Identification of DEGs in NAFLD-related HCC
The DEGs among HC, NASH, and NAFLD-related HCC in GSE164760 dataset were identified, respectively (Figure 2A-C). Next, the intersection among the three groups represented 1334 DEGs associated with disease progression (Figure 2D). These results illustrated that the cross DEGs had effects on the pathogenesis from NASH to NAFLD-related HCC.
Identification of Trait-related Genes by WGCNA
WGCNA was performed to identify key modules related to clinical traits in GSE164760 dataset. (Figure 3A). The power of β = 6 (scale-free R2=0.87) was selected as the soft thresholding parameter to construct a scale-free network (Figure 3B). Similar module clustering was constructed by using dynamic hybrid cutting (threshold = 0.25, Figure 3C, Supplementary Table S1-2). A total of 19 modules were identified (Figure 2D). The results in Figure 3D showed that the red module was the highest correlation module to NASH (NAS, R2 = 0.67, p < 1e-200) and HCC (R2 = 0.60, p= 3.1e-116). Figure 3E-F showed gene significance for NASH and HCC in red modules. Then, 116 trait-related genes obtained from WGCNA, 1334 DEGs from GSE164760 dataset, and survival-related genes from TCGA database were intersected, and finally 42 trait-survival-related genes were selected for further analysis. The results indicated that these genes were not only highly associated with NAFLD-related HCC but also with prognosis.
Construction of LASSO Logistic Regression and Diagnostic Value of TMEM126A
To further filtrate key genes, LASSO logistic regression was performed based on 42 trait-survival-related genes in TCGA database, and 8 hub genes were screened out for further analysis (Figure 4A, Supplementary Table S3). Figure 4B showed the risk score of selected genes(Supplementary Table S4). Subsequently, we chose genes with the top 3 risk score to construct the ROC curve, and TMEM126A had the highest diagnostic value compared with SRRT and GPR180. The area under curve (AUC) of TMEM126A was 0.866 showing favorable practical functions for the diagnosis (Figure 4C). Considering TMEM126A had the most diagnosis value and the highest risk score, it was chosen for further exploration. Figure 4D-E demonstrated that high TMEM126A expression was significantly correlated with overall survival (OS, p = 0.002) rate and disease-free survival (DFS, p = 0.016) rate in the GEPIA database.
High TMEM126A Expression is Correlate with Clinicopathologic Features and poor prognosis in patients with HCC
Next, further exploration was performed to explore the relationship between TMEM126A expression and HCC in TCGA and GTEx databases. The median of TMEM126A expression was selected as the cut-off value to divide the patients into two groups, and high TMEM126A was associated with T stage (p=0.024) and histologic grade (p < 0.001) (Table 1). Surprisingly, TMEM126A expression was significantly higher in HCC tissues than in adjacent HCC samples (p < 0.001) by using TCGA database (Figure 4F). Meanwhile, the different expression of TMEM126A in normal samples of GTEx combined adjacent HCC tissues and HCC samples were analyzed and found that TMEM126A was high-expressed in HCC (p < 0.001) (Figure 4G). Then, among 50 HCC samples and matched adjacent samples, TMEM126A expression was significantly increased in tumor samples (p<0.001) (Figure 4H). In the HPA database, immunofluorescence staining of the subcellular distribution showed that TMEM126A was located in the cytosol (green) and nucleoplasm (blue), and the expression of TMEM126A was also abnormally elevated in HCC (Figure 4I-J).
High TMEM126A expression was significantly associated with pathologic stage, histologic grade, vascular invasion, and T stage, while it was not associated with other features (Figure 5A-C and Figure S1). Subsequently, univariate Cox analyses showed that high TMEM126A expression was significantly correlated with poor OS (Hazard ratio [HR] =2.154, 95% CI= 1.494-3.105, p < 0.001). Further estimation using multivariate Cox analysis using 271 complete data demonstrated that high TMEM126A expression might be an independent risk factor correlated with poor OS (HR =2.222, 95% CI= 1.387-3.559, p < 0.001, Table 2). Then, the nomogram used age, T, M, N classification, pathologic stage, histologic grade, vascular invasion, and TMEM126A to predict the 1, 3, 5-year OS in the TCGA dataset (Figure 5D).
TMEM126A-Related signaling Pathways Based on GSEA
GSEA was performed to identify the signaling pathways activated in HCC by comparing data sets that had low and high expression of TMEM126A. Figure 5E showed the results of GO(Supplementary Table S5). The results indicated that cell cycle, complement and coagulation cascades, and DNA replication were enriched in TMEM126A high expression phenotype, and primary bile acid biosynthesis, oxidative phosphorylation, and fatty acid degradation were enriched in TMEM126A low expression phenotype (Figure 5F, Supplementary Table S6).
Activation of the FA synthesis and suppression of the FAO are typically featured in NAFLD-related HCC. Considering that TMEM126A was highly associated with lipid metabolism, the correlation analysis was performed between TMEM126A and genes related to the FA biosynthesis and oxidation pathways (Figure 6A). In the FA biosynthesis pathway, TMEM126A was significantly correlated with triosephosphate isomerase (TPI1, p <0.001), acetyl-CoA carboxylase 1 (ACACA, p <0.001), NADH: ubiquinone oxidoreductase subunit AB1 (NDUFAB1, p <0.001), malonyl-CoA-acyl carrier protein transacylase (MCAT, p <0.001), and ATP-citrate lyase (ACLY, p <0.001). In the FAO pathway, TMEM126A was negatively correlated with enoyl-CoA hydratase domain-containing protein 2 (ECHDC2, p <0.001) and enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase (EHHADH, p <0.001), short-chain specific acyl-CoA dehydrogenase (ACADS, p <0.001), long-chain-fatty-acid--CoA ligase 1 (ACSL1, p <0.001), and long-chain specific acyl-CoA dehydrogenase (ACADL, p <0.001). These results indicated that TMEM126A might be involved in lipid metabolism reprogramming.
Further Validation of TMEM126A in Diagnosis and Prognosis
Subsequently, further validation was performed in GSE37031 and ICGC datasets. Consistent with our results, TMEM126A expression was abnormally up-regulated in NASH and HCC patients (Figure 6B-C). In addition, high TMEM126A expression was also significantly correlated with OS (p = 0.0017, Figure 6D). These outcomes suggested that TMEM126A had superior diagnostic and prognostic value.