M2-phenotype Macrophages Regulate Gemcitabine Resistance In Pancreatic Cancer Through The Exosome/circFGD6/HDAC9 Pathway

Background:Pancreatic cancer is extremely malignant,with a 5-year survival rate of less than 5%,a low surgical resection rate,and a very poor prognosis.Gemcitabine is the first-line chemotherapeutic agent for the clinical treatment of pancreatic cancer,and primary or acquired resistance to gemcitabine in pancreatic cancer patients can affect the efficacy.Increased expression of histone deacetylase in tumor cells deacetylates histones,increasing the positive charge of histone bands,causing histones to bind tightly to DNA,chromatin to become dense,and DNA to become relatively resistant to physicochemical factors,leading to chemoresistance.Competitive endogenous RNAs are one of the most important modes of post-transcriptional regulation of genes.miRNAs prevent translation of target proteins and promote degradation of target mRNAs;cyclic RNAs act as miRNA "sponges",adsorbing miRNAs and blocking their action.M2 macrophages are the most common phenotype of tumor-associated macrophages in the tumor microenvironment,which can promote tumor growth,angiogenesis,invasion and metastasis,and chemotherapy tolerance.In-depth study of the molecular mechanisms of gemcitabine resistance in pancreatic cancer and the search for targets regulating chemotherapy sensitivity are important to improve the treatment outcome and prognosis of pancreatic cancer patients.Object:The aim of this study was to explore the mechanism of M2-type macrophage induced chemoresistance in pancreatic cancer and to provide a theoretical basis for enhancing chemo-sensitivity in pancreatic cancer.Methods:MTT was used to detect the effects of macrophage conditioned medium,gemcitabine,and histone deacetylase inhibitor SAHA on the growth of pancreatic cancer cell line PANC-1.The degree of chromatin densification was detected by micrococcal nuclease MNase digestion.High-throughput sequencing was used to detect the gene expression of pancreatic cancer cells PANC-1 after treatment with macrophage conditioned medium.TIMER database was used to analyze the relationship between gene expression and macrophage infiltration.A bioinformatics approach was used to analyze the competing endogenous RNA mechanisms regulating gene expression.Competing endogenous RNA mechanisms regulating gene expression were verified by luciferase reporter gene assay.Exosomes from M2 macrophages were collected by ultracentrifugation,and the extracted exosomes were identified using transmission electron microscopy as well as molecular marker Western Blot assay.Dil-labeled exosomes were used and used to incubate PANC-1 cells,and the uptake of exosomes by pancreatic cancer cells was observed using fluorescence microscopy.Real-time PCR was used to detect the expression of genes after treatment of pancreatic cancer cells with M2-type macrophage exosomes.Results:Macrophage conditioned medium was able to promote the growth of pancreatic cancer cells;gemcitabine was able to inhibit the growth of pancreatic cancer cells;when macrophage conditioned medium was combined with gemcitabine treatment,macrophage conditioned medium was able to weaken the inhibitory effect of gemcitabine on the growth of pancreatic cancer cells.The chromatin of pancreatic cancer cells showed heterochromatinization and increased denseness after treatment with macrophage conditioned medium.The histone deacetylase inhibitor SAHA inhibited the growth of pancreatic cancer cells and showed no chemo-sensitizing effect in the culture system without the addition of macrophage conditioned medium,while SAHA had a chemo-sensitizing effect in the culture system with the addition of macrophage conditioned medium.m2-type macrophages could upregulate the expression of HDAC9,circFGD6 in pancreatic cancer cells.circFGD6 regulates HDAC9 expression through adsorption of hsa-miR-520g-3p,hsamiR-494-3p,hsa-miR-495-3p,hsa-miR-520h.Histone deacetylase inhibitors were able to attenuate chemotherapy tolerance caused by overexpression of circFGD6.M2-type macrophage exosomes were able to upregulate circFGD6 expression in pancreatic cancer cells,whereas deletion of exosomes attenuated the upregulation of circFGD6 expression in pancreatic cancer cells by MCM.Conclusion:M2-type macrophages deliver circFGD6 to pancreatic cancer cells via the exosome pathway;circFGD6 adsorbs miRNAs targeting histone deacetylase HDAC9 via the ceRNA mechanism:hsa-miR-520g-3p,hsa-miR-494-3p,hsa-miR-495-3p,hsa-miR520h,which upregulates HDAC9 expression,deacetylates histones,leading to chromatin aggregation(heterochromatinization)and chemoresistance.