Fig 1.
The NAFLD Dataset and GSE135251 Dataset Were Processed in Batches.
A-b. boxplot of NAFLD dataset before (A) and after (B) batch effect removal treatment. C-d. PCA plot of NAFLD dataset before (C) and after (D) batch effect removal treatment. E-f. boxplot plot of dataset GSE135251 before (E) and after (F) removal of batch effect treatment. PCA: Principal Component Analysis, NAFLD: Nonalcoholic fatty liver disease.
Fig 2.
Differential Analysis of NAFLD Dataset and Dataset GSE135251.
A. Volcano plot of differentially expressed genes analysis of NAFLD dataset. B. Volcano plot of differentially expressed genes analysis of non-alcoholic fatty liver disease group (NAFLD) relative to normal Control group (Control) in dataset GSE135251. C. Venn diagram of differentially expressed genes in NAFLD dataset and differentially expressed genes in dataset GSE135251. D. Venn diagram of common differentially expressed genes (DEGs) and m6A related genes (m6A). E. Differential expression analysis of m6A-RDEGs in NAFLD dataset. F. Differential expression analysis of m6A-RDEGs in dataset GSE135251. G. Simple numerical heatmap of m6A-RDEGs in the NAFLD dataset. H. Simple numerical heat map of m6A-RDEGs in dataset GSE135251.
Fig 3.
Functional Enrichment Analysis (GO) of M6A-Related Differentially Expressed Genes.
A. GO functional enrichment analysis of m6A-related differentially expressed genes BP, CC, MF bar chart display. B-d. GO functional enrichment analysis results of m6A related differentially expressed genes BP(B), CC(C), MF(D) network diagram display. E. Bubble plot display of GO enrichment analysis results of m6A-related differentially expressed genes combined with logFC. In bubble plot (E), blue circles represent BP pathways, yellow circles represent CC pathways, and purple circles represent MF pathways. In the network diagram (B-D), yellow dots represent specific pathways and blue dots represent specific genes. The screening criteria for GO enrichment items were p. Adj < 0.05 and FDR value (q.value) < 0.05.
Fig 4.
WGCNA Analysis Identified Co-Expression Modules in NAFLD Dataset.
A. Demonstration of WGCNA analysis clustering of NAFLD dataset samples in dataset. B. Scale-free network display of sample module screening threshold in NAFLD dataset. C. Display of gene module clustering results in NAFLD dataset. D. The corresponding relationship between genes and modules in NAFLD dataset is displayed. E. The results of correlation analysis between NAFLD dataset gene cluster module and different groups are displayed. F-h. Venn diagram of NAFLD dataset M6A-related differentially expressed genes (m6A-RDEGs) with MEblue (F), MEturquoise (G) and MEyellow (H) module genes. WGCNA: Weighted gene co-expression network analysis. M6a-rdegs: m6A related differentially expressed genes. NAFLD: Nonalcoholic fatty liver disease.
Fig 5.
Key Genes Screening and LASSO Risk Model Construction.
A. The number of genes with the lowest error rate obtained by SVM algorithm. B. The number of genes with the highest accuracy obtained by the SVM algorithm. C. Diagnostic model diagram of LASSO regression model. D. Variable trajectory plot of LASSO regression model. E. Forest plot of key genes in LASSO regression model. F. Nomogram of key genes in NAFLD diagnostic model based on NAFLD dataset. G. Decision curve analysis (DCA) plot of key genes of nonalcoholic fatty liver disease (NAFLD) diagnostic model based on NAFLD dataset. H. Nomogram of key genes in non-alcoholic fatty liver disease (NAFLD) diagnosis model based on dataset GSE135251. I. Decision curve analysis (DCA) plot of key genes of non-alcoholic fatty liver disease (NAFLD) diagnostic model based on dataset GSE135251. SVM: Support Vector Machine. M6a-rdegs: m6A related differentially expressed genes, NAFLD: Nonalcoholic fatty liver disease, LASSO, Least Absolute Shrinkage and Selection Operator.
Fig 6.
Differential Analysis of ssGSEA Immune Characteristics of NAFLD Dataset.
A. The results of ssGSEA immune infiltration analysis between the NAFLD group and the normal group in the NAFLD dataset were shown. B. The results of correlation analysis of immune cell infiltration abundance in NAFLD dataset are presented. C. Dot plot of correlation between immune cells and key genes in NAFLD dataset. The symbol ns is equivalent to P ≥ 0.05, which is not statistically significant; The symbol * is equivalent to P < 0.05 and statistically significant. ssGSEA, single-sample gene-set enrichment Analysis. NAFLD: Nonalcoholic fatty liver disease.
Fig 7.
Construction of m6A Phenotype Score and Correlation Analysis of Key Genes.
A. Group comparison figure of key genes between NAFLD dataset m6A phenotype score Low group and High group. B. Group comparison diagram of key genes between Low group and High group of m6A phenotype score in GSE135251 dataset. C-g. ROC curve results of key genes RBM15 (C), IGF2BP2 (D), EIF3B (E), YTHDC1 (F), WTAP (G) in NAFLD dataset samples with m6A phenotype score Low group and High group are shown. H-l. The ROC curve results of key genes RBM15 (H), IGF2BP2 (I), EIF3B (J), YTHDC1 (K), WTAP (L) in the non-alcoholic fatty liver disease group samples with m6A phenotype score Low group and High group in GSE135251 dataset were shown. The closer the AUC in the ROC curve is to 1, the better the diagnostic effect is. When AUC was between 0.5 and 0.7, the accuracy was low. When AUC was 0.7-0.9, it had a certain accuracy. AUC > 0.9 had high accuracy.
Fig 8.
Construct mRNA-miRNA, mRNA-RBP, mRNA-TF and mRNA-drugs Interaction Networks.
A. The orange dots in the mRNA-miRNA interaction network of key genes are mrnas; Yellow dots are mirnas. B. The orange oval blocks in the mRNA-RBP interaction network of key genes are mrnas; Blue diamonds are RBP; The yellow oval blocks are both mRNA and RBP. C. The orange oval block in the mRNA-TF interaction network of key genes is mRNA; Purple hexagonal blocks are transcription factors (TFS). D. The orange oval blocks in the mRNA-drugs interaction network of key genes are mrnas; Green triangles are specific molecular compounds (drugs). RBP: RNA binding protein; TF: Transcription factors.
Fig 9.
High-Fat Diet-Induced Mouse Model of Non-Alcoholic Fatty Liver Disease (NAFLD)
(A) Body weight gain. (B) Liver TG content. (C) Liver TC content, (D) Liver AST content. (E) Liver ALT content. n=6 in each group. (F) H&E-stained liver tissues(X200). (G) Representative Oil Red O staining of mouse livers in each group (X200). *p < 0.05, **p < 0.01 and ***p < 0.001 compared with the control group (CON).
Fig 10.
Verification of The 5 Dignostic Hubgenes Using Real-Time Quantitative PCR (NAFLD samples= 6, control samples = ).
*p < 0.05, **p < 0.01 and ***p < 0.001.
Fig 11.
Using Western Blotting to Validate 5 Significantly Changed Genes (NAFLD Samples= 6, Control Samples = 6).
*p < 0.05, **p < 0.01 and ***p < 0.001.