Research On Regulation Of Autophagy In Tumor-associated Fibroblasts By Exosomes From Triple-negative Breast Cancer

BackgroundBreast cancer is the world’s leading cause of cancer in women(23%of all cancer cases in women)and accounts for 14%of all cancer deaths.According to estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER 2)expression,breast cancer can be divided into five types:luminal-A,luminal-B,luminal-B HER2+,HER2 Nonluminal and triple-negative breast cancer(TNBC).Among them,TNBC is a special type of breast cancer,named triple negative breast cancer due to the negative expression of ER,PR and Her2 in its cells,which accounts for about 12-17%of the incidence of breast cancer in women.According to the guidelines for the treatment of NCCN for breast cancer,TNBC is mainly treated with surgery combined with chemotherapy(not suitable for endocrine therapy and her2-targeted therapy),but the 5-year survival rate of such patients is significantly lower than that of other types of breast cancer patients.Therefore,it is particularly important to find a more effective treatment for TNBC patients.TNBC cells do not exist in isolation but in a "tumor microenvironment," including cancer-associated fibroblasts(CAFs),immune cells,vascular epithelial cells and cytokines.Among them,substance exchange and energy transfer are conducted between CAFs and TNBC cells through paracrine,which plays an important role in the biological behaviors of cancer cell proliferation,invasion and metastasis.Studies have found that exosome is considered as a transmission medium of paracytosis,that is,cancer cells release exosomes carrying their own proteins,microRNA and other substances,which are absorbed by other cells in the microenvironment,thus"directing" other cells to "serve" cancer cells.MicroRNA is a small non-coding RNA that regulates the expression of target genes by inhibiting gene transcription and promoting mRNA degradation.Micrornas are highly conserved in organisms and are involved in the regulation of many cell life activities,including apoptosis,differentiation and proliferation.In recent years,more and more studies have shown that microrna-mediated transcription and post-transcriptional regulation play an important role in autophagy,which has become a research hotspot.Currently,it is only beginning to clarify whether the expression and function of mirnas in fibroblasts in the tumor microenvironment are affected by their interaction with cancer cells.There is growing evidence that micrornas are involved in the conversion of NFs to CAFs,and vice versa,and that micrornas released from CAFs can affect various properties of cancer cells.Overall,the published results suggest an interaction between CAFs and cancer cells that may contribute to tumor invasiveness.ObjectiveTo explore the effect of TNBC-derived exosomes on autophagy of CAFs,and to screen microRNAs that could potentially target and inhibit autophagy of CAFs.Methods:1.Primary CAFs and NFs were extracted by collagenase digestion and identified by immunofluorescence Western blot;2.CAFs were co-cultured with triple negative breast cancer cells(MDA-MB-231 BT549 BT20)by Transwell and changes in autophagy protein were detected;3.Exosomes from triple negative breast cancer cells(MDA-MB-231 BT549 BT20)were extracted by exo-spin kit and qualitative quantified by electron microscopy,Namosight flow cytometry,etc.4.The changes of autophagy protein were detected after CAFs were co-cultured with exosomes from triple negative breast cancer cells(MDA-MB-231 BT549 BT20).5.CAFs were co-cultured with exosomes from triple negative breast cancer cells(MDA-MB-231 BT549 BT20)and then transcriptome sequencing was performed.6.To analyze the effect of exosomes from triple negative breast cancer cells MDA-MB-231 BT549 BT20)on the mRNA levels of CAFs,and to conduct GO Analysis and metabolic Pathway Analysis on the mRNA levels with significant differences;7.The effect of exosomes from triple negative breast cancer cells(MDA-MB-231 BT549 BT20)on miRNA levels in CAFs was analyzed,and the intersection of significantly different miRNA was selected for target gene prediction.After that,GO Analysis and Pathway Analysis were performed,and mirnas that might target and regulate autophagy were screened through gene annotationResults1.Immunofluorescence staining showed that the expression of α-SMA in the cytoplasm of CAFs was significantly higher than that of NFs.Western blot results showed that Vimentin,an mesenchymal marker,was detected in both CAFs and NFs,but E-cadherin,an epithelial marker,was not.Both Vimentin were highly expressed,but significantly lower than NFs in CAFs.