Hypoxic Tumor-derived Exosomal Hsa_circ₀048117 Facilitates M2 Macrophage Polarization In Esophageal Squamous Cell Carcinoma

BackgroudThe oncogenesis and evolvement of esophageal squamous cell carcinoma?ESCC?is a comprehensive process related to multiple factors,multiple genetic changes and multiple stages of development.Tumor cells must rely on the support of the host to be able to form tumors,including various tissue cells,inflammatory cells,lymphatic vessels,blood vessels that contribute to tumor growth and metastasis,and these together constitute the microenvironment for tumorigenesis and development.Recent studies on tumor microenvironment have gradually revealed the important role of hypoxia in the development of various malignant tumors.Studies have confirmed that under hypoxic conditions,a variety of hypoxic response genes participate in the proliferation,differentiation,invasion and migration of esophageal squamous carcinoma cells through a variety of signaling pathways.Hypoxia in malignant tumors such as non-small cell lung cancer,glioma,colorectal cancer and pancreatic cancer can promote the development of malignant tumors by affecting the polarization of tumor-associated macrophages?TAMs?,but this mechanism is in ESCC related research has not been reported.Exosomes are widely found in various body fluids such as blood,urine,saliva,cerebrospinal fluid,and breast milk,consisting of m RNA,micro RNA,lnc RNA,circ RNA,DNA fragments,and proteins.Tumor cells secrete non-coding RNA and proteins with special functions into the tumor microenvironment in the way of exosomes,which affects tumor cell proliferation,apoptosis,invasion and migration.Circular RNAs are a class of non-coding RNAs that are widely present in eukaryotic organisms and do not contain a 5'end cap and a 3'end Poly tail.They are continuous closed loop structures formed by covalent bonds.Circular RNA was found to function as a competitive endogenous RNA?ce RNA?,which can be combined with a specific miRNA and adsorb miRNAs to play the role of miRNA"sponge".Through high-throughput second-generation sequencing combined with bioinformatics analysis,it has been confirmed that many circular RNAs were involved in the oncogenesis and progression of ESCC,and participated in the regulation of tumor cell proliferation,differentiation,invasion and migration,and formation of the tumor microenvironment.Studies have found that the expression abundance of circular RNA in exosomes is significantly higher than that in the producer cells,so we chose exo-circular RNA as the breaking point to screen for circular RNAs related to the polarization of tumor-associated macrophages in the hypoxic microenvironment,combined with bioinformatics analysis,molecular biology and cell function experiments to analyze its effect in ESCC,providing a new sight and theoretical evidence to fundamental research of ESCC.Objectives:1.Collecting and enriching exosomes from the culture medium of tumor cells under hypoxic and normoxic conditions,and performing high throughput sequencing to screen the target circular RNA and verify it in serum of ESCC.2.To clarify the biological function of Hsa_circ₀048117 and to verify the relationship between Hsa_circ₀048117/miR-140 and their joint effects to TAMs in ESCC.3.To explore the molecular mechanism of Hsa_circ₀048117 in exosomes affecting macrophage polarization and its effect on the development of esophageal squamous cell carcinoma.Methods:1.Collection and identification of exosomes?1?Cell treatment,collection of culture medium and serum:the tumor cell supernatants under hypoxia and normoxia culture were collected for exosomes extraction after 12 hours and 24 hours;2ml of peripheral venous blood from 12 ESCC patients and5 healthy adults were drawn into an EDTA tube,all the ESCC patients had not received any anti-tumor therapy.?2?Extraction and enrichment of exosomes:Use exosomes extraction kit to extract and enrich exosomes according to protocols for cell culture medium and serum,refrigerated at-80°C for extraction of RNA.?3?Identification and quantitative analysis of exosomes using transmission electron microscopy,nanoparticle tracking system and exosomal quantification kit.2.Screening and functional identification of circular RNAs of interest in exosomes?1?RNA extraction,reverse transcription and Sanger sequencing:total RNA in exosomes was extracted with miRNeasy Mini Kit,quantified using Nano Drop ND-2000and tested for RNA integrity by Agilent Bioanalyzer 2100.The RNA is reverse transcribed into double-stranded c DNA,the labeled circular RNA is hybridized with the chip,and the original image is obtained after scanning.?2?Data analysis:Standardize and follow-up processing after extracting the original data.Differential genes and differential c RNA screening using fold change values,GO function enrichment analysis and KEGG signal pathway analysis and unsupervised