, apoptosis, and ECM synthesis were measured after treatment with EVs and miR-129-5p inhibitor or SOX4 overexpression. Expressions of SOX4 and Wnt/β-catenin pathway-related proteins were determined. Results: hBMSC-EVs promoted DNPCs proliferation, inhibited apoptosis, increased expressions of ECM synthetic genes, and reduced expressions of ECM degrading genes. hBMSC-EVs carried miR-129-5p into DNPCs. Silencing miR-129-5p in EVs partially inverted the effect of EVs on DNPCs proliferation and ECM synthesis. miR-129-5p targeted SOX4. SOX4 overexpression annulled the effect of EVs on DNPCs proliferation and ECM synthesis. Expressions of Wnt1 and β-catenin were decreased in EVs-treated DNPCs, while silencing miR-129-5p in EVs promoted expressions of Wnt1 and β-catenin. Conclusion: hBMSC-EVs promoted DNPCs proliferation and ECM synthesis by carrying miR-129-5p into DNPCs to target SOX4 and deactivating the Wnt/β-catenin axis.

Keywords:  SRY-box transcription factor 4; Wnt/β-catenin; degenerated nucleus pulposus cells; extracellular matrix; extracellular vesicles; human bone marrow mesenchymal stromal cells; intervertebral disk degeneration; miR-129-5p

DOI:  https://doi.org/10.3389/fphys.2021.723220
Comput Math Methods Med. 2021 ;2021 3562942

Identification of Potential Osteoporosis miRNA Biomarkers Using Bioinformatics Approaches.

Wei Lu, Qiang Wang, Yi Xue, Jie Gu, Ping Yao, Yufan Ge, Yiming Miao, Jun Chen.

Osteoporosis is a degenerative osteoarthropathy commonly found in old people and postmenopausal women. Many studies showed that microRNAs (miRNAs) can regulate the expression of osteoporosis-related genes and are abnormally expressed in patients with osteoporosis. miRNAs therefore have the potential to serve as biomarkers of osteoporosis. In this study, the limma package was used for the differential expression analysis of mRNA expression profiles and 357 significantly differentially expressed genes (DEGs) were obtained. Metascape was used for functional enrichment analysis of DEGs. The result revealed that DEGs were mainly enriched in signaling pathways like MAPK6/MAPK4. Based on the STRING database, the protein-protein interaction (PPI) network of DEGs was constructed. MCODE was used to analyze the functional subsets, and a key functional subset composed of 9 genes was screened out. In addition, the miRNA-mRNA regulatory interaction network (RegIN) was analyzed by the CyTargetLinker plugin, which generated 55 miRNA-mRNA regulatory interactions. Through literature searching, the osteoporosis-related gene FOXO1 in the key functional subset was determined to be the main object of the study. In qRT-PCR assay, the expression of the predicted miRNAs was tested in peripheral blood mononuclear cells of mice with osteoporosis, in which 13 miRNAs were remarkably highly expressed. All in all, this study, based on bioinformatics analysis and testing assay of miRNA expression, determined the potential biomarkers of osteoporosis.

DOI: https://doi.org/10.1155/2021/3562942
Bioengineered. 2021 Nov 16.

DNA methyltransferase 3 beta mediates the methylation of the microRNA-34a promoter and enhances chondrocyte viability in osteoarthritis.

Shouliang Xiong, Yong Zhao, Tiantong Xu.

  Osteoarthritis (OA) is characterized by destruction of articular cartilage with as an imbalance between synthesis and degradation of extracellular matrix (ECM). In the current study, we explored the role of microRNA-34a (miR-34a) and the behind epigenetic mechanism in the degradation of ECM in OA. Using miRNA-based microarray analysis, we found that miR-34a was overexpressed in cartilage tissues of OA patients relative to patients with acute traumatic amputations. Moreover, its expression was positively correlated with the ECM degradation and inflammation. Mechanistically, miR-34a targeted MCL1, and possible target genes of miR-34a were enriched in the PI3K/AKT pathway. Furthermore, DNMT3B inhibited miR-34a by promoting miR-34a methylation. Functional experiments using CCK-8, flow cytometry, Safranin O staining, RT-qPCR, ELISA, western blot, and HE staining revealed that miR-34a inhibitor suppressed ECM degradation and inflammatory response of chondrocytes and cartilage tissues. By contrast, downregulation of DNMT3B and MCL1 reversed the repressive effects of miR-34a inhibitor in vitro and in vivo. Altogether, our findings establish that silencing of miR-34a by DNMT3B could effectively reduce chondrocyte ECM degradation and inflammatory response in mice by targeting MCL1 and mediating the downstream PI3K/AKT pathway. This present study revealed that miR-34a knockdown might develop a novel intervention for OA treatment.

