Let-7i-5p functions as a putative osteogenic differentiation promoter by targeting CKIP-1.
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ABSTRACT: Supplementary information
The online version of this article (10.1007/s10616-020-00444-1) contains supplementary material, which is available to authorized users.
SUBMITTER: Zhang Y
PROVIDER: S-EPMC7817755 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundOsteoporosis (OP) is a metabolic disease that involves microstructure destruction and fracture damage. The present study probed into the significance of miR-215-5p in OP progression.MethodsSerum samples were collected from surgical patients and healthy controls. qRT-PCR analysis was utilized to determine the miR-215-5p level in clinical samples and human bone mesenchymal stem cells (hBMSCs) induced by β-glycerol phosphate. A dual luciferase reporter assay was exploited to examine the targeted relationship between miR-215-5p and XIAP. The mineralization and calcium deposition of hBMSCs were assessed by detection of ALP activity, Alizarin red staining, and osteoblast marker expression. Protein expression was determined by western blot analysis.ResultsMiR-215-5p was significantly reduced in patients with OP and increased in hBMSCs treated with β-glycerophosphate. Enhanced miR-215-5p level triggered augment in osteoblast markers (Alkaline phosphatase/ ALP, Osteocalcin/ OCN, and Runt-Related Transcription Factor 2/ Runx2), which was accompanied by the increase of ALP activity in hBMSCs and accumulation of Calcium. Functional experiments show that XIAP was a target of miR-215-5p and negatively modulated by miR-215-5p. XIAP expression levels were increased in OP samples, and decreased XIAP in β-glycerophosphate-treated hBMSCs inhibited its' osteogenic differentiation. Functional loss and acquisition experiments depicted that miR-215-5p promoted the differentiation of hBMSCs by inhibiting the XIAP level, playing a protective role in the pathogenesis of OP.Conclusionsβ-glycerophosphate promoted the osteogenic differentiation of hBMSCs, increased miR-215-5p level, and decreased XIAP. miR-215-5p stimulated osteogenic differentiation of hBMSCs by targeting XIAP, shedding new insights for the detection and therapy of OP.
| S-EPMC9387055 | biostudies-literature
Project description:Background and purposeTrauma-induced osteonecrosis of the femoral head (TIONFH) is a major complication of femoral neck fractures. Degeneration and necrosis of subchondral bone can cause collapse, which results in hip joint dysfunction in patients. The destruction of bone metabolism homeostasis is an important factor for osteonecrosis. MicroRNAs (miRNAs) have an important role in regulating osteogenic differentiation, but the mechanisms underlying abnormal bone metabolism of TIONFH are poorly understood. In this study, we screened specific miRNAs in TIONFH by microarray and further explored the mechanism of osteogenic differentiation.DesignBlood samples from patients with TIONFH and patients without necrosis after trauma were compared by microarray, and bone collapse of necrotic bone tissue was evaluated by micro-CT and immunohistochemistry. To confirm the relationship between miRNA and osteogenic differentiation, we conducted cell culture experiments. We found that many miRNAs were significantly different, including miR-93-5p; the increase in this miRNA was verified by Q-PCR. Comparison of the tissue samples showed that miR-93-5p expression increased, and alkaline phosphatase (ALP) and osteopontin (OPN) levels decreased, suggesting miR-93-5p may be involved in osteogenic differentiation. Further bioinformatics analysis indicated that miR-93-5p can target bone morphogenetic protein 2 (BMP-2). A luciferase gene reporter assay was performed to confirm these findings. By simulating and/or inhibiting miR-93-5p expression in human bone marrow mesenchymal stem cells, we confirmed that osteogenic differentiation-related indictors, including BMP-2, Osterix, Runt-related transcription factor, ALP and OPN, were decreased by miR-93-5p.ConclusionOur study showed that increased miR-93-5p in TIONFH patients inhibited osteogenic differentiation, which may be associated with BMP-2 reduction. Therefore, miR-93-5p may be a potential target for prevention of TIONFH.
