A short PIWIL2 isoform PL2L60A induces functional and transcriptional changes indicative of a procancerous phenotype

Background: RNA-binding PIWIL proteins are known as key contributors to germ cell development and stem cell maintenance, and deregulation of PIWIL expression is associated with various types of cancer. PIWIL2 is usually expressed in the testes, where it acts as a translational regulator and is considered a tumor-barrier protein. In contrast, short PIWIL2 isoforms transcribed from intragenic promoters are associated with testicular tumors called seminomas, suggesting the opposite roles of PIWIL2 and PL2L60A in oncogenesis. The underlying mechanisms are unclear. Methods: To investigate the effects of the full-length PIWIL2 gene and its short isoform, PL2L60A, we constructed isogenic knock-in (KI) cell lines using CRISPR/Cas9 technology and compared their transcriptional profiles using RNA sequencing. Analysis of KI cell proliferation, migration, and apoptosis was performed using the MTS proliferation assay, wound healing assay, flow cytometry, and caspase activation assay. Results: PL2L60A-expressing cells had higher proliferative and migratory activities and were less prone to apoptosis than PIWIL2-expressing cells. Transcriptomics analysis of the obtained cell lines by RNA-seq revealed that PL2L60A induced genes involved in angiogenesis, wound healing, cell proliferation, extracellular matrix remodeling, and cancer-promoting signaling; however, the mTORC1-related genes were repressed, which is typical for seminomas. Conclusion: Our results indicate that the oncogenic function of PL2L60 is based on the stimulation of molecular pathways typical for seminoma, suggesting PL2L60A as a possible driver of seminoma initiation and/or progression. © The Author(s), under exclusive licence to Springer Nature B.V. 2025.