hierarchical clustering of differential genes,The target circular RNA was screened based on the combined results of ce RNA_score and expression values.?3?q RT-PCR was used to verify the expression level of the target circular RNA in cells and serum.Sanger generation sequencing confirmed the reverse insertion site.RNase R tolerance test verified its circular structure.Designed si RNA or over-expressed lentivirus and a series of cell phenotyping identification experiments were performed to verify the cellular functions of the circular RNA of interest.3.The underlying molecular mechanism of Hsa_circ₀048117 promoting the polarization of macrophages to M2 type?1?Screening miRNAs that bind to Hsa_circ₀048117 by bioinformatics analysis,pull-down assay was conducted to detect the enrichment of corresponding miRNAs and determine the binding of miR-140 to Hsa_circ₀048117.?2?Ago2-RIP:RNA immunoprecipitation using Ago2 antibodies in macrophages,RIP-q PCR to detect whether Hsa_circ₀048117 binds to AGO2 protein to determine its potential to be a ce RNA.?3?In situ hybridization of Hsa_circ₀048117 and miR-140 in macrophages by designing synthetic probes to detect the localization and expression of both in cells.?4?Dual-Luciferase report experiment:Constructed mutant and wild-type vectors of Hsa_circ₀048117,co-transfected miR-140 mimics and controls with them,and corresponding luciferase activity was tested to confirm the direct interaction between Hsa_circ₀048117 and miR-140.?5?According to the results of the bioinformatics analysis,find the potential target genes of miR-140 involved in the polarization of macrophages.The luciferase report experiment determined TLR4 as the downstream target gene of miR-140,plasmid transfection was used to verify the regulation effect of Hsa_circ₀048117 and miR-140 to TLR4 and flow-cytometry after transfection was conducted to explore their joint effects on macrophage polarization in ESCC.4.The effect of exo-Hsa_circ₀048117 on macrophage polarization and invasion/migration of tumor cells and its correlation with clinical pathological stage?1?Exosomes dye:Use DIL live cell tracer to fluorescently stain exosomes to observe the exosomes entering macrophages;?2?Co-culture of macrophages and exosomes:Macrophages and exosomes secreted by hypoxia/normoxia treated Eca-109cells were co-cultured,and the situation of polarization of macrophages in each group was detected by flow cytometry;?3?The effect of exo-Hsa_circ₀048117 on invasion and migration ability of tumor cells:24-well Transwell plates were used to establish a co-culture system consisting of tumor cells,macrophages,and exosomes secreted by hypoxia/normoxia treated Eca-109 cells,observed the effect of macrophages on the invasion and migration ability of Eca-109 cells in each group.?4?The correlation between exo-Hsa_circ₀048117 from peripheral serum and clinical pathological stage:Collected the peripheral blood of 50 patients with ESCC and 12 normal healthy volunteers,extracted the exosomes and detected the expression of Hsa_circ₀048117,and its correlation with clinical pathological data was analyzed.Results:1.Extraction,enrichment and identification of exosomesThe treated samples under transmission electron microscopy?TEM?clearly showed a vesicle-like structure with a diameter of about 100 nm.The results of the nanoparticle tracking system?NTA?showed that the concentration of particles in the exosome extracts of each group was between 1-10×10¹⁰particles/ml.The particles are arranged according to size,and the median particle size is between 100-120nm,and the percentage of these particle sizes exceeds 98%.The results of Western blotting showed that exosome markers CD63 and TSG101 in the samples was obvious expressed.The above results suggest that the purity and abundance of exosomes are satisfied.2.Hsa_circ₀048117 promotes M2 macrophage polarizationHigh throughput sequencing results revealed that after hypoxia treatment,there were 526 and 229 circular RNAs in 12h group were found upregulated and downregulated?Fold change?2 and p<0.05?,respectively,and 175 and 276 circular RNAs in 24h group were found upregulated and downregulated,respectively.Based on the expression level,MRE number,ce RNA_score,expression value and p value,the first5 RNAs were selected for screening and then verified in cells and serum.It was found that the expression of Hsa_circ₀048117 was stable and the change trend was consistent,so it was selected as our study object.GO function enrichment analysis suggested that Hsa_circ₀048117 may be involved in the polarization of macrophages.Phorbol-12-myristate-13-acetate?PMA?was used to induce THP-1 monocytes differentiate into macrophages,the expression of CD14 and CD206 on the surface of macrophages were significantly increased after Hsa_circ₀048117 was overexpressed,indicating that Hsa_circ₀048117 was capable of favoring M2 macrophage