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Project description:BackgroundClear cell renal cell carcinoma (ccRCC) is one of the best-characterized and most pervasive renal cancers. The present study aimed to explore the effects and potential mechanisms of let-7i-5p in ccRCC cells.MethodsUsing bioinformatics analyses, we investigated the expression of let-7i-5p in The Cancer Genome Atlas (TCGA) database and predicted biological functions and possible target genes of let-7i-5p in ccRCC cells. Cell proliferation assay, wound healing assay and transwell invasion assay were conducted to characterize the effects of let-7i-5p in ccRCC cells. To verify the interactions between let-7i-5p and HABP4, dual-luciferase reporter assay, quantitative real-time polymerase chain reaction, and western blotting were conducted. Rescue experiments were used to investigate the relationship between let-7i-5p and HABP4.ResultsTCGA data analysis revealed that ccRCC tissues had significantly increased let-7i-5p expression, which was robustly associated with poor overall survival. Further verification showed that ccRCC cell proliferation, migration and invasion were inhibited by let-7i-5p inhibitor but enhanced by let-7i-5p mimics. Subsequently, HABP4 was predicted to be the target gene of let-7i-5p. TCGA data showed that ccRCC tissues had decreased expression of HABP4 and that HABP4 expression was negatively correlated with let-7i-5p. Further verification showed that downregulation of HABP4 expression promoted cell proliferation, migration and invasion. The dual-luciferase reporter gene assay suggested that the let-7i-5p/HABP4 axis was responsible for the development of ccRCC.ConclusionOur results provide evidence that let-7i-5p functions as a tumor promoter in ccRCC and facilitates cell proliferation, migration and invasion by targeting HABP4. These results clarify the pathogenesis of ccRCC and offer a potential target for its treatment.
| S-EPMC8005230 | biostudies-literature
Project description:This study was aimed to explore the differential expression of long noncoding RNAs (lncRNA)-PCAT1, miR-145-5p and TLR4 in osteogenic differentiation via the Toll-like receptor (TLR) signalling pathway and consequently determine the potential molecular mechanism. The mRNAs and pathways related to the osteogenic differentiation in human adipose-derived stem cells (hADSCs) were analysed by bioinformatics. The MiRanda and TargetScan database were employed to detect the potential binding sites of miRNAs on lncRNAs and mRNAs. The differential expression of lncRNA-PCAT1, miR-145-5p and TLR4 were detected by qRT-PCR. Rrelated protein expression was analysed by Western blot. The targeted relationships between lncRNA-PCAT1, miR-145-5p and TLR4 were verified by dual-luciferase reporter assay. Alkaline phosphatase (ALP) activity and ARS staining assays were used to measure the impacts exerted by lncRNA PCAT1, miR-145-5p and TLR4 mRNA on osteogenic differentiation. After the induction of osteoblast differentiation, the expression of lncRNA-PCAT1 and TLR4 increased, while the expression of miR-145-5p decreased. Dual-luciferase reporter assay confirmed the targeted relationship between lncRNA-PCAT1, miR-145-5p, and TLR4. LncRNA-PCAT1 negatively regulated miR-145-5p and positively regulated TLR4. Knockdown of lncRNA-PCAT1 or TLR4 decreased the expression of osteogenic differentiation-related proteins, reduced the ALP and ARS levels and the activity of the TLR signalling pathway. MiR-145-5p could reverse the effects of PCAT1 and TLR4 in hADSCs osteogenic differentiation. LncRNA-PCAT1 negatively regulated miR-145-5p, which promoted TLR4 expression to promote osteogenic differentiation by activating the TLR signalling pathway.
