MicroRNA-29a-3p regulates osteoblast differentiation and peri-implant osseointegration in a rat model of hyperlipidemia by modulating Frizzled 4 expression

doi:10.7518/hxkq.2019.02.013

Abstract

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 environ-ment. 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. Conclusion 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.

Key words: microRNA-29a-3p, hyperlipidemia, Frizzled 4, dual luciferase reporter gene, osteointegration

CLC Number:

R783

Fei Liu,Zhifeng Wang,Fangfang Liu,Jinzhao Xu,Qibo Liu,Jing Lan. MicroRNA-29a-3p regulates osteoblast differentiation and peri-implant osseointegration in a rat model of hyperlipidemia by modulating Frizzled 4 expression[J]. West China Journal of Stomatology, 2019, 37(2): 200-207.

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引物名称	引物序列
ALP上游引物	5’-TGAGCGACACGGACAAGAAG-3’
ALP下游引物	5’-GCCTGGTAGTTGTTGTGAGCAT-3’
Runx2上游引物	5’-CACAAGTGCGGTGCAAACTT-3’
Runx2下游引物	5’-AATGACTCGGTTGGTCTCGG-3’
Fzd4上游引物	5’-GGAAGGACCAGGTGACGAAG-3’
Fzd4下游引物	5’-GGAATATGATGGGGCGCTCA-3’
mmu-miR-29a-3p	5’-UAACCGAUUUCAAAUGGUGCUA-3’
GAPDH上游引物	5’-TGATGGGTGTGAACCACGAG-3’
GAPDH下游引物	5’-CCCTTCCACGATGCCAAAGT-3’

血脂指标	对照组	实验组
TC	1.25±0.22	3.25±0.45*
TG	0.73±0.12	1.29±0.24*
HDL	0.91±0.16	1.21±0.95
LDL	0.29±0.06	0.65±0.12*