Keywords:  DNMT3B; MCL1; Osteoarthritis; PI3K/AKT pathway; microRNA-34a

DOI:  https://doi.org/10.1080/21655979.2021.2005308
Hum Cell. 2021 Nov 18.

Global miRNA expression of bone marrow mesenchymal stem/stromal cells derived from Fanconi anemia patients.

Ilgin Cagnan, Mustafa Keles, Ayse Gokce Keskus, Melike Tombaz, Ozge Burcu Sahan, Fatima Aerts-Kaya, Duygu Uckan-Cetinkaya, Ozlen Konu, Aysen Gunel-Ozcan.

  Fanconi anemia (FA) is a rare genetic disorder characterized by genomic instability, developmental defects, and bone marrow (BM) failure. Hematopoietic stem cells (HSCs) in BM interact with the mesenchymal stem/stromal cells (MSCs); and this partly sustains the tissue homeostasis. MicroRNAs (miRNAs) can play a critical role during these interactions possibly via paracrine mechanisms. This is the first study addressing the miRNA profile of FA BM-MSCs obtained before and after BM transplantation (preBMT and postBMT, respectively). Non-coding RNA expression profiling and quality control analyses were performed in Donors (n = 13), FA preBMT (n = 11), and FA postBMT (n = 6) BM-MSCs using GeneChip miRNA 2.0 Array. Six Donor-FA preBMT pairs were used to identify a differentially expressed miRNA expression signature containing 50 miRNAs, which exhibited a strong correlation with the signature obtained from unpaired samples. Five miRNAs (hsa-miR-146a-5p, hsa-miR-148b-3p, hsa-miR-187-3p, hsa-miR-196b-5p, and hsa-miR-25-3p) significantly downregulated in both the paired and unpaired analyses were used to generate the BM-MSCs' miRNA-BM mononuclear mRNA networks upon integration of a public dataset (GSE16334; studying Donor versus FA samples). Functionally enriched KEGG pathways included cellular senescence, miRNAs, and pathways in cancer. Here, we showed that hsa-miR-146a-5p and hsa-miR-874-3p were rescued upon BMT (n = 3 triplets). The decrease in miR-146a-5p was also validated using RT-qPCR and emerged as a strong candidate as a modulator of BM mRNAs in FA patients.

Keywords:  Bone marrow; Fanconi anemia; Mesenchymal stem/stromal cells; Non-coding RNAs; miRNA

DOI:  https://doi.org/10.1007/s13577-021-00626-9
Int J Med Sci. 2021 ;18(16): 3674-3683

miR-23b mediates TNF-α-Inhibited Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Targeting Runx2.

Xuefei Sun, Mingwei Li, Jinghao Ban, Zhidan Li.

  Periodontitis is the most prevalent oral infection disease, which causes the destruction of periodontal supporting tissues and eventual tooth loss. This study aimed to investigate the molecular mechanism of miRNA-23b (miR-23b) in regulating the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in an inflammatory environment. Results revealed that tumor necrosis factor-α (TNF-α), a notoriously inflammatory cytokine, remarkably attenuated the osteogenic differentiation of hPDLSCs, which were partially rescued by SKL2001 (Wnt/β-catenin agonist). We further explored the underlying roles of miRNAs involved in TNF-α-inhibited osteogenesis of hPDLSCs. The miR-23b significantly increased with TNF-α stimulation, which was abolished by SKL2001. Similar to the effect of TNF-α, miR-23b agonist (agomir-23b) dramatically reduced the expression of runt-related transcription factor 2 (Runx2) and suppressed the osteogenic differentiation of hPDLSCs. The inhibition of miR-23b significantly increased Runx2, which is the major transcription factor during osteogenesis, thereby indicating that miR-23b was an endogenous regulator of Runx2 in hPDLSCs. Bioinformatic analysis and dual luciferase reporter assays confirmed that Runx2 was a target gene of miR-23b. Furthermore, the gain function assay of Runx2 revealed that the Runx2 overexpression efficiently reversed the suppression of the osteogenic differentiation of hPDLSCs with miR-23b agonist, suggesting that the suppressing effect of miR-23b on osteogenesis was mediated by Runx2 inhibition. Our study clarified that miR-23b mediated the TNF-α-inhibited osteogenic differentiation of hPDLSCs by targeting Runx2. Therefore, the expanded function of miR-23b in the osteogenesis of hPDLSCs under inflammatory conditions. This study might provide new insights and a novel therapeutic target for periodontitis.

