miR-29c-3p targeted dishevelled 2 on osteogenesis differentiation of rat bone marrow mesenchymal stem cells in high-fat environment
10.3760/cma.j.issn.1002-0098.2018.10.009
- VernacularTitle: 高脂环境下微RNA-29c-3p靶向调节蓬乱蛋白2对大鼠骨髓间充质干细胞成骨分化的影响
- Author:
Feilong WANG
1
;
Duoduo LI
2
;
Xin HUANG
1
;
Fei LIU
1
;
Qibo LIU
1
;
Jing LAN
1
Author Information
1. Department of Prosthodontics, School of Stomatology, Shandong University & Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Jinan 250012, China
2. Department of Prosthodontics, School of Stomatology, Shandong University & Shandong Provincial Key Laboratory of Oral Tissue Regeneration, Jinan 250012, China (Present address: Department of Prosthodontics, Stomatological Hospital of Tai'an, Tai'an Shandong 271000, China)
- Publication Type:Journal Article
- Keywords:
MicroRNAs;
Bone marrow;
Mesenchymal stem cells;
Dishevelled 2
- From:Chinese Journal of Stomatology
2018;53(10):694-700
- CountryChina
- Language:Chinese
-
Abstract:
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.
