Mechanisms of miR‑128‑3p in inhibiting osteoblast differentiation from bone marrow‑derived mesenchymal stromal cells.

The authors' previous study demonstrated that miR‑128 may exert an inhibitory effect on the osteogenic differentiation of bone marrow‑derived mesenchymal stem cells (BM‑MSCs), but its downstream mechanisms remain to be elucidated. The aim of the present study was to investigate the microRNA (miRNA/miR) and mRNA profiles of differentiated and undifferentiated BM‑MSCs and explore new downstream targets for miR‑128. The sequencing datasets of GSE107279 (miRNA) and GSE112318 (mRNA) were downloaded from the Gene Expression Omnibus database. The differentially expressed miRNAs (DEMs) and genes (DEGs) were identified using the DESeq2 method. The target genes of DEMs were predicted by the miRwalk 2.0 database. The hub target genes of miR‑128 were screened by constructing the protein‑protein interaction (PPI) network and module analysis. The expression levels of miR‑128 and crucial target genes were validated by reverse transcription‑quantitative (RT‑q) PCR before or after transfection of miR‑128 mimics to BM‑MSCs. The miRNA expression profile analysis identified miR‑128 as one of the significantly downregulated DEMs (total 338) in differentiated BM‑MSCs compared with the undifferentiated control. A total of 103 predicted target genes of miR‑128‑3p were overlapped with upregulated DEGs. By calculating the topological properties of each protein in the PPI network, 6 upregulated genes (KIT, NTRK2, YWHAB, GAB1, AXIN1 and RUNX1; fold change was the highest for NTRK2) were considered to be hub genes. Of these, 4 were enriched in module 4 (RUNX1, KIT, GAB1 and AXIN1; RUNX1 was particularly crucial as it can interact with the others), while one was enriched in module 7 (YWHAB). The expression levels of miR‑128 and these 6 target genes during the osteogenic differentiation were experimentally confirmed by RT‑qPCR. In addition, the expression levels of these 6 genes were significantly reversed after transfection of miR‑128‑3p mimics into rat BM‑MSCs compared with the miR‑control group. These findings indicated that miR‑128‑3p may inhibit the osteoblast differentiation of BM‑MSCs by downregulation of these 6 genes, particularly RUNX1, YWHAB and NTRK2.