α-SMA was expressed in both,but significantly higher in CAFs than in NFs.In conclusion,the above results indicate that the fibroblasts of cancer tissues are CAFs phenotype,with phenotypes such as α-SMA(+),Vimentin(+)and E-cadherin(-),which are consistent with other studies and can meet the needs of further research.2.Western Blot results of CAFs co-cultured with triple-negative breast cancer cells in Transwell showed that Beclin 1,P62 and LC3b autophagy protein levels of CAFs were significantly increased after 48h co-cultured with other three types of breast cancer cells in 0.4um Transwell compartment,respectively,compared with those of non-co-cultured with breast cancer cells.3.The Exo-spin exosome extraction kit adopted in this experiment was used for efficient exosome separation.The results of transmission electron microscopy showed that exosomes with a diameter of 30-120 nm were round or oval"cup-shaped" membranous vesicles,which could be distributed individually or aggregated into clusters.The membrane structure of the vesicle can be seen on the periphery,with a low electron density component in the center.The scatterplot of Nanosight particles suggests that exosomes are relatively concentrated in diameter,most of which are between 30-120nm.The specific marker proteins CD63 and CD81 of exosomes were detected by flow cytometry,and the results showed that all three exosomes expressed CD63 and CD81.In conclusion,exosomes can be extracted from the exosome kit of exo-spin.4.The results of Western Blot after co-culture of CAFs and exosomes from triple-negative breast cancer cells showed that,compared with the control group of CAFs cells,Beclin 1,P62 and LC3b levels of CAFs cells were significantly increased after co-culture of CAFs cells with exosomes from other three breast cancer cells for 48h.5.Through the whole transcriptome sequencing technology,we obtained the expression matrix of mRNA and microRNA.6.To further understand the genetic differences between the treatment group and the control group,and combine them with phenotypes.The software used this time was DEGseq,and the screening conditions for significantly different genes were:1.|log2FC|>1,2.FDR<0.05.Through analysis,it was found that there were 936 significantly different genes in CAFs+20 VS CAFs,763 significantly different genes in CAFs+549 VS CAFs,and 647 significantly different genes in CAFs+231 VS CAFs.7.In order to accurately classify genes functionally,GO Analysis can assign genes with significant differences to different functional classifications.Through GO analysis,we found that these three groups of significantly different genes are closely related to biological processes such as inflammation,regulation of cell cycle,catabolism of collagen,cell proliferation and regulation of cell physiological processes.Moreover,these significantly different genes mainly involved in the analysis of Molecular Function(MF)and Cellular Component,such as chromosomes and extracellular matrix,and concentrated on their functions.8.The relevant genes in the results of Pathway analysis are related in the actual signaling Pathway,and there is a direct interaction between genes.The purpose of signal pathway analysis is to find the signal pathway with significant enrichment of differentially expressed genes based on KEGG database.In terms of KEGG pathway analysis,The screened differential genes were mainly involved in PI3K-Akt signaling pathway,ecm-receptor interaction,Cytokine-Cytokine receptor interaction and TNF signaling Pathway and other pathways closely related to the occurrence and development of tumors.9.Through analysis,it was found that there were 116 significantly different miRNA intersections in the three groups(CAFs+20 VS CAFs,CAFs+549 VS CAFs,and CAFs+231 VS CAFs).Miranda and RNAhybird software were used to predict the relationship between miRNA and differential mRNA.We finally took the intersection of the above two prediction software as the final target gene prediction result,and obtained a total of 3 9,785 target genes.The gene function and metabolic pathway of the target genes were analyzed,and the results of these target genes were consistent with those of mRNA.We annotated these target genes and screened out 35 mirnas related to the targeted regulation of autophagy through gene description.Conclusion4.When TNBC cells were co-cultured with CAFs cells,the expression of autophagy protein in CAFs cells was significantly increased.5.When TNBC exosomes acted on CAFs cells,the expression of autophagy protein in CAFs cells was significantly increased.6.After the effect of TNBC exosomes on CAFs cells,the mRNA and miRNA of CAFs cells changed significantly,and the significantly different mRNA and miRNA showed the trend of autophagy,so as to screen out the miRNA that might target and regulate the autophagy of CAFs.