Mohamed, R. H. | Search bioRxiv

bioRxiv2026

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

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.

biochemistry↗

bioRxiv2025

Host aging induces a senescent-like phenotype in neutrophils and altered transcriptional responses to Streptococcus pneumoniae

Aging drives increased susceptibility to respiratory infections by Streptococcus pneumoniae (pneumococci). Polymorphonuclear leukocytes (PMNs) are among the first responders in the lung following pneumococcal infection and are required for bacterial clearance. However, PMN antimicrobial function declines with age. To identify mechanisms underlying this decline, we performed RNA sequencing on PMNs in the lungs of young and old mice following pulmonary infection with S. pneumoniae. We observed significant transcriptomic differences across host age. Transcriptional analysis followed by functional validation revealed that in infected mice, PMNs from aged hosts failed to upregulate several effector activities including glycolysis and subsequent mitochondrial reactive oxygen species (ROS) production, which are necessary for bacterial killing by PMNs. Analysis of potential transcription factors controlling these changes indicated differential regulation by E2f2 in aged mice, which was linked to lower PMN differentiation resulting in more immature PMNs in the lungs of aged mice compared to young controls. Conversely, PMNs in aged mice displayed a higher senescence-associated secretory phenotype (SASP) score and upregulated pathways involved in cellular senescence. Follow-up functional characterization found that in uninfected hosts, PMNs in aged mice expressed higher levels of SASP factors IL-10, TNF, and ROS, had lower incidence of apoptosis, and had a higher proportion of cells positive for senescence-associated {beta}-galactosidase, features of a senescent-like phenotype. In conclusion, host aging is associated with altered PMN phenotypes, including a shift toward senescent-like energy-deficient cells, which may contribute to impaired host defense and represent potential targets for improved interventions against infection in older adults.

immunology↗