IGF2BP1-HAX-1 positive feedback loop-mediated HAX-1 overexpression blocks autophagic flux and promotes chemoresistance in nasopharyngeal carcinoma
Autophagic flux is associated with chemoresistance, the leading cause of chemotherapeutic failure. Here, we showed that HAX-1 promotes chemoresistance by effectively blocking the fusion of autophagosomes with lysosomes. Mass spectrometric and functional studies demonstrated that HAX-1 recruited NEDD4 to promote Rab7a degradation and inhibited the binding of Rab7a with SNAREs by competitively binding to it. Furthermore, HAX-1 could bind to IGF2BP1 mRNA, thereby contributing to its stability and translation. Moreover, IGF2BP1 enhanced HAX-1 m6A methylation, thereby enhancing its stability. Via in vivo and in vitro experiments, we confirmed the positive role of the IGF2BP1-HAX-1 feedback loop in chemoresistance. Our data provide evidence that HAX-1, IGF2BP1, and SQSTM1 levels are useful predictors of clinical outcome and chemoresistance risk. In addition, our data provide new insights into the clinical applications of therapies related to autophagic flux and its associated molecular network in targeting cisplatin chemoresistance in nasopharyngeal carcinoma. Overall design: Guide RNA (sgRNA) preparation: sgRNA was designed using the CRISPR online tool (https://zlab.bio/guide-design-resources). The effective targeting site for HAX-1 is 5'-GCTGAGGACTATGGAACCTT-3'.Plasmid construction: The CRISPR-SaCas9-KKH system was selected for this study because it can be packaged and delivered through a single AAV vector and has known PAM sequences (50-NNNRRT-30) that are compatible with larger sgRNA numbers. sgRNA was inserted into the BsaI site of the pCMV_SaCas9-KKH_hU6 plasmid using PCR and Gibson assembly techniques. Finally, the plasmid was transfected into the CNE2 cells for high-throughput RNA sequencing analysis.