Keywords:  Osteogenic differentiation; Runx2; TNF-α; hPDLSCs; miR-23b

DOI:  https://doi.org/10.7150/ijms.64312
J Orthop Res. 2021 Nov 19.

Circulating miRNAs associated with bone mineral density in healthy adult baboons.

Ellen E Quillen, Jaydee Foster, Anne Sheldrake, Maggie Stainback, Jeremy Glenn, Laura A Cox, Todd L Bredbenner.

  MicroRNAs (miRNAs) regulate gene expression post-transcriptionally and circulate in the blood, making them attractive biomarkers of disease state for tissues like bone that are challenging to interrogate directly. Here, we report on five miRNAs-miR-197-3p, miR-320a, miR-320b, miR-331-5p, and miR-423-5p-associated with bone mineral density (BMD) in 147 healthy adult baboons. These baboons ranged in age from 15 to 25 years (45-75 human equivalent years) and 65% were female with a broad range of BMD values including a minority of osteopenic animals. miRNAs were generated via RNA sequencing from buffy coats collected at necropsy and areal BMD (aBMD) measured postmortem via dual-energy X-ray absorptiometry (DXA) of the lumbar vertebrae. Differential expression analysis controlled for the underlying pedigree structure of these animals to account for genetic variation which may drive miRNA abundance and aBMD values. While many of these miRNAs have been associated with the risk of osteoporosis in humans, this finding is of interest because the cohort represents a model of normal aging and bone metabolism rather than a disease cohort. The replication of miRNA associations with osteoporosis or other bone metabolic disorders in animals with healthy aBMD suggests an overlap in normal variation and disease states. We suggest that these miRNAs are involved in the regulation of cellular proliferation, apoptosis, and protein composition in the extracellular matrix throughout life; and age-related dysregulation of these systems may lead to disease. These miRNAs may be early indicators of progression to disease in advance of clinically detectible osteoporosis.

Keywords:  bone mineral density; miRNA; nonhuman primate

DOI:  https://doi.org/10.1002/jor.25215
Stem Cells Int. 2021 ;2021 9778207

Sox9-Increased miR-322-5p Facilitates BMP2-Induced Chondrogenic Differentiation by Targeting Smad7 in Mesenchymal Stem Cells.

Yongsheng Zeng, Chengcheng Du, Pengcheng Xiao, Yiting Lei, Piao Zhao, Zhenglin Zhu, Shengqiang Gao, Bowen Chen, Shengwen Cheng, Wei Huang, Chen Zhao.

Bone morphogenetic protein 2 (BMP2) induces effective chondrogenesis of mesenchymal stem cells (MSCs) by promoting Sox9 expression. However, BMP2 also induces chondrocyte hypertrophy and endochondral ossification by upregulating Smad7 expression, which leads to the disruption of chondrogenesis. In addition, Smad7 can be inhibited by Sox9. Therefore, the underlying mechanism is not clear. Currently, an increasing number of studies have shown that microRNAs play a pivotal role in chondrogenic and pathophysiological processes of cartilage. The purpose of this study was to determine which microRNA is increased by Sox9 and targets Smad7, thus assisting BMP2 in maintaining stable chondrogenesis. We found that miR-322-5p meets the requirement through next-generation sequencing (NGS) and bioinformatic analysis. The targeting relationship between miR-322-5p and Smad7 was confirmed by dual-luciferase reporter assays, qPCR, and western blotting (WB). The in vitro study indicated that overexpression of miR-322-5p significantly inhibited Smad7 expression, thus causing increased chondrogenic differentiation and decreased hypertrophic differentiation, while silencing of miR-322-5p led to the opposite results. Flow cytometry (FCM) analysis indicated that overexpression of miR-322-5p significantly decreased the rate of early apoptosis in BMP2-stimulated MSCs, while silencing of miR-322-5p increased the rate. A mouse limb explant assay revealed that the expression of miR-322-5p was negatively correlated with the length of the BMP2-stimulated hypertrophic zone of the growth plate. An in vivo study also confirmed that miR-322-5p assisted BMP2 in chondrogenic differentiation. Taken together, our results suggested that Sox9-increased miR-322-5p expression can promote BMP2-induced chondrogenesis by targeting Smad7, which can be exploited for effective tissue engineering of cartilage.

