Dataset Information

Hypoxia-responsive miRNA-21-5p inhibits Runx2 suppression by targeting SMAD7 in MC3T3-E1 cells.

ABSTRACT: Sustained hypoxia inhibits osteogenesis and osteoblast differentiation by downregulating the expression of runt-related transcription factor 2 (Runx2). MicroRNAs (miRNAs) have been shown to regulate osteogenesis and osteoblast differentiation. In the present study, we profiled miRNAs, with microRNA array and quantitative real-time polymerase chain reaction (RT-PCR) methods, in mouse osteoblast (MC3T3-E1) cells under hypoxia. Then, we investigated regulation by miRNA-21-5p on the expression of Runx2 and other osteoblast differentiation-associated markers via gain-of-function and loss-of-function strategies. We found that expression of miRNA-21-5p, miRNA-210-5p, and other eight miRNAs was upregulated significantly in hypoxia-treated MC3T3-E1 cells. miRNA-21-5p overexpression downregulated the expression of the mRNA and protein of suppressor of mothers against decapentaplegic (SMAD7) markedly, the 3'-untranslated region (3'-UTR) of which was highly homologous with the miRNA-21-5p sequence. miRNA-21-5p overexpression upregulated the protein expression of Runx2 in hypoxia-treated MC3T3-E1 cells, although mRNA expression of Runx2 and other osteoblast differentiation-associated molecules (eg, osteocalcin, procollagen type 1 amino-terminal propeptide, P1NP) were not regulated by it; such upregulation was SMAD7-dependent. In conclusion, hypoxia-responsive miRNA-21-5p promoted Runx2 expression (at least in part) by targeting the 3'-UTR and downregulating SMAD7 expression. Our study suggests a protective role of miRNA-21-5p in promoting osteoblast differentiation under hypoxia.

SUBMITTER: Li L

PROVIDER: S-EPMC6766843 | biostudies-other | 2019 Oct

REPOSITORIES: biostudies-other

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Hypoxia-responsive miRNA-21-5p inhibits Runx2 suppression by targeting SMAD7 in MC3T3-E1 cells.

Li Lujun L Jiang Dianming D

Journal of cellular biochemistry 20190520 10

Sustained hypoxia inhibits osteogenesis and osteoblast differentiation by downregulating the expression of runt-related transcription factor 2 (Runx2). MicroRNAs (miRNAs) have been shown to regulate osteogenesis and osteoblast differentiation. In the present study, we profiled miRNAs, with microRNA array and quantitative real-time polymerase chain reaction (RT-PCR) methods, in mouse osteoblast (MC3T3-E1) cells under hypoxia. Then, we investigated regulation by miRNA-21-5p on the expression of Ru ...[more]

PMID: 31106445

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Project description:ObjectiveMicroRNAs (miRNAs) are well-recognized for their abilities to regulate gene expression post-transcriptionally in plants and animals. Recently, miRNA-messenger RNA (mRNA) regulatory relationships have been confirmed during biological processes, including osteogenic differentiation. This study aimed to find out more candidate miRNA-mRNA pairs involved in the osteogenic differentiation of MC3T3-E1 cells.MethodsAn MC3T3-E1-based microarray dataset (accessioned as GSE46400) downloaded from the Gene Expression Omnibus included MC3T3-E1 cells with or without 14-day osteoblast differentiation osteoblast induction. Multiple miRNA-mRNA prediction databases were searched by differentially expressed genes (DEGs) to obtain pairs of a miRNA-DEG regulatory network. The MC3T3-E1 cells were cultured and incubated in the osteogenic differentiation medium for 14 days. The expressions of candidate miRNAs and mRNAs were determined by real-time quantitative PCR(RT-qPCR) in MC3T3-E1 cells. The miRNA-mRNA interactions were verified by dual-luciferase reporter gene assays and experiments using mimics miRNA or their inhibitors.ResultsWe identified 715 upregulated DEGs and 603 downregulated DEGs between MC3T3-E1 cells with and without osteoblast induction by analyzing the raw data of the GSE46400 dataset. There were 7 overlapped miRNA-mRNA pairs identified during osteogenic differentiation of MC3T3-E1 cells, including mmu-miR-204-5p-Arhgap11a, mmu-miR-211-5p-Arhgap11a, mmu-miR-24-3p-H2afx, mmu-miR-3470b-Chek2, mmu-miR-3470b-Dlgap5, mmu-miR-466b-3p-Chek1, and mmu-miR-466c-3p-Chek1. The Arhgap11a, H2afx, Chek2, Dlgap5, and Chek1 were hub genes downregulated in MC3T3-E1 cells after osteogenic differentiation, verified by RT-qPCR results. The RT-qPCR also determined declined expressions of miR-204-5p and miR-24-3p concomitant with elevated expressions of miR-211-5p, miR-3470b, miR-466b-3p, and miR-466c-3p in the MC3T3-E1 cells, with osteoblast induction compared with undifferentiated MC3T3-E1 cells. Dual-luciferase reporter gene assays demonstrated Arhgap11a as the target of miR-211-5p. MiR-211-5p upregulation by its mimic increased Arhgap11a expression in MC3T3-E1 cells.ConclusionOur study characterizes miR-211-5p targeting Arhgap11a promotes the osteogenic differentiation of MC3T3-E1 cells, which provides novel targets to promote the osteogenesis process during bone repair.

Dataset Information

Hypoxia-responsive miRNA-21-5p inhibits Runx2 suppression by targeting SMAD7 in MC3T3-E1 cells.

Publications

Hypoxia-responsive miRNA-21-5p inhibits Runx2 suppression by targeting SMAD7 in MC3T3-E1 cells.

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