| S-EPMC6237555 | biostudies-literature
Project description:Osteoporosis (OP), a common systemic bone metabolism disease with a high incidence rate, is a serious health risk factor. Osteogenic differentiation balance is regulated by bone marrow mesenchymal stem cells (BMSCs) and plays a key role in OP occurrence and progression. Although, LIM homeobox 8 (Lhx8) has been identified to affect BMSCs osteogenic differentiation, its roles in OP and the associated mechanism remains unclear. Here, we aimed to elucidate the role and mechanism of Lhx8 in the osteogenic differentiation of BMSCs. BMSCs isolated from wild type and OP Sprague-Dawley rats were cultured and confirmed via flow cytometry and microscopy. Based on dual-luciferase reporter assay, BMSCs were transfected with miR-142-5p mimics and miR-NC (negative control). Real-time quantitative reverse transcription polymerase chain reaction and Western blot analyses were performed to determine the role of Lhx8 in BMSCs osteogenic differentiation. Lhx8 expression was significantly reduced in OP, whereas that of miR-142-5p, a possible Lhx8 regulator, was significantly upregulated. Dual-luciferase reporter assay demonstrated that miR-142-5p exerted a direct targeted regulatory effect on Lhx8. Moreover, miR-142-5p mimics significantly inhibited BMSCs osteogenic differentiation as well as Lhx8 expression in vitro, indicating that miR-142-5p may be involved in BMSCs osteogenic differentiation via Lhx8 expression regulation and may serve as a potential diagnostic target for OP. Overall, these findings indicated that miR-142-5p inhibits BMSCs osteogenic differentiation by suppressing Lhx8. These may serve as a foundation for further studies on OP treatment based on miR-142-5p targeting.
| S-EPMC10559276 | biostudies-literature
Project description:BackgroundThe aim of this study was to evaluate, the existence of a signature of differentially expressed microRNAs (miRNAs) during osteogenic differentiation of bone marrow MSCs from OA and healthy donors and to describe their possible implication in joint regeneration through modulation of molecular mechanisms involved in homeostatic control in OA pathophysiology.MethodsFollowing phenotypic assessment of BM-MSCs obtained from OA diagnosed patients (n = 10) and non-OA (n = 10), total small RNA was isolated after osteogenic induction for 1, 10 and 21 days, miRNA profiles were generated using a commercial expression array of 754 well-characterized miRNAs. MiRNAs, with consistent differential expression were selected for further validation by quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis.ResultsA total of 246 miRNAs were differentially expressed (fold change ≥ ± 2, P ≤0.05) between OA and non-OA BM-MSC samples; these miRNAs showed variable interactions depending on the cell and differentiation status. Two miRNAs, hsa-miR-210 and hsa-miR-335-5p out of 21 used for validation showed a significant downregulated expression during induced osteogenesis. In particular hsa-miR-335-5p, a critical regulator in bone homeostasis, was further studied. hsa-miR-335-5p downregulation in OA-MSCs, as well as their host coding gene, MEST, were also assessed.ConclusionsTo our knowledge, this study represents the most comprehensive assessment to date of miRNA expression profiling in BM-MSCs from OA patients and their role during osteogenic differentiation. We describe the existence of a correlation between miR-335-5p expression and OA indicating the putative role of this miRNA in OA features. These findings, may contribute to our understanding of the molecular mechanisms involved in MSCs mediated homeostatic control in OA pathophysiology that could be applicable in future therapeutic approaches.