DOI: https://doi.org/10.1155/2021/9778207
Front Cell Dev Biol. 2021 ;9 747057

MiR-181a Targets RSPO2 and Regulates Bone Morphogenetic Protein - WNT Signaling Crosstalk During Chondrogenic Differentiation of Mesenchymal Stromal Cells.

Svitlana Melnik, Nina Hofmann, Jessica Gabler, Nicole Hecht, Wiltrud Richter.

  Mechanisms of WNT and bone morphogenetic protein (BMP) signaling crosstalk is in the focus of multiple biological studies, and it also has been discovered to play important roles in human mesenchymal stromal cells (MSC) that are of great interest for neocartilage engineering due to their high chondrogenic differentiation potential. However, MSC-derived chondrocytes undergo hypertrophic degeneration that impedes their clinical application for cartilage regeneration. In our previous study, we established that several microRNAs (miRs) are differentially expressed between articular chondrocytes (AC) - and MSC-derived neocartilage, with miR-181a being the most prominent candidate as key microRNA involved in the regulation of a balance between chondral and endochondral differentiation. The aim of this study was the identification of precise mRNA targets and signaling pathways regulated by miR-181a in MSC during chondrogenesis. MiR-181a was upregulated during chondrogenesis of MSC, along with an increase of the hypertrophic phenotype in resulting cartilaginous tissue. By in silico analysis combined with miR reporter assay, the WNT signaling activator and BMP signaling repressor RSPO2 was suggested as a target of miR-181a. Further validation experiments confirmed that miR-181a targets RSPO2 mRNA in MSC. It was found that in human MSC miR-181a activated BMP signaling manifested by the accumulation of SOX9 protein and increased phosphorylation of SMAD1/5/9. These effects, together with the concomitant reduction of canonical WNT signaling induced by miR-181a mimic, were in accordance with the effects expected by the loss of RSPO2, thus indicating the causative link between miR-181a and RSPO2. Moreover, we observed that a tight correlation between miR-181a and miR-218 expression levels in healthy human cartilage tissue was disrupted in osteoarthritis (OA) highlighting the importance of the WNT-BMP signaling crosstalk for preventing OA.

Keywords:  BMP signaling; RSPO2; WNT signaling; chondrogenesis; mesenchymal stromal cells (MSC); miR-181a; microRNA; osteoarthritis (OA)

DOI:  https://doi.org/10.3389/fcell.2021.747057
J Clin Lab Anal. 2021 Nov 19. e23916

Microarray analysis of circRNAs sequencing profile in exosomes derived from bone marrow mesenchymal stem cells in postmenopausal osteoporosis patients.

Miao Fu, Liping Fang, Xi Xiang, Xijing Fan, Junqi Wu, Jinhua Wang.

