Fig 1.
Single cell transcriptome analysis showed differences between normal and osteoporosis samples. (A) The relationship between nCount RNA and nFeature RNA of the two groups of samples. (B) The relationship between nCount_RNA and mitochondrial gene ratio in the two groups of samples. (C) The relationship between nCount RNA and ribosome gene ratio of the two groups of samples. (D) Standard deviation distribution in PCA. (E) The relationship between average and standard variance of gene expression. (F) The distribution of samples in a two-dimensional PCA plot. (G) Cell population distribution shown by PCA dimensionality reduction of all samples, normal sample, and the osteoporosis sample.
Fig 2.
Cell type clustering and trajectory analysis of scRNA data.
(A) Cell types distribution shown by t-SNE dimensionality reduction. (B) Cell types distribution as shown by UMAP dimensionality reduction. (C) Two-dimensional projections of Pseudo-time analysis of different cell types. (D) Pseudo-time analysis results. (E) The gene function classification.
Fig 3.
The MIF signal path network analysis.
(A) The heatmap of the MIF signal network. (B) The receptor and ligand interaction of CD74 and CXCR4 in the MIF signal network was shown. (C) The receptor and ligand interaction of CD74 and CD44 in the MIF signal network was shown. (D) The receptor of ACKR3 in the MIF signal network. (E) Network centrality scores of MIF are shown.
Fig 4.
The identification of DEGs and functional enrichment analysis.
(A) The volcano Plot showed DEGs between the OP samples and normal bone tissues. (B) The heatmap showed the expression levels of DEGs in different sample groups. (C) The Venn Diagram showed the overlap genes of oxidative stress-related genes (OS) and DEGs. GO (D) and KEGG (E) pathway functional enrichment analysis of DEGs.
Fig 5.
Cell components and correlation analysis.
(A) The relative percentage of different immune cell types in the treat and control group. (B) The LASSO coefficient distribution. (C) The cross-validation curves. (D) The ROC curve of CHRM2 to distinguish the osteoporosis from the control group. (E) The GSVA functional enrichment analysis.
Fig 6.
The expression levels of CHRM2 and OGD-related factors at 0, 7, 14 and 21 days after OGD induction.
The ALP staining (A), alizarin red staining (B), western blot (C) showed protein levels of CHRM2 and OGD-related factors (OPN and RUNX2) at 0, 7, 14, and 21 days after OGD induction, normalized with GAPDH. (D) The mRNA expression levels of OGD-related factors (OPN and RUNX2) was detected by RT-qPCR and normalized with GAPDH. The results were shown as mean ±SD. *p < 0.05; **p < 0.01; ***p < 0.001; ns: no significance.
Fig 7.
The effects of CHRM2 knockdown on osteogenic differentiation and cell proliferation.
(A) The expression of CHRM2 was significantly reduced in cells knocked down with Si-CHRM2. (B) The RT-qPCR results showed that the expression of CHRM2 in cells with Si-CHRM2 knockdown decreased significantly. (C) The cell proliferation were measured by OD value at 450 nm, and the observation time points were at 0, 24, 48, 72 and 96 hours. (D) The protein expression levels of osteogenic markers RUNX2, OPN, and Osterix in Si-CHRM2 knockdown cells and control cells by western blot. (E) Flow cytometry of cell cycle in the control group and the experimental group. (F) ALP staining in the Si-CHRM2 experimental group. (G) Alizarin red staining. *p < 0.05; **p < 0.01; ***p < 0.001; ns: no significance.
Fig 8.
CHRM2 binding mRNA expression profile.
(A) Box plot of FPKM values showing gene expression levels between BMSC1-IP and BMSC1-input groups. (B) Violin plots of FPKM values further demonstrated the distribution of gene expression between BMSC1-IP and BMSC1-input groups. (C) The heat map shows the correlation between BMSC1-IP and BMSC1-input samples. *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 9.
GO functional annotation and KEGG pathway enrichment analysis of DEGs.
BP (A), CC (B), and MF (C) enrichment analysis of GO in DEGs. (D) KEGG pathway enrichment analysis results of DEGs. (E) Thesignificant DEGs between the IgG and IP group were shown, which indicated the highest log2FC of COL4A2. *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 10.
Effect of CHRM2 knockdown on the COL4A2 expression level.
(A) The protein expression level of COL4A2 in the Si-CHRM2 and control cells. (B) The results of RT-qPCR showed the expression of COL4A2 mRNA in si-CHRM2 knockout at different time points (0 h, 6 h, 12 h). (C) Immunofluorescence staining images showed COL4A2 (red) staining results at different time points (0 h, 6 h, 12 h) in the control and Si-CHRM2 groups. *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 11.
The expression level of COL4A2 at different periods and the effect of COL4A2 knockdown on cells.
(A) The protein expression level of COL4A in the OGD model at 0, 7, 14, and 21 days. (B) The mRNA expression levels of COL4A2 at different time points of OGD (0, 7, 14, and 21 days); (C) The protein expression levels of COL4A2 and CHRM2 in the cells of the control group and the Si-COL4A2 group. (D) The relative mRNA expression level of CHRM2 in the control and si-COL4A2 experimental groups. (E) Immunofluorescence staining images showed the expression and localization of CHRM2 (green) and COL4A2 (red) proteins in cells of the control and si-COL4A2 groups, while DAPI (blue) was used for nuclear staining. (F) FISH and IF images further showed the expression changes of CHRM2 mRNA and COL4A2 mRNA. *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 12.
Effect of COL4A2 knockdown on osteogenic differentiation and cell proliferation.
(A) The expression levels of Osterix, RUNX2, and OPN in the control and si-COL4A2 groups by western blot. (B) The mRNA expression levels of OPN and RUNX2 in the control group and the si-COL4A2 groups. (C) Cell proliferation assay (CCK-8) showed OD values at 450 nm at different time points (0, 24, 48, 72 and 96 hours) in the control group and the si-COL4A2 group. (D) The cell cycle distribution in the control and si-COL4A2 groups was analyzed by flow cytometry. (E) ALP staining in the control and si-COL4A2 group. (F) The alizarin red staining showed mineralized nodules formation in the control and si-COL4A2 groups. *p < 0.05; **p < 0.01; ***p < 0.001.