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Effects of inhibiting PDK?1 expression in bone marrow mesenchymal stem cells on osteoblast differentiation in vitro.

ABSTRACT: Osteoblasts are the main functional cells in bone formation, which are responsible for the synthesis, secretion and mineralization of bone matrix. The PI3K/AKT signaling pathway is strongly associated with the differentiation and survival of osteoblasts. The 3?phosphoinositide?dependent protein kinase?1 (PDK?1) protein is considered the master upstream lipid kinase of the PI3K/AKT cascade. The present study aimed to investigate the role of PDK?1 in the process of mouse osteoblast differentiation in vitro. In the BX?912 group, BX?912, a specific inhibitor of PDK?1, was added to osteoblast induction medium (OBM) to treat bone marrow mesenchymal stem cells (BMSCs), whereas the control group was treated with OBM alone. Homozygote PDK1^flox/flox mice were designed and generated, and were used to obtain BMSCs^{PDK1flox/flox}. Subsequently, an adenovirus containing Cre recombinase enzyme (pHBAd?cre?EGFP) was used to disrupt the PDK?1 gene in BMSCs^{PDK1flox/flox}; this served as the pHBAd?cre?EGFP group and the efficiency of the disruption was verified. Western blot analysis demonstrated that the protein expression levels of phosphorylated (p)?PDK1 and p?AKT were gradually increased during the osteoblast differentiation process. Notably, BX?912 treatment and disruption of the PDK?1 gene with pHBAd?cre?EGFP effectively reduced the number of alkaline phosphatase (ALP)?positive cells and the optical density value of ALP activity, as well as the formation of cell mineralization. The mRNA expression levels of PDK?1 in the pHBAd?cre?EGFP group were significantly downregulated compared with those in the empty vector virus group on days 3?7. The mRNA expression levels of the osteoblast?related genes RUNX2, osteocalcin and collagen I were significantly decreased in the BX?912 and pHBAd?cre?EGFP groups on days 7 and 21 compared with those in the control and empty vector virus groups. Overall, the results indicated that BX?912 and disruption of the PDK?1 gene in vitro significantly inhibited the differentiation and maturation of osteoblasts. These experimental results provided an experimental and theoretical basis for the role of PDK?1 in osteoblasts.

SUBMITTER: Bai Y

PROVIDER: S-EPMC7751487 | biostudies-literature | 2021 Feb

REPOSITORIES: biostudies-literature

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Effects of inhibiting PDK‑1 expression in bone marrow mesenchymal stem cells on osteoblast differentiation <i>in vitro</i>.

Bai Yiguang Y Zhang Qiong Q Zhou Quan Q Zhang Yanan Y Nong Haibin H Liu Mingfu M Shi Zhuohua Z Zeng Gaofeng G Zong Shaohui S

Molecular medicine reports 20201210 2

Osteoblasts are the main functional cells in bone formation, which are responsible for the synthesis, secretion and mineralization of bone matrix. The PI3K/AKT signaling pathway is strongly associated with the differentiation and survival of osteoblasts. The 3‑phosphoinositide‑dependent protein kinase‑1 (PDK‑1) protein is considered the master upstream lipid kinase of the PI3K/AKT cascade. The present study aimed to investigate the role of PDK‑1 in the process of mouse osteoblast differentiation ...[more]

PMID: 33300048

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Project description: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.

Dataset Information

Effects of inhibiting PDK?1 expression in bone marrow mesenchymal stem cells on osteoblast differentiation in vitro.

Publications

Effects of inhibiting PDK‑1 expression in bone marrow mesenchymal stem cells on osteoblast differentiation <i>in vitro</i>.

Similar Datasets

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