| Objective:Breast cancer of women overtakes lung cancer for the first time as the most common cancer in worldwide.For locally advanced breast cancer,neoadjuvant chemotherapy may improve the feasibility and success of surgery.However,not all patients achieve complete pathological remission after neoadjuvant chemotherapy,and the reasons for this remain to be explored.Exosomes are a class of small membranous vesicles containing complex RNA and proteins,approximately 30-150 nm in diameter.Exosomes are specific membranous vesicles by cells and are involved in intercellular communication.Studies have found that chemotherapy may promote the progression of cancer,chemotherapy is administered in multiple cycles,and the surviving tumor cells reproliferate during the interval of treatment,which is the main reason for treatment failure in chemotherapy.In contrast,after received chemotherapy,dying tumor cells secrete a variety of cytokines and exosomes,which affect the tumor microenvironment and cause chemoresistance.The aim of this study was to investigate the changes of tumor cells and their exosomes after stimulated by chemotherapeutic drugs,and the relevant mechanisms of these exosomes regulated chemoresistance,so as to provide potential targets for improving the effect of chemotherapy.Methods:1.Collected serum exosomes of patients who received anthracene and paclitaxel neoadjuvant chemotherapy before,after one cycle and after four cycles of treatment,and changes in miRNA expression of exosomes during different chemotherapy periods were detected by miRNA sequencing technology.Analysis of the correlation between miR-378a-3p and miR-378 d expression in serum exosomes and chemotherapy response in patients before and after neoadjuvant chemotherapy using RT-PCR to validate primary sequencing results.Western blot was used to detect the expression of stemness-related proteins before and after neoadjuvant chemotherapy in tumor tissues of patients with poor efficacy of neoadjuvant chemotherapy.And the correlation of the expression of miR-378 and stemness-related genes was analyzed by TCGA database data.2.Construction of in situ NSG mouse models of CAL51 breast cancer cells,doxorubicin or PBS-induced exosomes secreted by CAL51 breast cancer cells were injected around the tumor after tumor formation,respectively,followed by tail vein injection of the chemotherapeutic drug doxorubicin 4mg/kg or PBS,changes of tumor size were measured and growth curves were plotted.CAL51,MDA-MB-231 and MCF7 breast cancer cells were treated with the doxorubicin,paclitaxel or PBS for 4hours,respectively,after that extracted and purified exosomes to treat corresponding tumor cells.The drug resistance of cells was tested by CCK8 assay;the CD44+/CD24-stem-like cell population content was detected by flow cytometry;3D tumor spheres were cultured to detect tumor stemness and treated with chemotherapeutic agents to tested the sensitivity of drugs.3.RT-PCR was performed to detect changes in miR-378a-3p and miR-378 d contents in exosomes secreted from CAL51,MDA-MB-231 and MCF7 breast cancer cells after treatment with doxorubicin,paclitaxel or PBS for 4 hours and in cells after co-culture of the above exosomes with the corresponding cells for 48 hours.After making breast cancer cells overexpress miR-378a-3p and miR-378 d,CCK8 assay was performed to detect drug resistance in transfected cells;cellular CD44+/CD24-stem-like cell population content and tumor stemness sphere-forming assay were performed to detect changes in tumor stem-like cellularization characteristics of transfected cells.4.GSEA analysis was performed using TCGA database gene expression data to analyze the pathways significantly enriched in miR-378 expression profile.The target genes of miR-378a-3p and miR-378 d were predicted by Mi Randa software.RT-PCR was performed to detect the changes of target genes after overexpressed miR-378a-3p and miR-378 d in CAL51 breast cancer cells,then screened for target genes.A dual luciferase reporter gene vector was constructed to detect whether DKK3 and NUMB were common target genes of miR-378a-3p and miR-378 d.5.Western Blot assay to detect protein expression of CAL51,MDA-MB-231 and MCF7 breast cancer cells transfected with miR-378a-3p and miR-378 d mimics/inhibitors;and the protein expression of miR-378a-3p and miR-378 d mimics and DKK3/NUMB overexpression plasmids.6.Three types of breast cancer cells were treated with chemotherapy-induced exosomes and then transfected with DKK3/NUMB overexpression plasmids,and the expression levels of DKK3,NUMB and related stemness pathway proteins were detected by western blot assay;and the stemness changes of the treated cells were detected by tumor stemness sphere-forming assay.7.Immunohistochemical staining of DKK3 and NUMB was performed on tumor tissues before and after treatment in patients with poor neoadjuvant chemotherapy efficacy.And the correlation analysis of miR-378 expression with DKK3 and NUMB gene expression was performed using TCGA database data.8.Western Blot assay was used to detect intracellular EZH2 and STAT3 protein expression levels after 4 hours of doxorubicin or paclitaxel chemotherapeutic drug effects.The expression levels of EZH2 and STAT3 were knocked down by small interfering RNA in CAL51 