   INTRODUCTION: Bone marrow-derived mesenchymal stem cells (BMSCs)-derived exosomes are involved in the modulation of tissue repair and regeneration. CircRNAs play important roles in BMSCs exosomes. The current study sought to explore the role of circRNAs in exosomes derived from BMSCs of postmenopausal osteoporosis (PMOP) patients and the underlying mechanisms.
METHODS: RNA was extracted from BMSCs exosomes of PMOP and a control group. RNA microarray and bioinformatics analyses were used to explore the expression profile and functions circRNAs. Differentially expressed circRNAs from 20 PMOP and 20 controls were analyzed using RT-qPCR.
RESULTS: A total of 237 upregulated and 279 downregulated circRNAs were identified in the current study. The top-10 most upregulated circRNAs in the PMOP group were hsa_circ_0069691, hsa_circ_0005678, hsa_circ_0006464, hsa_circ_0015813, hsa_circ_0000511, hsa_circ_0076527, hsa_circ_0009127, hsa_circ_0047285, hsa_circ_0027741, and hsa_circ_0090949. The top-10 most downregulated circRNAs were hsa_circ_0048669, hsa_circ_0090247, hsa_circ_0070899, hsa_circ_0087557, hsa_circ_0045963, hsa_circ_0090180, hsa_circ_0058392, hsa_circ_0040751, hsa_circ_0067910, and hsa_circ_0049484. RT-PCR verified dysregulation of 5 circRNAs including hsa_circ_0009127, hsa_circ_0090759, hsa_circ_0058392, hsa_circ_0090247, and hsa_circ_0049484. Moreover, a circRNA-microRNA-mRNA interaction network was developed based on differentially expressed circRNAs. Functional analysis showed that pathways involved in the regulation of autophagy, PI3K-Akt signaling, FoxO signaling, and MAPK signaling were associated with the differentially expressed circRNAs in PMOP patients.
CONCLUSION: The findings of this study show dysregulated circRNAs in BMSCs exosomes of PMOP patients, which may affect the progression of PMOP. These circRNAs can be used as predictive biomarkers and as therapeutic targets for the treatment of PMOP.

Keywords:  bone marrow mesenchymal stem cells; circular RNA; exosome; postmenopausal osteoporosis

DOI:  https://doi.org/10.1002/jcla.23916
Int Immunopharmacol. 2021 Nov 11. pii: S1567-5769(21)00954-1. [Epub ahead of print]101(Pt B): 108318

MicroRNA-497 inhibits inflammation in DSS-induced IBD model mice and lipopolysaccharide-induced RAW264.7 cells via Wnt/β-catenin pathway.

Mengjiao Zhang, Dongmei Yang, Honggang Yu, Qing Li.

   BACKGROUND AND AIMS: MicroRNA (miR)-497 is downregulated in several inflammatory diseases, excluding inflammatory bowel disease (IBD). The aim of this study is to evaluate whether miR-497 inhibits gut inflammation both in vivo and in vitro.
METHODS: The 3% dextran sulphate sodium (DSS) was used to induce experimental colitis, while 1 μg/ml lipopolysaccharide (LPS) was for RAW264.7 cell damage.Colitis severity was evaluated by disease activity index (DAI), colon length, histopathologic injury, etc. The nuclear transcription factor NF-κB activity in colon tissues was also estimated by western blot. Then, the quantitative real-time polymerase chain reaction (qRT-PCR) was performed to evaluate the expression levels of miR-497, pro-inflammatory cytokines and chemokines in colon tissues and RAW264.7 cells. Furthermore, the activity of Wnt/β-catenin pathway was determined by western blot and TOP/FOP-flash reporter assays.
RESULTS: The level of miR-497 was reduced in inflamed mucosa from IBD patients, mice with colitis and LPS-treated RAW264.7 cells. miR-497 knockout (miR-497 KO) mice were more susceptible to DSS-induced colitis, with increased inflammatory response, compared with control mice. Furthermore, the overexpression of miR-497 reduced the release of pro-inflammatory cytokines and chemokines in LPS-treated RAW264.7 cells. Finally, we found that miR-497 inhibited inflammation through Wnt/β-catenin pathway both in vitro and in vivo.
CONCLUSION: Our data indicate that miR-497 inhibits inflammation in DSS-induced IBD model mice and LPS-induced RAW264.7 cells by inhibiting the activation of NF-κB pathway and the release of cytokines, indicating that miR-497 plays a key role in the progression of IBD. Thus, therapeutic regulation of miR-497 expression may be beneficial for the treatment of IBD.

Keywords:  Animal model; IBD; Inflammatory response; Wnt/β-catenin; miR-497

DOI:  https://doi.org/10.1016/j.intimp.2021.108318
Am J Transl Res. 2021 ;13(10): 11126-11143

Screening of differentially expressed miRNAs during osteogenic/odontogenic differentiation of human dental pulp stem cells exposed to mechanical stress.

