Identification and functional validation of SPON2 as a novel biomarker for the diagnosis and prognosis in hepatocellular carcinoma

Background and Aims: Hepatocellular carcinoma (HCC) remains a global health challenge with poor survival, largely due to the absence of reliable biomarkers for early detection and prognosis. This study aimed to identify and functionally characterize novel biomarkers for HCC diagnosis and outcome prediction. Methods Transcriptomic profiles of HCC and adjacent normal tissues, along with single-cell RNA sequencing (scRNA-seq) data from six HCC patients, were obtained from public databases. The differentially expressed genes (DEGs) were identified and functionally annotated. HCC tumor cell-specific genes were determined using the “FindAllMarkers” function. Genes that were upregulated in both HCC tissues and tumor cells were considered candidate biomarkers. Its role in promoting proliferation, migration, and invasion via WNT signaling was validated through qRT-PCR, Western blotting, CCK-8, EdU, colony formation, wound healing, and transwell assays. Results SPON2 showed high diagnostic accuracy in distinguishing HCC patients from healthy controls across four independent cohorts, and its elevated expression was linked to poor overall survival (OS) and increased metastatic potential. At the single-cell level, SPON2⁺ tumor cells showed higher invasion and migration scores. Functional assays confirmed that SPON2 promoted malevolent behavior of HCC cells. Moreover, SPON2 expression exhibited a robust positive correlation with the activation of WNT signaling pathway; while SPON2 knockdown suppressed WNT-related protein expression. Conclusion Integrative transcriptomic and single-cell analyses identified SPON2 as a novel HCC biomarker. SPON2 upregulation predicts poor prognosis and promotes tumor aggressiveness by activating the WNT pathway, offering new diagnostic and therapeutic insights.