Uncovering the Multifaceted Role of the Kinetochore Scaffold 1 (KNL1) in Hepatocellular Carcinoma: Integrative Bioinformatics Analysis and CRISPR-Cas9 Experimental Study

Abstract

Hepatocellular carcinoma (HCC) is a highly aggressive malignancy characterized by pronounced heterogeneity, chromosomal instability, and limited therapeutic options. Kinetochore scaffold 1 (KNL1), a core mitotic checkpoint component, is implicated in multiple cancers, but its role in HCC remains unclear. Here, we integrated bulk and single-cell transcriptomic analyses with functional in vitro validation to elucidate the biological and clinical significance of KNL1 in HCC. KNL1 was markedly overexpressed in HCC tumors, particularly those harbouring TP53 mutations, and correlated with increased tumor break load, suggesting a link to structural chromosomal instability. Weighted gene co-expression network analysis (WGCNA) and hallmark gene set enrichment identified a KNL1-associated G2M checkpoint network, in which KNL1 ranked among the top 10 hub genes. A KNL1-G2M network score, stratified patients into high- and low-score groups, with high scores correlates with poorer overall survival, increased proliferation, and higher chromosomal instability. Immune deconvolution (CIBERSORTx) revealed that high-score tumors were enriched in T-regulatory cells and M0 macrophages, and had elevated TIDE and exclusion scores, indicating potential resistance to immune checkpoint blockade. Single-cell RNA-seq confirmed preferential activation of the KNL1-G2M module in malignant hepatocytes and highlighted increased extracellular matrix remodeling and immunomodulatory interactions. CRISPR-Cas9-mediated KNL1 downregulation in HepG2 cells induced modest G2/M arrest, suppressed proliferation, reduced survivin and Bcl2 expression, and produced ultrastructural changes consistent with early apoptosis. Collectively, our results implicate KNL1 and its associated G2/M network in tumor progression-related processes, including chromosomal instability, proliferative activity, and immune modulation in HCC. While further mechanistic and clinical studies are required, KNL1 and its associated network may represent a biologically relevant vulnerability and a potential biomarker of aggressive disease behavior in hepatocellular carcinoma.