The Effects And Mechanisms Of Adipocytes In Regulating The Radiosensitivity Of Pancreatic Cancer

Objective:Pancreatic cancer is a highly lethal malignancy with poor prognosis.The insidious onset of the patient is usually at an advanced stage when discovered.Advanced pancreatic cancer has lost the opportunity to be completely removed by surgery and requires adjuvant chemotherapy and radiation therapy.Its poor radiosensitivity limits the effectiveness of its radiotherapy.The pancreas is in a adipose-rich anatomical location,and the occurrence of pancreatic cancer is often accompanied by obesity.However,the interplay between abdominal fat and the radiosensitivity of pancreatic cancer cells remains largely unexplored.This study aims to elucidate the interplay between abdominal fat and the radiosensitivity of pancreatic cancer cells.Methods:Human mature adipocytes isolated from Abdominal adipose tissue were co-cultured with pancreatic cancer cells(PANC-1&SW1990)in the transwell culture system.Survival fraction of pancreatic cancer cells after X-ray exposure was measured by colony-forming unit assay.DNA damage was measured by Comet assay and immunofluorescence were conducted to evaluate DNA damage after irradiation.Cell apoptosis and cell cycle analysis were performed using flow cytometry assay.Mito-tracker Red,JC-1 and ER-tracker were used to exam the damage of mitochondria and ER.Seahorse Cell Energy Metabolism Analyzer detects the changes in energy metabolism of human pancreatic cancer cells after co-culture of adipocytes.BODIPY staining and immunofluorescence were used to detect whether the content of fatty acids and fatty acid binding protein in human pancreatic cancer cells in the co-culture system was changed.CRISPR/Cas9 technology was used to construct FABP4 knockout 3T3-L1 cells(mouse pre-fatty embryonic fibroblast).WB experiment confirmed that FABP4 was successfully knocked out.The differentiation medium was used to induce 3T3-L1 into mature adipocytes,and Oil red O stained mature adipocytes.Cell apoptosis,comet assay,immunofluorescence and cell cycle analysis was used to explore the effect of FABP4 on the radiosensitivity of pancreatic cancer cells.Co-IP combined with LC/MS/MS was used to discover the protein interacting with FABP4 in PANC-1 cells.RIP(RNA Binding Protein Immunoprecipitation)assay was used to detect the effect of FABP4 on the binding of eIF3a to the RNA of radiation damage repair-related genes.RNA-sequencing explored possible mechanism by which adipocyte exosomes affect the radiosensitivity of pancreatic cancer.Results:Co-culture with adipocytes promotes survival of pancreatic cancer cells after irradiation.Co-culture with adipocytes promotes the survival of pancreatic cancer cells after irradiation,while reducing DNA double-strand damage,the rate of apoptosis and G2/M phase block.Pancreatic cancer cells treated with exogenous FABP4 obtained results consistent with co-culture of adipocytes.142 FABP4-interacting protein was identified in PANC1 cells by applying immunoprecipitation using anti-FABP4 monoclonal antibody followed by LC/MS/MS.Through multi-dimensional protein profiling,it was found that eIF3a had the largest number of peptides among these 142 proteins,and 10 other eIF3a were also included in the identified protein.RIP results show that FABP4 promotes the RNA binding of eIF3a protein to DNA damage repair related genes.Small RNA sequencing results of adipocyte exosomes show that there are 330 differential microRNAs.Go and KEGG analysis of predicted target genes found that 14 kinds of miRNAs in 20 kinds of abundant miRNAs are related to radiation damage.Conclusion:1.Adipocyte microenvironment causes pancreatic cancer radiation resistance.2.Adipocytes affect pancreatic cancer radiosensitivity by secreting FABP4.3.The interaction of fabp4 and eIF3a promotes the expression of DNA damage repair proteins,thereby affecting the radiosensitivity of pancreatic cancer.3.Exosomes secreted by adipocytes may regulate the radiosensitivity of pancreatic cancer through miRNA.