Dataset Information

MicroRNA-22 contributes to dexamethasone-induced osteoblast differentiation inhibition and dysfunction through targeting caveolin-3 expression in osteoblastic cells.

ABSTRACT: Osteoporosis is a common complication of long-term use of glucocorticoids (GCs) characterized by the loss of bone mass and damage of the microarchitecture as well as osteoblast dysfunction. Previous studies have demonstrated that microRNA-22 (miR-22) is the negative modulator of osteogenesis that may target caveolin-3 (CAV3), which has been reported to enhance bone formation and inhibit the progression of osteoporosis as well as apoptosis. The present study aimed to investigate whether miR-22 may be involved in dexamethasone (DEX)-induced inhibition of osteoblast differentiation and dysfunction by regulating CAV3 expression. Reverse transcription-quantitative PCR (RT-qPCR) was performed to measure the expression of miR-22 and western blotting was performed to determine protein levels. The results demonstrated that miR-22 expression was upregulated in DEX-treated osteoblastic cells compared with the control group. In addition, miR-22 mimic aggravated, whereas miR-22 inhibitor mitigated DEX-induced damage in osteoblastic cells compared with the control groups. Additionally, CAV3 was identified as the target of miR-22 in osteoblasts using RT-qPCR, western blotting and dual-luciferase reporter gene assay analysis. The results also demonstrated that silencing of CAV3 blocked the beneficial effects of miR-22 inhibitor against DEX-induced cell damage and apoptosis in osteoblasts, as evidenced by the increased expression levels of cleaved caspase-3, Bax and alkaline phosphatase activity as well as decreased cell viability and Bcl-2 levels. Collectively, these results indicate a novel molecular mechanism by which miR-22 contributes to DEX-induced osteoblast dysfunction and apoptosis via the miR-22/CAV3 pathway.

SUBMITTER: Li P

PROVIDER: S-EPMC7903452 | biostudies-literature | 2021 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

MicroRNA-22 contributes to dexamethasone-induced osteoblast differentiation inhibition and dysfunction through targeting caveolin-3 expression in osteoblastic cells.

Li Peng P Mao Weiwei W Zhang Shuai S Zhang Liang L Chen Zhirong Z Lu Zhidong Z

Experimental and therapeutic medicine 20210208 4

Osteoporosis is a common complication of long-term use of glucocorticoids (GCs) characterized by the loss of bone mass and damage of the microarchitecture as well as osteoblast dysfunction. Previous studies have demonstrated that microRNA-22 (miR-22) is the negative modulator of osteogenesis that may target caveolin-3 (CAV3), which has been reported to enhance bone formation and inhibit the progression of osteoporosis as well as apoptosis. The present study aimed to investigate whether miR-22 ma ...[more]

PMID: 33732309

Similar Datasets

Project description:Dickkopf-1 (DKK1) is a powerful antagonist of canonical WNT signaling pathway, and is regarded as a biomarker for osteoporosis. Its expression is highly correlated with bone mass and osteoblasts maturation. In this study, mouse primary bone marrow cells and osteoblast cell lines were used. Luciferase reporter assay and western blotting methods were employed to validate if miRNA-433-3p epigenetically regulated DKK1 translation. Rat bone marrow derived osteoblasts were infected with lentivirus vector in which miR-433-3p was constructed. The authors constructed lentivirus mediated miRNA-433-3p stable expression and examined the alkaline phosphatase (ALP) activity and mineral deposition level in vitro. In situ hybridization method was used to observe miR-433-3p in primary osteoblasts. We built up an OVX rat model to mimic postmenopausal osteoporosis, and found aberrant circulating miR-433-3p and miR-106b, which were not reported previously. Results showed that miR-433-3p potentially regulated DKK1 mRNA, Furthermore, the correlation of serum DKK1 with circulating miR-433-3p level was significant (r = 0.7520, p = 0.046). In the luciferase reporter assay, we found that miR-433-3p siRNA decreased luminescence signal, indicating direct regulation of miR-433-3p on DKK1 mRNA. When the miR-433-3p binding site in DKK1 3'UTR was mutant, such reduction was prohibited. Western blotting result validated that miR-433-3p inhibited over 90% of DKK1 protein expression. Similarly, the change of protein expression was not observed in mutant group. The stable expression of lentivirus mediated miR-433-3p increased ALP activity and mineralization both in human and rat derived immortalized cells. We found that primary osteoblasts had higher miR-433-3p level compared with immortal cells through real-time PCR, as well as in situ hybridization experiment. Conclusively, our findings further emphasized the vital role of miR-433-3p in DKK1/WNT/?-catenin pathway through decreasing DKK1 expression and inducing osteoblasts differentiation.

