BMSC-Exo miR-122-5p facilitates osteogenic differentiation of MC3T3-E1 cells through specifically suppressing SPRY2-Reference-Cited by-同舟云学术

BMSC-Exo miR-122-5p facilitates osteogenic differentiation of MC3T3-E1 cells through specifically suppressing SPRY2

Published:2024-09-13 Issue: Volume: Page:
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Author:

Chen Zhiyu¹,Fan Yizhe¹,Yang Chengyi¹,Wang Chenhao¹,Wang Peng¹,Cheng Shaolei¹,Wang Yikai¹,You Wulin¹

Affiliation:

1. Nanjing University of Chinese Medicine Wuxi Affiliated Hospital: Wuxi Hospital of Traditional Chinese Medicine

Abstract

Background Bone Marrow Mesenchymal Stem Cells-Exosomes (BMSC-Exo) possess the ability to facilitate bone remodeling, and this mechanism has always been of great interest in the field. Our study aimed to elucidate the impacts of BMSC-Exo on MC3T3-E1, the murine embryonic osteogenic progenitor cells, and the interaction behind. Methods We initially extracted and characterized exosomes from BMSCs. Following treatment with GW4869, a compound that inhibits exosome production and release, BMSCs produced exosomes (BMSC-Exo). These were subsequently combined in culture with MC3T3-E1 cells. Upon an application of Phalloidin and PKH26 staining, we observed morphology of the cellular actin fibers and the uptake of exosomes. To evaluate the osteogenic potential of the cells, we utilized Alizarin Red S (ARS) and Alkaline Phosphatase (ALP) staining. Additionally, we measured expressions of osteogenic factors RUNX2, ALP, OSX, OCN, and OPN through qRT-PCR and Western blot analyses. Afterwards, we intervened with BMSC-Exo with a lentivirus over-expressing miR-122-5p and co-cultured it with MC3T3-E1 cells. To further assess osteogenic differentiation, we conducted additional ARS & ALP staining, along with qRT-PCR and Western blot assays. With the help of dual-luciferase reporter assay, we found that miR-122-5p interacts specifically with SPRY2. Ultimately, we treated MC3T3-E1 cells with a lentivirus over-expressing miR-122-5p and a plasmid over-expressing OE-SPRY2. Osteogenic differentiation was then assessed using ARS & ALP staining, qRT-PCR, and Western blot. Results Our laboratory outcomes demonstrated that exosomes derived from BMSC-Exo are instrumental in the advancement of calcified nodule genesis within MC3T3-E1 cells, concurrently amplifying the transcriptional and translational expressions of osteogenic markers (RUNX2, ALP, OSX, OCN, and OPN). These excreted exosomes from the BMSCs modified by a miR-122-5p-over-expressing lentivirus are found to further accelerate osteogenic differentiation of the cells. Moreover, our application of dual-luciferase reporter gene system has elucidated a specific interplay between miR-122-5p and SPRY2. Furthermore, overexpressing of SPRY2 negates the miR-122-5p-induced osteogenic differentiation. Conclusions BMSC-Exo facilitates osteogenic differentiation in MC3T3-E1 cells by suppressing SPRY2, a process mediated by miR-122-5p.

Publisher

Springer Science and Business Media LLC

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