ABSTRACT
OBJECTIVE@#This work aimed to study and identify the influence and target gene of microRNA-29a-3p (miR-29a-3p) in the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) in a high-fat environment in vitro and in vivo.@*METHODS@#1) In vitro: BMSCs were randomly allocated into two groups and were then induced to undergo osteogenic differentiation in a normal or high-fat environment. Next, a miR-29a-3p mimic/inhibitor was transfected into the two groups of cells. The mRNA expression levels of alkaline phosphatase (ALP), Runt related gene 2 (Runx2), and miR-29a-3p and the protein expression levels of ALP and Runx2 were detected before and after transfection through reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) and Western blot analyses. Moreover, Frizzled (Fzd) 4 was predicted as the target gene of miR-29a-3p by using an online database (Target Scan, MiRNA.org). The interactive relationship between miR-29a-3p and Fzd4 was confirmed through dual-luciferase assays. 2) In vivo: Rats were randomly divided into two groups and fed with a standard or high-fat diet. Titanium implants were grown in rats. Then, the expression levels of miR-29a-3p, ALP, and Runx2 were detected in bone tissues surrounding implants. Moreover, hard tissue sections were subjected to methylene blue-acid magenta staining and observed under microscopy to study bone formation around implants. In addition, miR-29a-3p-overexpressing lentiviral vectors were transfected into rats, and the expression levels of ALP, Runx2, and miR-29a-3p in bone tissues surrounding implants were detected at 3 and 10 days after transfection.@*RESULTS@#The expression levels of ALP, Runx2, and miR-29a-3p and the osteogenic differentiation of BMSCs were suppressed in high-fat groups in vitro and in vivo.@*CONCLUSIONS@#MiR-29a-3p plays a positive role in the regulation of BMSCs in a high-fat environment. It can increase ALP and Runx2 expression levels in bone tissues surrounding implants in hyperlipidemia models. This result implies that miR-29a-3p can promote implant osseointergration in a rat model of hyperlipidemia.
Subject(s)
Animals , Rats , Cell Differentiation , Dental Implants , Hyperlipidemias , MicroRNAs , Osseointegration , Osteoblasts , Osteogenesis , Random AllocationABSTRACT
Objective@#To study the different expression of 4 microRNA (miRNA, miR) during the osteogenesis differentiation of bone marrow mesenchymal stem cell (BMSC) cultured in high-fat or normal environment and to explore the relationship of these miRNAs with disheveled 2 during osteogenesis differentiation.@*Methods@#BMSC were cultured with 2 ml normal osteogenic induction (control group) and high-fat osteogenic induction (high-fat group) respectively. On the 3rd, 5th, 7th,14th, 21st day, quantitative real-time PCR (qPCR) was used to analyze expression levels of four miRNAs (miR-21-5p, miR-29c-3p, miR-138-5p and miR-351-5p), mRNA of disheveled 2, osteogenic related factors such as alkaline phosphatase (ALP), Runt-related transcription gene 2 (Runx2). And the protein was detected by Western blotting. After BMSC were transfected by 50 μl 50 nmol/L miRNA mimics/inhibitors/negative controls respectively, BMSC were put on osteogenic induction, on the 1st, 3rd, 5th, 7th day, ALP activity was detected. On the 7th day, ALP staining was to observe the degree of osteogenesis differentiation, and Western blotting was adopted to analyze the expression of dishevelled 2 and other osteogenic related factors, while qPCR was used to analyze the expression of disheveled 2 mRNA. After 293T cells were co-transfected with disheveled 2 wild-type/mutant firefly luciferase reporter plasmid with either negative control (NC) or a mimic of these four miRNAs respectively for 48 h, luciferase activities were measured.@*Results@#On the 21th day, the expressions of miR-21-5p, miR-29c-3p, miR-138-5p and miR-351-5p in high-fat groups were higher by 20%, 60%, 340% and 4 420% respectively than those in control groups (P<0.05). The expression of ALP and Runx2 in BMSC decreased after BMSC transfected miR-21-5p and miR-29c-3p mimics, while increased after transfected miR-21-5p and miR-29c-3p inhibitors. The expression of disheveled 2 decreased by 35% after transfected by miR-29c-3p mimic, while it increased by 269% after transfected by miR-29c-3p inhibitor (P<0.05). Transfection of the miR-29c-3p mimics significantly decreased the luciferase activity of wild-type 3'-UTR compared with NC control (P<0.05). There were no statistical significances among other groups.@*Conclusions@#miRNAs had better expression during osteogenesis differentiation of BMSC in high-fat environment; miR-29c-3p could negatively regulate the osteogenesis differentiation of BMSC by targets on dishevelled 2.