polarization.3.Hsa_circ₀048117 could upregulate TLR4 via acting as miR-140 sponge to promote M2 macrophage polarizationUsing AGO2 antibody for RNA immunoprecipitation?RIP?in macrophages,Hsa_circ₀048117 was significantly enriched by AGO2 antibody,suggesting that Hsa_circ₀048117 has the potential to be a ce RNA.Using the Circular RNA Interactome and miRanda miRNA target prediction tools,31 miRNAs that may bind to Hsa_circ₀048117 were found.The top 10 miRNAs were selected based on the context score,and Hsa_circ₀048117 related RNA was purified by circ RIP.It was found that miR-140 was specifically enriched,FISH Detection showed that Hsa_circ₀048117 and miR-140 were co-localized.Through a pull down experiment of the biotin-coupled miR-140 mimic,we observed that Hsa_circ₀048117 was significantly enriched compared to the control group.The luciferase reporter experiment showed that miR-140mimics reduced luciferase activity by at least 50%after binding to Hsa_circ₀048117.The target site of Hsa_circ₀048117 was mutated and the corresponding miRNA was transfected into macrophages,but the luciferase activity did not change significantly.The luciferase reporting experiments of miR-140 and TLR4 also confirmed that miR-140 can target the 3'-untranslated region?3'-UTR?of TLR4 and inhibit its expression.Overexpression of Hsa_circ₀048117 in macrophages can upregulate the expression of TLR4,and the expression of TLR4 decreased after miR-140 was overexpressed.The above experimental results confirm that Hsa_circ₀048117 and TLR4 can competitively bind to miR-140,which are a pair of competitive endogenous RNAs.Hsa_circ₀048117up-regulates the expression of TLR4,the target gene of miR-140,and promoted M2macrophage polarization through targeted adsorption of miR-140.4.Exo-Hsa_circ₀048117 promotes M2 macrophage polarization and invasion and metastasis of ESCCThe exosomes were dyed with DIL live cell tracer to observe their entry into macrophages.It was found that Hsa_circ₀048117 and TLR4 in macrophages were upregulated after co-culture with exosomes by hypoxic tumor cells,the results of flow-cytometry revealed CD14 and CD206 of macrophages were significantly increased.However,when Hsa_circ₀048117 in tumor cells was interfered with si RNA,the corresponding exosomes did not show a M2 macrophage polarization promotion after co-culture,the proportion of CD14⁺/CD206⁺macrophages decreased significantly compared to the control,suggesting hypoxia contributed to the synthesis of Hsa_circ₀048117 which was packaged in exosomes and then secreted,after phagocytosed by macrophages,the highly expressed Hsa_circ₀048117 promotes M2macrophage polarization by upregulating TLR4.After the establishment of co-culture system including tumor cells,macrophages and exosomes.Transwell invasion and migration experiments confirm that the macrophages co-cultured with exosomes from hypoxia-stimulated tumor cells can in turn increase the invasion and migration ability of tumor cells.The expression of Arg1,IL-10and TGF-?in the lower chamber medium was detected by Elisa.It was found that exosomes secreted by hypoxic tumor cells can promote the secretion of Arg1,IL-10 and TGF-?by macrophages,thereby enhancing tumor cells invasion and migration capabilities.Comparing the expression of Hsa_circ₀048117 in the exosomes from serum of tumor patients and healthy volunteers,it was found that the expression of Hsa_circ₀048117 in the serum exosomes of tumor patients was significantly higher than that of normal people,and that exo-Hsa_circ₀048117 was positively related with T stage,N stage,and TNM grade of ESCC patients.Conclusion:In this study,we preliminarily investigated and explored the role of hypoxia,exosomes,and circular RNA in the development of ESCC.We identified differentially expressed circular RNAs in the exosomes secreted by tumor cells after hypoxia treatment.Hsa_circ₀048117 was selected as the research object.It was found that exosomes can carry Hsa_circ₀048117 into TAMs and induce them differentiated towards M2 type.Bioinformatic analysis,molecular biology and cell functions experiments confirmed that Hsa_circ₀048117 can be used as miR-140"sponge"in macrophages,and competes with TLR4 to bind miR-140,and macrophages that overexpress Hsa_circ₀048117 can up-regulate TLR4 expression in them,and promote the transformation to M2 type.M2macrophages could secrete Arg1,IL-10 and TGF-?which then enhance the invasion and migration ability of tumor cells,and promote the metastasis of esophageal squamous carcinoma cells.The expression of Hsa_circ₀048117 in exosomes of peripheral blood of ESCC patients was significantly higher than that in normal healthy people.Higher exo-Hsa_circ₀048117 predicted an advanced T and N stage and positively correlated with TNM grade.This study may also provide some theoretical basis and references for the future exploration of the role of circular RNA in exosomes in ESCC.