Identification and Validation of Novel Biomarkers for Diagnosis and Prognosis of Hepatocellular Carcinoma

Abstract

Introduction: Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide due to poor survival outcome. Thus, there is an urgent need to identify effective biomarkers for early diagnosis and prognosis prediction. Methods: A total of 389 differentially expressed genes (DEGs) between HCC samples and normal were selected based on the Robust Rank Aggregation (RRA) method. We combined DEGs expression and clinical traits to construct a gene co-expression network through WGCNA. Forty hub genes were selected from the key module. Among them, YWHAB, PPAT, NOL10 were eventually identified as prognostic biomarkers using multivariate Cox regression model. Biomarkers expression pattern was investigated by informatic analysis and verified by RNA-seq of 32 patients with HCC. DiseaseMeth 2.0, MEXPRESS, and Tumor Immune Estimation Resource (TIMER) were used to assess the methylation and immune status of biomarkers. GSVA, CCK8, colony formation assay, Edu imaging kit, wound-healing assay, and xenograft tumor model were utilized to investigate the effects of biomarkers on proliferation, metastasis of HCC cells in vitro, and in vivo. The Kaplan–Meier (KM) plotter and ROC curves were used to validate the prognostic and diagnostic value of biomarker expression. Results: All the selected biomarkers were upregulated in HCC samples and higher expression levels were associated with advanced tumor stages and T grades. The regulation of YWHAB, PPAT, NOL10 promoter methylation varied in tumors, and precancerous normal tissues. Immune infiltration analysis suggested that the abnormal regulations of these biomarkers were likely attributed to B cells and dendritic cells. GSVA for these biomarkers showed their great contributions to proliferation of HCC. Specific inhibition of their expression had strong effects on tumorigenesis in vitro and in vivo. ROC and KM curves confirmed their usefulness of diagnosis and prognosis of HCC. Conclusions: These findings identified YWHAB, PPAT, and NOL10 as novel biomarkers and validated their diagnostic and prognostic value for HCC.