Yani He, Xiaoyue Guan, Yang Du, Guanzhi Liu, Yingxue Li, Zhichen Wei, Chen Shi, Jianmin Yang, Tiezhou Hou.

MicroRNAs (miRNAs) have been demonstrated as crucial transcriptional regulators in proliferation, differentiation, and tumorigenesis. The comprehensive miRNA profiles of osteogenic/odontogenic differentiation of human dental pulp stem cells (hDPSCs) under the condition of mechanical stress remains largely unknown. In this study, we aimed to discover the miRNA expression profiles of hDPSCs exposed to mechanical stress under the osteogenic/odontogenic process. We found that mechanical stress (0.09 MPa and 0.18 MPa, respectively, 30 min/day) significantly promoted the proliferation of hDPSCs since the fifth day. The expressions of DSPP, DMP1, and RUNX2 were significantly increased on day 7 in the presence of 0.09 MPa and 0.18 MPa mechanical stress. On day 14, the expression levels of DSPP, DMP1, and RUNX2 were decreased in the presence of mechanical stress. Among 2578 expressed miRNAs, 5 miRNAs were upregulated and 3 miRNAs were downregulated. Six hub target genes were merged in protein-protein interactions (PPI) network analysis, in which existed only one sub-network. Bioinformatics analysis identified an array of affected signaling pathways involved in the development of epithelial and endothelial cells, cell-cell junction assembly, Rap1 signaling pathway, regulation of actin cytoskeleton, and MAPK signaling pathway. Our results revealed the miRNA expression profiles of osteogenic/odontogenic differentiation of hDPSCs under mechanical stress and identified eight miRNAs that were differentially expressed in response to the mechanical stress. Bioinformatics analysis also showed that various signaling pathways were affected by mechanical stress.

Keywords: DPSCs; mechanical stress; miRNAs; odontogenic; osteogenic differentiation
J Cell Physiol. 2021 Nov 19.

CircRNA-miRNA networks in regulating bone disease.

Wei Huang, Yongyao Wu, MingXin Qiao, Zhuojun Xie, Xiao Cen, Xinqi Huang, Zhihe Zhao.

  Circular RNA (circRNA) is a class of endogenous noncoding RNA (ncRNA), presenting as a special covalent closed loop without a 5' cap or 3' tail, maintaining resistance to RNA exonuclease and keeping high stability. Although lowly expressed in most situations, circRNA makes an active difference in regulating physiological or pathological processes by modulating gene expression by regulation of transcription, protein, and miRNA functions through various mechanisms in particular tissues. Recent studies have demonstrated the roles of the miRNA-circRNA network in the development of several bone diseases such as osteoporosis, a multiple-mechanism disease resulting from defective bone quality and low bone mass, osteoarthritis, whose main pathomechanism is inflammation and articular cartilage degradation, as well as osteosarcoma, known as one of the most common bone cancers. However, the specific mechanism of how circRNA along with miRNA influences those diseases is not well documented, showing potential for the development of new therapies for those bone diseases.

Keywords:  circRNA; miRNA; osteoarthritis; osteoporosis; osteosarcoma

DOI:  https://doi.org/10.1002/jcp.30625
Biochem Genet. 2021 Nov 18.

Integrative Analysis of the Expression of microRNA, Long Noncoding RNA, and mRNA in Osteoarthritis and Construction of a Competing Endogenous Network.

Juntan Li, Xiang Gao, Wannan Zhu, Xu Li.

  This study aimed to identify potential core microRNAs (miRNAs), long noncoding RNAs (lncRNAs), and mRNAs in osteoarthritis (OA) to construct a competing endogenous RNA (ceRNA) and co-expression network. Differentially expressed miRNAs (DEMis) in the dataset GSE143514 comprising five OA and three normal tissues were identified using the DEseq package. Core miRNAs were identified as DEMis overlapping with those reported by the human microRNA disease database. LncRNAs were predicted by the miRNA-lncRNA interactions network from the encyclopedia of RNA interactomes (ENCORI). MiRNet and ENCORI were employed to predict the mRNAs which overlapped with the differentially expressed mRNAs from the dataset GSE114007 to obtain overlapping mRNAs. MiRNA-lncRNA and miRNA-mRNA interactions were integrated to construct the ceRNA network. A total of 143 DEMis were identified in OA and normal tissues, from which hsa-miR-451a, hsa-miR-370-5p, hsa-miR-34a-5p, hsa-miR-210-3p, and hsa-miR-101-3p were assessed as core miRNAs using overlapping analyses. These RNAs may represent potential therapeutic targets for the treatment of OA.

