Identification of key genes involved in collagen hydrogel-induced chondrogenic differentiation of mesenchymal stem cells through transcriptome analysis: the role of m6A modification

Abstract

Collagen hydrogel has been shown promise as an inducer for chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), contributing to the repair of cartilage defects. However, the precise molecular mechanism underlying this phenomenon remains poorly elucidated. Here, we induced chondrogenic differentiation of BMSCs using collagen hydrogel and identified 4451 differentially expressed genes (DEGs) through transcriptomic sequencing. Our analysis revealed that DEGs were enriched in the focal adhesion pathway, with a notable decrease in expression levels in the collagen hydrogel group compared to the control group. Protein-protein interaction network analysis suggested that actinin alpha 1 (ACTN1) and actinin alpha 4 (ACTN4), two proteins also involved in cytoskeletal recombination, may be crucial in collagen hydrogel-induced chondrogenic differentiation of BMSCs. Additionally, we found that N6-methyladenosine RNA methylation (m6A) modification was involved in collagen hydrogel-mediated chondrogenic differentiation, with fat mass and obesity-associated protein (FTO) implicated in regulating the expression of ACTN1 and ACTN4. These findings suggest that collagen hydrogel might regulate focal adhesion and actin cytoskeletal signaling pathways through down-regulation of ACTN1 and ACTN4 mRNA via FTO-mediated m6A modification, ultimately driving chondrogenic differentiation of BMSCs. In conclusion, our study provides valuable insights into the molecular mechanisms of collagen hydrogel-induced chondrogenic differentiation of BMSCs, which may aid in developing more effective strategies for cartilage regeneration.

Fig. 1

Volcano plot of screening differential expressed genes (DEGs). A Analysis of DEGs in C7 vs. B7 group; (B) Analysis of DEGs in C14 vs. B14 group; (C) Analysis of DEGs in T7 vs. B7 group; (D) Analysis of DEGs in T14 vs. B14 group. C: collagen hydrogel group, B: Blank control group, T: TGF‐β group

Fig. 2

Venn diagram illustrating the differentially expressed genes (DEGs) comparison between 7 days and 14 days. A DEGs comparison between C7 vs. B7 and C14 vs. B14 groups using the collagen hydrogel treatment; (B) DEGs comparison between T7 vs. B7 and T14 vs. B14 groups using TGF-β treatment. C Collagen hydrogel group, B: Blank control group, T: TGF-β group

Fig. 3

Gene ontology (GO) and KEGG pathway enrichment analysis for differentially regulated genes. (A) GO enrichment of DEGs in collagen hydrogel vs. Blank control group; (B) GO enrichment of DEGs in TGF‐β vs. Blank control group; (C) KEGG pathway enrichment of DEGs in collagen hydrogel vs. Blank control group; (D) KEGG pathway enrichment of DEGs in TGF‐β1 vs. Blank control group

Fig. 4

Analysis of key signaling pathway genes. A Gene expression levels in the focal adhesion signaling pathway; (B) Protein-protein interaction (PPI) network analysis for the genes involved in the focal adhesion signaling pathway

Fig. 5

Correlation of 28 N6-Methyladenosine modifiers. A Gene expression levels of 28 modulators involved in N6-Methyladenosine RNA methylation; (B) The correlation among 28 m6A modification regulators; (C) The correlation of two essential genes and 28 m6A modification regulators; (D) The correlation analysis between FTO and ACTN1; (E) The correlation analysis between FTO and ACTN4; (F) The correlation analysis between ACTN1 and ACTN4

Fig. 6

Prediction of m6A site(s) for key genes. (A) Prediction of m6A sites in the ACTN1 gene using SRAMP. (B) Prediction of m6A sites in the ACTN1 gene using SRAMP

Fig. 7

Verification of expression levels of key genes and key m6A modification regulator. A RT-qPCR analysis confirmed the expression levels of ACTN1. B RT-qPCR analysis confirmed the expression levels of ACTN4. C RT-qPCR analysis confirmed the expression levels of FTO. These data represent the means ± SD of three independent experiments; *p < 0.05, **p < 0.01, and ***p < 0.001. D Western blot analysis was used to measure the protein levels of ACTN1, ACTN4, and FTO. Representative images from one of three independent experiments are shown, and the quantitative data are presented as the means ± SD; *p < 0.05, **p < 0.01, and ***p < 0.001