CRISPR-Cas9 knockout screening identifies KIAA1429 as an essential gene in Ewing sarcoma

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Abstract
Background: Ewing sarcoma (ES) is an aggressive childhood bone and soft tissue cancer. KIAA1429 is one type of N6-methyladenosine (m6A) writer that plays a tumor-progressive role in various cancers, but the role of KIAA1429 in ES remains to be elucidated. The aim of the study was to investigate the role of KIAA1429 in ES. Methods: We performed a multi-omic screen including CRISPR-Cas9 functional genomic and transcriptomic approaches, and identified that KIAA1429 played a significant role in ES progression. Gene knockdown, quantitative real-time PCR (Q-RT-PCR), immunoblotting, CellTiter-Glo assays, clonogenic assays, a subcutaneous xenograft model and immunohistochemistry were used to assess the functional role of KIAA1429 in ES. We mainly conducted RNA sequencing (RNA-seq) in ES cells to analyze the downstream regulatory mechanism of KIAA1429. An integrative analysis of chromatin immunoprecipitation sequencing (ChIP-seq) and RNA-seq indicated the upstream regulatory mechanism of KIAA1429. Results: In vitro and in vivo CRISPR-Cas9 knockout screening identified KIAA1429 as an ES-dependent gene. Genetic suppression of KIAA1429 inhibited ES cell proliferation and tumorigenicity both in vitro and in vivo. Further studies revealed that KIAA1429 promotes ES tumorigenesis by regulating the ribosome-associated cell cycle and cancer-related inflammation. Interestingly, we found that STAT3 was a target of KIAA1429 and that a STAT3 inhibitor reduced KIAA1429 transcript levels, indicating positive feedback between KIAA1429 and STAT3. Finally, we found that NKX2-2 bound to the KIAA1429 promoter and transactivated KIAA1429. Conclusion: Our study systematically analyzed ES-dependent epigenetic/transcriptional regulatory genes and identified KIAA1429 as a biomarker of tumor progression in ES, providing a potential therapeutic target for treating ES.

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