When cells are exposed to x-irradiation, they go through two-step malignant transformation. The exposure of x-irradiation is considered as the first step, and in this study, we are here to elucidate what happens in the second step using bulk RNA sequencing, whole-genome sequencing, and single cell RNA sequencing.. X-irradiation induced two-step malignant transformation

Tumourigenesis is initiated by various factors and its progression is also affected by numerous elements. In 1980, an experiment using x-irradiation was performed by Little’s group to study the initiation and progression of tumourigenesis. Although tumourigenesis was reproduced in vitro, there were some missing parts in the puzzle. In the present study, we revisited Little’s study and elucidated some unanswered questions beneath this experiment using whole-genome sequencing, RNA sequencing, and single-cell RNA sequencing. When the genomic alterations were examined, the changes were not as striking as expected. However, the transcriptomic changes were very dynamic. The principal component analysis and admixture analysis showed distinctively different transcriptome profile of samples according to focus generation status and x-irradiation exposure. To investigate what drives focus generation, differentially expressed genes (DEGs) of focus cells were initially analysed. As a result, significant up-regulation of DNA repair pathway related genes were discovered. Additionally, we were able to determine that error-prone repair system was activated due to overloaded error-free repair system in focus cells. Furthermore, to study primary changes in cells that were experiencing malignant transformation, DEGs of x-irradiated non-focus cells were examined. Tgf genes were found discovered as decreased DEGs and down-regulated DEGs were enriched with TGF- signaling pathway were found as one of the enriched pathways using down-regulated DEGs. Myc, one of the increased DEGs of non-focus cells, showed a gradual increment as Tgf gene showed a gradual decrease. Tgf genes and Myc are known to orchestrate each other and they both were found as DEGs of transformed focus cells as well. As Tgf genes exhibit down-regulation in early tumourigenesis, non-focus cells can be considered as at the very initial stage of malignant transformation process. Moreover, as a decrement of Tgf genes occur in early stage tumourigenesis, and similar phenomenon was observed in non-focus and focus cells, it may be considered as one of the factors that contributes to two-step malignant transformation. displayed further down-regulation as malignant transformation proceeded which may be though as their decreased expression led the cells to transform further. Then, to overcome the heterogeneity of each sample, we used single-cell RNA sequencing approach. With in vitro experiment, the role of TGF- gene decrement was investigated. When TGF- signal was down-regulated, focus was formed without x-irradiation and DNA repair related genes, error-prone system as well as error-free system, were elevated. Taken together, we believe the factors that contribute to the second step of two-step hypothesis are Tgf down-regulation and increased error-prone DNA repair system and we are confident that these findings will extend the understanding of tumourigenesis.