cells,and then treated with 4 hours of doxorubicin or paclitaxel chemotherapeutic drugs.Then the exosomes of treated tumor cells were collected.Using RT-PCR to detect the changes of miR-378a-3p and miR-378 d contents in the cells and exosomes.And after CAL51 cells treated by the above exosomes,CCK8 assay to detect the drug resistance of the cells,and tumor stemness sphereforming assay or the CD44+/CD24-stem-like cell population assay to detect the stemness of the tumor cells.9.Detected whether STAT3 could bind to the promoter regions of miR-378a-3p and miR-378 d by ChIP assay.10.Mouse models of in situ NSG of CAL51 breast cancer cells was constructed,and after tumor formation,doxorubicin or PBS-induced exosomes secreted by CAL51 breast cancer cells were injected around the tumor,and then divided into four groups,which were treated with tail vein injection of doxorubicin,PBS,oral tazemetostat or doxorubicin combined with tazemetostat,to measure the change of tumor size and plot the growth curve.Results:1.Small RNA sequencing results showed increased expression of miR-378a-3p,miR-378 c,miR-378 d and miR-4488 in serum exosomes after chemotherapy.And among them,miR-378a-3p and miR-378 d were associated with poor response to chemotherapy.Patients with poor efficacy of neoadjuvant chemotherapy showed increased expression of stemness-related proteins in tumor tissues after treatment.Analysis of TCGA database data revealed a positive correlation between miR-378 expression and expression of β-catenin,NOTCH1 and EZH2 stemness-related genes.2.In vivo experiments in NSG mice showed that tumors became resistant to chemotherapy after injection of doxorubicin-induced exosomes around the tumors.The tumor cell exosomes induced by doxorubicin and paclitaxel chemotherapy induced increased drug resistance and stemness characteristics in the recipient cells.3 Doxorubicin and paclitaxel chemotherapy induced an increase in miR-378a-3p and miR-378 d content in the exosomes of tumor cells.The expressions of miR-378a-3p and miR-378 d were also increased in the cells after treated the corresponding cells by chemotherapy-induced exosomes.Moreover,breast cancer cells overexpressing miR-378a-3p and miR-378 d showed increased drug resistance and content of stem-like cell population.4.GSEA analysis was performed and miR-378 high expression was found to be significantly enriched to WNT and NOTCH stemness pathways.The target genes of miR-378a-3p and miR-378 d were predicted by Mi Randa software are DKK3 and NUMB,and DKK3 and NUMB were verified to be common target genes of miR-378a-3p and miR-378 d by RT-PCR and dual luciferase reporter assay.5.DKK3 and NUMB protein expression decreased and stemness pathway protein expression increased after transfection of miR-378a-3p and miR-378 d mimics in three breast cancer cells,while expressions after transfection of miR-378a-3p and miR-378 d inhibitors were opposite.The activation of the stemness pathway by miR-378a-3p and miR-378 d targeting DKK3 and NUMB was inhibited after simultaneous transfection of miR-378a-3p and miR-378 d mimics and DKK3/NUMB overexpression plasmids.6.Treatment of three breast cancer cells with chemotherapy-induced exosomes followed by transfection with DKK3/NUMB overexpression plasmids revealed that the increase in stemness caused by exosomes was suppressed;and the corresponding protein expression content was also suppressed.7.The expression of DKK3 and NUMB was reduced in tumor tissues after treatment in breast cancer patients with poor efficacy of neoadjuvant chemotherapy.The analysis of TCGA database data revealed that miR-378 expression was negatively correlated with the expression of DKK3 and NUMB genes.8.CAL51 cells showed increased intracellular protein expression of EZH2 and STAT3 after 4 hours treat by doxorubicin or paclitaxel chemotherapeutic drugs.And after knocking down the expression levels of EZH2 and STAT3 with small interfering RNA and stimulation of cells with doxorubicin or paclitaxel chemotherapeutic drugs,the levels of miR-378a-3p and miR-378 d were decreased in the cells and exosomes.And CAL51 cells treated with the above exosomes,the cellular resistance induced by chemotherapy-induced exosomes disappeared,and the increased stemness of tumor cells also disappeared.9.ChIP experiments verified that STAT3 could bind to the promoter regions of miR-378a-3p and miR-378 d and regulated downstream.10.In vivo experiments in NSG mice revealed that inhibitior of EZH2 reversed chemoresistance caused by chemotherapy-induced exocytosis.And the combination of tazemetostat and doxorubicin enhances the efficacy of doxorubicin.Conclusions:1.Tumor cells secrete and deliver exosomes with high expression of miR-378a-3p and miR-378 d after chemotherapy,thereby enhancing drug resistance in chemotherapy residual cells.miR-378a-3p and miR-378 d directly target DKK3 and NUMB,thereby activating the WNT/β-catenin and NOTCH stemness pathways,which in turn enhance the stemness characteristics of tumor leading to increased drug resistance2.The EZH2/STAT3 pathway is activated in tumor cells after chemotherapy,and STAT3 can bind with the promoter region of miR-378a-3p and miR-378 d,which causes high expression of miR-378a-3p and miR-378 d in intracellular and exosomes. |
PDF Full Text Request