Project description:BackgroundThe aim of this study was to investigate using myogenic differentiation of adipose stem cells for the treatment of female pelvic floor dysfunction (PFD) and aimed to further study the influences of microRNA-124-3p (miR-124-3p) in the process of myogenic differentiation of adipose-derived stem cells (ADSCs) through targeting Caveolin-1 (Cav1) during PFD in Sprague Dawley (SD) rats.MethodsThe ADSCs were separated from 6-8-week-old female SD rats (n=25) and were cultivated. Then, we observed the cell status and conducted fat and osteogenic experiments. We then constructed an ADSC-green fluorescent protein (GFP) stable transfer strain. Flow cytometry was used to identify the positive rates of CD44, CD90, and CD45 in ADSCs and ADSC-GFP. Real-time quantitative polymerase chain reaction (qRT-PCR) and western blotting were used to mRNA and protein expression levels. Myogenic differentiation of ADSCs was measured with immunofluorescence methods. A dual-luciferase reporter assay was executed to affirm whether Cav1 was a target of miR-124-3p.ResultsThe isolated ADSCs cells were in good condition under the microscope. The results of flow cytometry showed that the positive rate of CD44 and CD90 was high, and the positive rate of CD45 was low in ADSCs and ADSC-GFP. Under normal culture conditions, ADSCs-GFP cells can be massively adipated and osteogenic. After 5-Aza induced ADSC-GFP myogenic differentiation, the level of miR-124-3p was significantly increased. We found that MiR-124-3p mimics promoted the myogenic differentiation of ADSCs. Moreover, we discovered that Cav1 was a target gene of miR-124-3p and was negatively regulated by miR-124-3p. The results of leak point pressure (LPP), hematoxylin and eosin (HE), and Masson showed that the collagen fiber content of the PFD group was lower than that of the control group; the collagen fiber content of ADSC-GFP, 5-Aza, or miR-124-3p mimics were increased after intervention. Furthermore, the outcomes qRT-PCR, western blotting, and immunofluorescence suggested that miR-124-3p facilitated the survival ADSC-GFP fat transplantation by regulating many key factors in vivo.ConclusionsThese results proofed that miR-124-3p could accelerate myogenic differentiation of ADSCs by down-regulating Cav1 to improve PFD in SD rats, which will pave the way for therapeutic delivery of miRNA targeting PFD disease.

Project description:Osteogenic differentiation is an important process of new bone formation, microRNA-409-3p (miR-409-3p) has been reported to be up-regulated in the osteogenic differentiation of human bone marrow mesenchymal stem cells (MSCs). The present study aimed to investigate the regulatory effect of miR-409-3p on osteogenic differentiation of MSCs and its molecular mechanism. The expression of miR-409-3p in osteoblast (human skull osteoblast, HCO) and bone marrow-derived MSCs (MSC-A, MSC-B, MSC-U) were detected by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The binding of miR-409-3p to suppressor of cancer cell invasion (SCAI) in MSC-B was investigated by performing a dual-luciferase reporter gene assay. MSC-B was selected to transfect with miR-409-3p analog/complementary sequence (cs), miR-409-3p analog + SCAI and miR-409-3p cs + small interfering (si)-SCAI, as well as control, respectively. The alkaline phosphatase (ALP) activity, Alizarin Red staining, and the expression of osteogenic markers (ALP, osteocalcin (OCN), osteopontin (OPN), runt-related transcription factor 2 (RUNX2)) in MSC-B during osteoblastic differentiation were tested by RT-qPCR and Western blotting, respectively. Additionally, the Wnt/β-catenin pathway was inhibited by dickkopf-related protein 1 (DKK-1) to get the roles of miR-409-3p during the osteoblastic differentiation of MSC-B when transfected with miR-409-3p analog. The expression of miR-409-3p in HCO was higher than that in these three MSCs and showed an increasing time-dependent trend on the 0 and 21st day of osteoblastic differentiation. MiR-409-3p directly regulated SCAI by targeting SCAI 3'UTR. Further, miR-409-3p suppressed SCAI expression, but SCAI up-regulation suppressed the osteoblastic differentiation, as well as reduced the relative mRNA/protein expression of Wnt/β-catenin signaling pathway-related genes (Axis inhibition protein 1 (AXIN1), β-catenin, Lymphoid Enhancer Binding Factor 1, Cellular-myelocytomatosis (c-myc) and cyclin D1). Importantly, disruption of Wnt signaling also blocked miR-409-3p induced osteoblastic differentiation of MSCs. Therefore, miR-409-3p promotes osteoblastic differentiation through the activation of the Wnt/β-catenin pathway by down-regulating SCAI expression.

Dataset Information

MicroRNA-22 contributes to dexamethasone-induced osteoblast differentiation inhibition and dysfunction through targeting caveolin-3 expression in osteoblastic cells.

Publications

MicroRNA-22 contributes to dexamethasone-induced osteoblast differentiation inhibition and dysfunction through targeting caveolin-3 expression in osteoblastic cells.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets