Mettl3-mediated m6A RNA methylation regulates the fate of bone marrow mesenchymal stem cells and osteoporosis

Nat. commun | Mettl3-mediated m6A RNA methylation regulates the fate of bone marrow mesenchymal stem cells and osteoporosis

Article introduction:
Recently, Zhou Xuedong and Yuan Quan, from the West China Hospital of Sichuan University, and the Lin Shuibin team of the First Affiliated Hospital of Sun Yat-sen University jointly developed a new study on Mettl3-mediated methylation of m6A RNA to regulate the fate of bone marrow mesenchymal stem cells and osteoporosis. mechanism. The study, titled Mettl3-mediated m6A RNA methylation regulates the fate of bone marrow mesenchymal stem cells and osteoporosis, was published online November 14 in Nature Communications (IF: 12.353).

Research ideas
The research team first constructed a conditional knockout of MSCs and knocked into Mettl3 model mice using CRISPR-Cas9 technology. Micro-CT and staining showed knockdown resulted in low bone mass and high bone marrow fat, and m6A overall methylation in MSCs. At the same time, Mettl3 overexpression prevented osteoporosis caused by estrogen deficiency compared with model rats who underwent oophorectomy; Mettl3 deletion was found by m6A MeRIP-seq and RNA-seq techniques. The global down-regulation of PTH-regulated gene expression in MSCs suggests that the PTH/Pth1r signal axis may be a pathway in which Mettl3-mediated methylation of m6A affects the fate of MSCs. The PTH intermittent injection experiment also confirmed the conclusion that m6A affects the osteogenesis and fat differentiation of MSCs by regulating the PTH/Pth1r signal axis. In addition, overexpression of Pth1r can largely reverse the oblique differentiation of MSCs into adipocytes and improve osteogenesis, further confirming that PTH/Pth1r signaling pathway is a new mechanism of mettl3-mediated m6A modification to control MSCs lineage distribution.

research content
1. Mettl3 knockout in MSCs can lead to low bone mass, high bone marrow fat <br> Using CRISPR-Cas9 technology to construct MSCs conditionally knockout Mettl3 model mice, femoral paraffin section immunostaining and WB validate Mettl3 Knockout is successful. The results showed that Mettl3 knockout mice had significant bone mass loss and accumulation of bone adipose tissue compared with the control group, and their skeletal characteristics were similar to those of osteoporosis. At the same time, LC-MS/MS detection showed that the overall level of m6A methylation in MSCs decreased.

2. Deletion of Mettl3 in MSCs leads to decreased osteogenic potential and increased adipogenic differentiation <br> Next, MSCs were isolated from Mettl3 knockout mice to verify their in vitro osteogenesis and adipogenic potential. Alkaline phosphatase staining activity was attenuated, calcification was reduced, and osteogenic markers Runx2, Sp7, Alp and Bglap were down-regulated, indicating that the missing MSCs were not osteogenic. In contrast, an increase in the intensity of oil red O staining and a significant high expression are potential factors for fat formation, including Pparγ, Cebpα, Adipoq, Plin1 and CD36.

3. Mettl3 overexpression prevents osteoporosis caused by estrogen deficiency <br> To confirm whether Mettl3 overexpression can enhance bone health or prevent bone disease, conditionally knocked Mettl3 into MSCs in MSCs. Overexpression. Osteoporosis experimental model was established by oophorectomy. Compared with the control, Mettl3 overexpression increased the number of osteoblasts in ovariectomized mice, but did not affect osteoclast activity. H&E staining and histomorphometric analysis showed that the ovariectomized mice had excessive accumulation of bone marrow adipocytes, whereas the ovariectomized Mettl3 knock-in mice had a smaller increase in bone marrow adipocytes.

4. m6A regulates the translation of parathyroid hormone receptor-1 (Pth1r) <br> The m6A MeRIP-seq technique is used to analyze the specific situation of m6A in MSCs. The results show that the m6A peak is mainly located in the CDS and 3'UTR regions, and mainly Located near the stop codon. Pth1r was also found to be a key regulator of lineage distribution in MSCs and osteoblast precursors, with high enrichment and specific m6A peaks near its translation stop codon. RNA-seq revealed a global down-regulation of parathyroid hormone (PTH)-regulated gene expression in Mettl3-deficient MSCs, suggesting that the PTH/Pth1r signal axis may be a pathway in which Mettl3-mediated m6A methylation affects the fate of MSCs.

5. m6A is essential for PTH function
Mettl3 knockout mice showed no significant enhancement of bone remodeling after PTH treatment, demonstrating that knockdown of Mettl3 attenuates PTH-induced osteogenesis. In addition to being significantly enriched during osteogenesis, PTH also inhibits adipocyte differentiation and promotes bone formation. Intermittent injection of PTH significantly reduced the accumulation of MAT in Mettl3 knockout mice, but it was difficult to reverse the higher bone marrow fat content in Mettl3-deficient mice; Pth1r overexpression improved the intensity of oil red O staining caused by m6A reduction, significantly Increase ALP activity and calcium nodule formation. Overexpression of Pth1r can largely reverse the oblique differentiation of MSCs into adipocytes and improve osteogenesis, further confirming that PTH/Pth1r signaling pathway is a new mechanism of mettl3-mediated m6A modification to control MSCs lineage distribution.

to sum up
Mettl3-mediated m6A RNA methylation modification in eukaryotes has a broad functional impact on stabilizing homeostasis, and any disturbance in m6A levels can lead to dysfunction or disease. Studies here have confirmed that the deletion of Mettl3 in bone marrow mesenchymal stem cells destroys the cell fate of mice, leading to a pathological phenotype of osteoporosis such as reduced bone mass of osteogenic potential and increased bone marrow fat with enhanced osteogenic potential. The effective specific regulation of m6A on bone marrow mesenchymal stem cells was revealed. In addition, the increased function of Mettl3 prevents postmenopausal osteoporosis caused by estrogen deficiency and establishes the indispensable role of Mettl3 in determining the fate of bone marrow MSCs, thus ensuring bone health. The reduction of epigenetic modification of m6A in early bone marrow mesenchymal stem cells inhibits the translation of mammalian Pth1r and blocks its synthesis of PTH during bone accumulation. These functional defects cause the delicate differentiation of MSCs to tilt toward the fat lineage, leading to severe bone loss and excessive MAT accumulation. The experimental data enriched the evidence of m6A regulating stem cell differentiation, revealing the functional link between improperly regulated m6A modification and bone pathological disorder, thus providing a possibility for the development of new strategies for osteoporosis treatment.
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