Dysregulated HIC1 and RassF1A expression in vitro alters the cell cytoskeleton and exosomal Piwi-interacting RNA

HIC1 and RassF1A methylation, which cause loss of gene function, are found in various cancers, including renal cell carcinoma (RCC), and could alter cell stiffness and the content of extracellular vesicles (EVs). These physiological changes may provide a tumoral survival advantage and thus could serve as cellular biomarkers for monitoring cell transformation, although direct associations between these changes and cell transformation remain to be established. As we found HIC1 and RassF1A methylation and expression changes in RCC samples, we examined the effects of gain and loss of HIC1 and RassF1A expression on cell DNA content, cytoskeletal structure, and Piwi-interacting RNA (piRNA) expression in EVs. We found HIC1 and RassF1A hypermethylation and abnormal expression in RCC patient samples was independent of the somatic mutations found in publicly available data. Cell stiffness was reduced in accordance with disrupted cytoskeleton conformation after knockdown of HIC1 or RassF1A. Gain or loss of HIC1 expression induced instability in genomic content, abnormal RassF1A expression disturbed cytoskeletal structure, and the abnormal expression of either gene altered piRNA content in EVs. These results suggest a causal relationship between abnormal tumor suppressor gene expression, cell stiffness, and piRNA expression.