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Project description:Calcific aortic valve disease (CAVD) is the most common heart valve disorder, yet its mechanism remains poorly understood. Valve interstitial cells (VICs) are the prevalent cells in aortic valve and their osteogenic differentiation may be responsible for calcific nodule formation in CAVD pathogenesis. Emerging evidence shows microRNA (miRNA, or miR) can function as important regulators of many pathological processes, including osteogenic differentiation. Here, we aimed to explore the function of miR-449c-5p in CAVD pathogenesis. In this study, we demonstrated the role of miR-449c-5p in VICs osteogenesis. MiRNA microarray assay and qRT-PCR results revealed miR-449c-5p was significantly down-regulated in calcified aortic valves compared with non-calcified valves. MiR-449c-5p overexpression inhibited VICs osteogenic differentiation in vitro, whereas down-regulation of miR-449c-5p enhanced the process. Target prediction analysis and dual-luciferase reporter assay confirmed Smad4 was a direct target of miR-449c-5p. Furthermore, knockdown of Smad4 inhibited VICs osteogenic differentiation, similar to the effect observed in up-regulation miR-449c-5p. In addition, animal experiments proved indirectly miR-449c-5p could alleviate aortic valve calcification. Our data suggested miR-449c-5p could function as a new inhibitory regulator of VICs osteogenic differentiation, which may act by targeting Smad4. MiR-449c-5p may be a potential therapeutic target for CAVD.
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Project description:ObjectivesCasein kinase 2 interacting protein-1 (CKIP-1) is a versatile player involved in various biological processes. However, whether CKIP-1 mediates the osteogenic/cementogenic differentiation of periodontal ligament cells (PDLCs) under Porphyromonas gingivalis (Pg) stimulation remains unknown.Material and methodsThe effect of Pg on PDLC differentiation was first verified. CKIP-1 expression in Pg-infected PDLCs or in PDL of apical periodontitis (AP) mice was detected. The changes of CKIP-1 during PDLC differentiation was also determined. PDLC differentiation capacity in CKIP-1 knockout (KO) mice and CKIP-1-silenced PDLCs with or without Pg stimulation were further studied. Inhibitor was finally applied to verify the involvement of p38 signaling pathway in PDLC differentiation.ResultsThe suppression effect of Pg on PDLC differentiation was demonstrated. CKIP-1 increased in the PDL of AP mice and Pg-induced PDLCs, and decreased gradually during PDLC differentiation. Increased OSX and RUNX2 expression in PDL were observed in CKIP-1 KO mice. Also, CKIP-1 silencing facilitated and rescued Pg-inhibited PDLC differentiation. Inhibitor for p38 signaling pathway blocked CKIP-1 silencing-facilitated PDLC differentiation.ConclusionsCKIP-1 mediated the osteogenic/cementogenic differentiation of PDLCs partially through p38 signaling pathway, which may provide evidence for the regeneration of periodontal hard tissues damaged by Pg.
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Project description:Myeloma bone disease (MBD) is one of the clinical features of multiple myeloma, which contributes to the attenuation of osteoblast function. Bone marrow mesenchymal stem cells exhibit a high potential for differentiation into osteoblasts. A number of studies have reported that microRNAs (miRs) serve a vital role in mesenchymal stem cell (MSC) osteogenesis; however, the role of miR-221-5p in the osteogenic differentiation of MBD-MSCs remains unclear. The present study revealed that the osteogenic differentiation capacity of MBD-MSCs was reduced compared with that of normal (N)-MSCs. Further experiments demonstrated that miR-221-5p expression was downregulated in N-MSCs following osteoblast induction while no obvious alterations in expression levels were observed in MBD-MSCs. The inhibition of miR-221-5p promoted the osteogenic differentiation of MBD-MSCs. Bioinformatics, luciferase reporter assays, reverse transcription-quantitative PCR and western blotting assays indicated that smad family member 3 (smad3) was a direct target of miR-221-5p in MBD-MSCs. A negative association was identified between the expression levels of smad3 and miR-221-5p. Investigations of the molecular mechanism indicated that suppressed miR-221-5p could regulate the osteogenic differentiation of MBD-MSCs by upregulating smad3 expression. It was also identified that the PI3K/AKT/mTOR signaling pathway was activated following miR-221-5p inhibition, and this increased the osteogenic differentiation capacity of MBD-MSCs. The present study may improve the understanding regarding the role of miR-221-5p in the regulation of osteogenic differentiation, and may contribute to the development of a novel therapy for MBD.
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