Involvement of MiRNA-211-5p and Arhgap11a Interaction During Osteogenic Differentiation of MC3T3-E1 Cells

Abstract

Objective: MicroRNAs (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.

Methods: An 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.

Results: We 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.

Conclusion: Our 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.

Keywords: Arhgap11a; MC3T3-E1 cell; miR-211-5p; microRNA; osteogenic differentiation.

Figure 1

Identification of Differentially expressed genes (DGEs) between MC3T3-E1 cells with and without osteoblast induction. (A) The volcano plot; red dots reflect upregulated DGEs (n = 715), blue dots reflect downregulated DGEs (n = 603), and black dots reflect non-DGEs. (B) The heatmap showing expression diversity of 20 representative DGEs in the GSE46400; the color from blue to red shows the expression from low to high.

Figure 2

Functional enrichment analyses of DEGs between MC3T3-E1 cells with and without osteoblast induction. (A) Significant enrichment of DEGs in the BP, CC, and MF categories (the top 15 GO terms for each category are listed). (B) Pathways significantly enriched by the DEGs in the KEGG database; red indicates small p-value, and blue indicates large p-value; the size of the bubbles indicates the degree of enrichment, and larger bubbles reflect larger generation.

Figure 3

Identification of microRNA (miRNA)-DEG regulatory network during osteogenic differentiation of MC3T3-E1 cells. (A) Overlapped putative miRNAs targeting DEGs among the TargetScan, miRTarBase, miRDB, and miRanda databases. (B) Construction of protein-protein interaction (PPI) network.

Figure 4

The mRNA expressions and cellular levels of osteoblast markers, ALP, OSX, OCN, and Runx2, were determined by RT-qPCR (A) and ELISA methods (B) in MC3T3-E1 cells with osteoblast induction and undifferentiated MC3T3-E1 cells. The *p < 0.05 compared with undifferentiated MC3T3-E1 cells. All measurement data, showing as mean ± standard deviation (SD), are representative of three independent experiments and analyzed by t-test.

Figure 5

The expressions of miR-204-5p, miR-211-5p, miR-24-3p, miR-3470b, miR-466b-3p, miR-466c-3p (A), mRNA expressions of Arhgap11a, H2afx, Chek2, Dlgap5, and Chek1 (B) were determined by RT-qPCR in MC3T3-E1 cells with osteoblast induction and undifferentiated MC3T3-E1 cells. (C), Immunoblots of Arhgap11a, H2afx, Chek2, Dlgap5, and Chek1 proteins, normalized to GADPH, in MC3T3-E1 cells with osteoblast induction and undifferentiated MC3T3-E1 cells. The *p < 0.05 compared with undifferentiated MC3T3-E1 cells. All measurement data, showing as mean ± SD, are representative of three independent experiments and analyzed by t-test.

Figure 6

MiR-211-5p targeting Arhgap11a was involved in the osteogenic differentiation of MC3T3-E1 cells. (A) Dual-luciferase reporter gene assays were performed to confirm miR-211-5p-Arhgap11a, miR-3470b-Chek2, miR-3470b-Dlgap5, and miR-466-Chek1 interactions. (B) RT-qPCR was performed to determine mRNA expressions of miR-211-5p and Arhgap11a in MC3T3-E1 cells undergoing 14-day osteoblast differentiation after miR-211-5p mimic and inhibitor transfections. (C) Immunoblots of Arhgap11a, normalized to GADPH, in MC3T3-E1 cells undergoing 14-day osteoblast differentiation after miR-211-5p mimic and inhibitor transfections. (D) The cellular levels of osteoblast markers, ALP, OSX, OCN, and Runx2 were determined by ELISA methods in MC3T3-E1 cells undergoing 14-day osteoblast differentiation after miR-211-5p mimic and inhibitor transfections. All measurement data, showing as mean ± standard deviation, are representative of three independent experiments and analyzed by t-test. *Means P < 0.05.