N6-methyladenosine-mediated CELF2 Regulates CD44 Alternative Splicing Affecting Tumorigenesis via ERAD Pathway in Pancreatic Cancer

Abstract

Background: Alternative splicing (AS) of genes has been found to affect gene stability, and its abnormal regulation can lead to tumorigenesis. CELF2 is a vital splicing factor to participate in mRNA alternative splicing. Its downregulation has been confirmed to promote the occurrence and development of pancreatic cancer (PC). However, the regulatory role and mechanisms in PC has not been elucidated.Methods: 80 patients diagnosed with PC were included in this study. The relationhip between CELF2 expression and clinicopathological characteristics was analyed. RNA immunoprecipitation qRT-PCR and RNA pull-down experiments were performed to confirm the potential mechanisms of CELF2 abnormal expression in PC. AS model of PC was established by TCGA database. Functional experiments in vitro and in vivo were conduced to reveal the effects of CELF2 or CD44 on PC cells proliferation, migration and invasion. Transcriptomic analysis was conducted to reveal the mechanism of CELF2-mediated CD44 AS in PC. Results: CELF2 was downregulated in PC tissues, which affected tumor TNM stage and tumor size, and low expression of CELF2 indicated a poor prognosis of PC. In vivo and in vitro experiments showed that abnormal expression of CELF2 affected the stemness, apoptosis, and proliferation of PC cells. Furthmore, we also found that CELF2 was targeted by ALKBH5 for m6A modification, leading to CELF2 degradation by YTHDF2. Bioinformatic analysis of AS model based on the TCGA database indicated that CELF2 could target CD44 to form different spliceosomes, thereby affecting the biological behavior of PC cells. The conversion of CD44s to CD44V is the key to tumorigenesis. Transcriptomic analysis was conducted to reveal the mechanism of CELF2-mediated CD44 AS in PC. We found that CELF2-mediated splicing of CD44 led to changes in the level of endoplasmic reticulum stress, further regulating the endoplasmic reticulum-associated degradation (ERAD) signaling pathway, thereby affecting apoptosis and cell stemness. In addition, ERAD signaling pathway inhibitor, EerI, could effectively reverse the effect of CD44 on tumors. Conclusions: This study indicates that CELF2 promotes AS of CD44, affecting the ERAD pathway and enhence the biological behavior of PC cells. CELF2 is expected to be a new target for targeted-drug development