Keywords:  Cartilage; Functional annotation; Osteoarthritis; ceRNA; miRNA

DOI:  https://doi.org/10.1007/s10528-021-10159-3
Am J Transl Res. 2021 ;13(10): 11065-11080

LncRNA HAGLR absorbing miR-214-3p promotes BMP2 expression and improves tibial fractures.

Jinlong Chen, Yishan Yang.

OBJECTIVE: To determine whether long-chain non-coding RNA (lncRNA) HAGLR can regulate BMP2 by absorbing microRNA-214-3p (miR-214-3p), and to explore its role and mechanism in tibial fracture (TF) healing.
METHODS: The HAGLR, miR-214-3p, and BMP2 expression levels in TF and in adjacent normal tissues were measured using quantitative real-time polymerase chain reaction (qRT-PCR). MC3T3-E1 osteoblasts were used to construct the in vitro model. HAGLR was localized subcellularly through RNA-fluorescence in situ hybridization (FISH). A dual-luciferase report experiment confirmed that miR-214-3p has a targeted relationship with HAGLR and BMP2. It was then divided into a HAGLR over-expression group, an miR-214-3p mimic group, a HAGLR+miR-214-3p mimic group, an sh-HAGLR group, a BMP over-expression group, an sh-HAGLR+over-expression BMP2 group, and a negative control group. The proliferation and apoptosis of the MC3T3-E1 osteoblasts were examined using MTT assays and flow cytometry. A TF model was established in male C57BL/6J mice. The serum alkaline phosphatase (ALP) and osteoprotegerin (OPG) levels in the sham group, the TF group, and the TF group that were injected with HAGLR were compared using ELISA. Hematoxylin-eosin (HE) staining was used to confirm the fracture healing in the mouse model.
RESULTS: Compared with the adjacent normal tissues in the TF patients, the HAGLR and BMP2 expressions decreased but the miR-214-3p expressions increased in the TF tissues (P<0.05). HAGLR, an endogenous sponge, absorbed the miR-214-3p, and the BMP2 expression was directly regulated by miR-214-3p. HAGLR increased the proliferative activity of the osteoblasts and decreased the apoptosis rate. The over-expression of miR-214-3p partly reversed the effect of HAGLR on the cells, decreased the proliferative activity, and increased the apoptosis rate (all P<0.05). The sh-HAGLR decreased the proliferative activity and increased the apoptosis rate. But after the over-expression of BMP2, the proliferative activity of the cells was higher, and the apoptosis rate was lower than it was in the sh-HAGLR group (all P<0.05). The over-expression of HAGLR can up-regulate the ALP and OPG levels in mouse models (P<0.05).
CONCLUSION: lncRNA HAGLR can regulate BMP2 to play a protective role in TF by absorbing miR-214-3p, and it is related to promoting the osteoblast proliferation, inhibiting apoptosis, and up-regulating the serum ALP and OPG levels to accelerate bone healing.

Keywords: Tibial fractures; apoptosis; lncRNA HAGLR; osteoblasts; proliferation
Front Immunol. 2021 ;12 768771

Role of microRNA Shuttled in Small Extracellular Vesicles Derived From Mesenchymal Stem/Stromal Cells for Osteoarticular Disease Treatment.

Eliana Lara-Barba, María Jesús Araya, Charlotte Nicole Hill, Felipe A Bustamante-Barrientos, Alexander Ortloff, Cynthia García, Felipe Galvez-Jiron, Carolina Pradenas, Noymar Luque-Campos, Gabriela Maita, Roberto Elizondo-Vega, Farida Djouad, Ana María Vega-Letter, Patricia Luz-Crawford.

