GSE212016
Effect of Bro40 and Bro40mut on gene expression of wild type H1299 non-small cell lung cancer cell line
We previously discovered WDR4 as a Cullin 4 ubiquitin ligase substrate adaptor, which associates with poor cancer prognosis. With the help of ubiquitylome analysis, we uncovered PTPN23 as a substrate of WDR4. While PTPN23 is a component of ESCRT complex that facilitates EGFR lysosomal trafficking and degradation, the high expression of WDR4 and low expression of PTPN23 in patient sample suggesting that WDR4-mediated PTPN23 ubiquitination might contribute to PTPN23 proteasomal degradation and lead to poor patient survival rate. To prevent PTPN23 from WDR4-mediated ubiquitination, we used fine mapping to figure out the binding site of these two proteins and identified a 40-amino acid region on PTPN23 (Bro1 domain) can bind with WDR4, we named this 40-amino acid region as Bro40. Structural docking of WDR4 and Bro1 showed that the N-term of Bro40 is the favorable contact site. Thus, 6 amino acids at the N-term of Bro40 were mutated into alanine, so called Bro40mut. Bro40 and Bro40mut were fused on a GFP-tag plasmid for functional test and animal experiment. The tumor suppressing function of Bro40 was confirm by migration/invasion assays and animal model. To understand how Bro40 regulates cancer progression at RNA level, we transfected Bro40 and Bro40mut into H1299 cells followed by RNA sequencing. Overall design: H1299 cells were transfected with Bro40 and Bro40mut for 24 hours, respectively. Comparative gene expression profiling analysis of high throughput RNA-seq data was obtained from 3 independent biological repeats. Bro40mut was set as control.