RNA binding protein Pumilio2 promotes chemoresistance of pancreatic cancer via focal adhesion pathway and interacting with transcription factor EGR1
Authors: Zhao Bangbo , Qin Cheng , Li Zeru , Wang Yuanyang , Yang xiaoying , Li Tianyu , Zhao Yutong , Wang Weibin , Zhao Yupei 
Published: November 2024

Abstract

Background
Pancreatic cancer (PC) has insidious onset, high malignancy and poor prognosis. Gemcitabine (GEM) is one of the first-line chemotherapy drugs for PC. However, resistance for GEM has always been a bottleneck problem leading to recurrence and death of PC patients. RNA-binding proteins (RBPs) are a kind of important proteins that regulate transportation, splicing, stability and translation of RNA. Abnormal expression of RBP often leads to a series of abnormal accumulation or degradation of downstream RNA resulting in various diseases. However, there is a lack of systematic study on whether RBPs play roles in GEM resistance of PC. Therefore, it is of great significance to explore RBPs and their specific molecular mechanisms that play an important role in GEM resistance of PC for further understanding and solving GEM resistance of PC.
Methods
RBPs closely related to GEM resistance of PC were screened based on transcriptome sequencing, siRNA library proliferation and GEM resistance test results. Relationship between expression level of PUM2 and clinicopathological variables was evaluated by immunohistochemical (IHC) staining of PC tissue chip. SRB proliferation assay, GEM drug resistance assay and transwell cell migration assay were used to detect the effects of PUM2 on the malignant biological behaviors of PC cells in vitro. Mice subcutaneous xenograft model was used to explore the effect of PUM2 in vivo. Furthermore, RIP-seq and RNA-seq were combined to explore the downstream mRNAs regulated by PUM2 in PC cells, and the regulation effect of PUM2 on downstream mRNAs was verified by qRT-PCR, Western Blot, RIP-qPCR, actinomycin D RNA stability assay, dual luciferase gene reporter assay and rescue experiments. Finally, transcription factors with mutual regulation relationship with PUM2 were screened by integrating data of RIP-seq, RNA-seq and JSAPAR database, and the regulatory relationship between the transcription factor EGR1 and PUM2 was verified by qRT-PCR, Western Blot, RIP-qPCR and rescue experiments.
Results
Several RBPs were found highly expressed in GEM resistant PC cell line. We screened out RNA-binding protein PUM2 as the most related RBP with GEM resistance of PC by siRNA library. IHC of PC tissue chip suggested that high expression of PUM2 was an independent risk factor for poor prognosis of PC patients. In vitro function experiments showed that PUM2 could promote proliferation, migration and resistance to GEM of PC cells. In vivo experiments showed that knockdown of PUM2 inhibited the growth of subcutaneous transplanted tumor in mice and increased sensitivity to GEM. Further, RNA-seq and RIP-seq were combined to explore the regulation role of PUM2 on downstream RNAs that promoted GEM resistance in PC. We found that PUM2 up-regulated mRNA stability of key genes (ITGA3, ADAM17, ASAP1, etc.) in the focal adhesion pathway. ITGA3 was verified to be the most significant downstream mRNA of PUM2 regulating GEM resistance in PC by rescue experiments in vitro, and PUM2 could stabilize ITGA3 mRNA by binding to PUM binding element (PBE) in the 3'UTR region of ITGA3 mRNA. Finally, we found the mutual regulation relationship between transcription factor EGR1 and PUM2, that is PUM2 binding to 3'UTR region of EGR1 mRNA, and EGR1 binding to promoter region of PUM2 gene, resulting in a cascade effect amplifying the role of PUM2 in PC chemoresistance.
Conclusions
RNA-binding protein PUM2 is closely related to the prognosis of PC patients. PUM2 promoted GEM resistance of PC by regulating mRNA stability of ITGA3 and other genes in focal adhesion pathway, and there was positive feedback regulation between PUM2 and transcription factor EGR1. The discovery of EGR1/PUM2/ITGA3 axis provided a solid experimental basis for the selection of chemotherapy regiments for PC patients and exploration of combined regimens to reverse GEM resistance in the future.