  Osteoarticular diseases (OD), such as rheumatoid arthritis (RA) and osteoarthritis (OA) are chronic autoimmune/inflammatory and age-related diseases that affect the joints and other organs for which the current therapies are not effective. Cell therapy using mesenchymal stem/stromal cells (MSCs) is an alternative treatment due to their immunomodulatory and tissue differentiation capacity. Several experimental studies in numerous diseases have demonstrated the MSCs' therapeutic effects. However, MSCs have shown heterogeneity, instability of stemness and differentiation capacities, limited homing ability, and various adverse responses such as abnormal differentiation and tumor formation. Recently, acellular therapy based on MSC secreted factors has raised the attention of several studies. It has been shown that molecules embedded in extracellular vesicles (EVs) derived from MSCs, particularly those from the small fraction enriched in exosomes (sEVs), effectively mimic their impact in target cells. The biological effects of sEVs critically depend on their cargo, where sEVs-embedded microRNAs (miRNAs) are particularly relevant due to their crucial role in gene expression regulation. Therefore, in this review, we will focus on the effect of sEVs derived from MSCs and their miRNA cargo on target cells associated with the pathology of RA and OA and their potential therapeutic impact.

Keywords:  mesenchymal stem cells; microRNA; osteoarthritis; rheumatoid arthritis; small extracellular vesicles

DOI:  https://doi.org/10.3389/fimmu.2021.768771
Front Genet. 2021 ;12 747274

MicroRNA Variants and HLA-miRNA Interactions are Novel Rheumatoid Arthritis Susceptibility Factors.

Shicheng Guo, Yehua Jin, Jieru Zhou, Qi Zhu, Ting Jiang, Yanqin Bian, Runrun Zhang, Cen Chang, Lingxia Xu, Jie Shen, Xinchun Zheng, Yi Shen, Yingying Qin, Jihong Chen, Xiaorong Tang, Peng Cheng, Qin Ding, Yuanyuan Zhang, Jia Liu, Qingqing Cheng, Mengru Guo, Zhaoyi Liu, Weifang Qiu, Yi Qian, Yang Sun, Yu Shen, Hong Nie, Steven J Schrodi, Dongyi He.

  Genome-wide association studies have identified >100 genetic risk factors for rheumatoid arthritis. However, the reported genetic variants could only explain less than 40% heritability of rheumatoid arthritis. The majority of the heritability is still missing and needs to be identified with more studies with different approaches and populations. In order to identify novel function SNPs to explain missing heritability and reveal novel mechanism pathogenesis of rheumatoid arthritis, 4 HLA SNPs (HLA-DRB1, HLA-DRB9, HLA-DQB1, and TNFAIP3) and 225 common SNPs located in miRNA, which might influence the miRNA target binding or pre-miRNA stability, were genotyped in 1,607 rheumatoid arthritis and 1,580 matched normal individuals. We identified 2 novel SNPs as significantly associated with rheumatoid arthritis including rs1414273 (miR-548ac, OR = 0.84, p = 8.26 × 10-4) and rs2620381 (miR-627, OR = 0.77, p = 2.55 × 10-3). We also identified that rs5997893 (miR-3928) showed significant epistasis effect with rs4947332 (HLA-DRB1, OR = 4.23, p = 0.04) and rs2967897 (miR-5695) with rs7752903 (TNFAIP3, OR = 4.43, p = 0.03). In addition, we found that individuals who carried 8 risk alleles showed 15.38 (95%CI: 4.69-50.49, p < 1.0 × 10-6) times more risk of being affected by RA. Finally, we demonstrated that the targets of the significant miRNAs showed enrichment in immune related genes (p = 2.0 × 10-5) and FDA approved drug target genes (p = 0.014). Overall, 6 novel miRNA SNPs including rs1414273 (miR-548ac, p = 8.26 × 10-4), rs2620381 (miR-627, p = 2.55 × 10-3), rs4285314 (miR-3135b, p = 1.10 × 10-13), rs28477407 (miR-4308, p = 3.44 × 10-5), rs5997893 (miR-3928, p = 5.9 × 10-3) and rs45596840 (miR-4482, p = 6.6 × 10-3) were confirmed to be significantly associated with RA in a Chinese population. Our study suggests that miRNAs might be interesting targets to accelerate understanding of the pathogenesis and drug development for rheumatoid arthritis.

Keywords:  SNP; miRNA; predispose; rheumatoid arthritis; susceptibility

DOI:  https://doi.org/10.3389/fgene.2021.747274