| Background:Breast cancer(BC)is a highly prevalent malignancy in women worldwide,and hormone receptor positive(HR+)is the most common BC subtype,accounting for about 70%of cases.Because of the strong dependence of BC on the estrogen-estrogen receptor axis,endocrine therapy(ET)has been the conventional treatment for HR+BC.Tamoxifen(TAM)is one of the most effective ET drugs for HR+BC and has been used clinically for more than 30 years,reducing the annual mortality rate of BC by about 31%.However,about 30%to 50%of patients still have disease progression due to TAM resistance,which seriously affects the survival of patients.Therefore,investigating the mechanism of TAM resistance is one of the priorities of BC research.Many studies have been conducted on TAM resistance signaling pathways,but the mechanisms of resistance are often extremely complex,and a fragmented understanding does not really solve this challenge.This makes it possible that even if a new drug with the appropriate mechanism is used in the clinic,a new drug resistance will soon emerge.Therefore,it is particularly important to integrate these factors into a mechanistic platform.In recent years,more and more researchers have realized that breast cancer stem cells(BCSCs)play an important central role in TAM drug resistance,and BSCSs have a strong multidirectional differentiation potential and the ability to self-renew.BCSCs may seem to be a promising platform,however,BCSCs and BCCs are always in a dynamic state of change,which makes them difficult to become precise therapeutic targets.Then,blocking the conversion of BCCs to BCSCs and regulating the homeostasis of BCSCs in TME seems to be a better strategy.Exosome(EXO)is a 30-200 nm diameter membranous vesicle released by almost all cell types,which mediates intercellular communication by delivering mRNAs,miRNAs,DNA and proteins,and it is a function of tumor-stromal interactions and epithelial-mesenchymal transition(EMT).transition(EMT).EXO was found to have a role in regulating the dynamics of tumor stem cells(CSCs)in TME,thereby inducing environmentally mediated therapeutic resistance.As a messenger of cellular interactions,EXO regulates BCSCs homeostasis in TME.Bone marrow mesenchymal stem cells(BMMSCs)are an important component of TME and play an important role in promoting tumor progression.Tumors select for BMMSCs through EXO,reprogramming their functional properties from normal nutrition to tumor promotion.The reprogrammed BMMSCs produce and release large amounts of EXO,which not only directly promote the growth of tumor cells,but also horizontally deliver to fibroblasts,endothelial cells and immune cells in TME,indirectly enhancing their tumor-promoting function,so BMMSCs/EXO have great potential to be explored.TCM treatment of BC emphasizes evidence-based therapy and treatment of the disease at its root.Since the action of TCM is multi-targeted,it is urgent to explore the reversal of TAM resistance in HR+BC patients from the integrated platform of BCSCs,which has a certain theoretical and practical basis.SGYS is a clinically effective formula for HR+BC,which is based on the experience of our famous veteran TCM doctors and theoretical understanding of the nature of HR+BC disease.The group’s preliminary basic research found that SGYS can inhibit several TAM resistance-related signaling pathways.This study intends to investigate the mechanism of TAM resistance and the reversal effect of SGYS from the perspective of TME-BCSCs-EXO,to explore the scientific essence of HR+BC liver qi stagnation and dysregulation of punching and reign,and to provide scientific data for the precise transformation of SGYS.Purpose:1.To clarify that BMMSCs/EXO and BCCs/EXO crosstalk in TME regulate BCSCs homeostasis and induce TAM resistance.2.To obtain the core targets of SGYS,the key gene sets related to TAM drug resistance,the differential miRNAs of BMMSCs/LCC9/EXO and BMMSCs/MCF-7/EXO and their target genes,respectively,using pharmacology of Chinese medicine network,bioinformatics analysis,and EXO miRNA sequencing,and to use the intersecting genes of the three as candidate target genes to screen SGYS key EXO miRNAs and their target genes for reversing TAM drug resistance.3.In vitro and in vivo validation of key miRNAs and their target genes that regulate homeostasis of BCSCs and induce TAM resistance delivered by BMMSCs/EXO to MCF-7 at three levels:molecular,cellular and animal.4.To explore at the clinical level that SGYS can reverse TAM resistance by regulating plasma EXO target miRNAs and their target gene protein levels,and to provide a basis for clarifying the efficacy of SGYS and the targets and mechanisms of its action.Methods:1.In vitro and in vivo investigation of BMMSCs/EXO and BCCs/EXO crosstalk in TME to regulate homeostatic induction of TAM resistance in BCSCs①MCF-7 and LCC9 cell lines and BMMSCs were used.MCF7/EXO and LCC9/EXO were obtained by differential centrifugation.The two types of EXO were co-cultured with BMMSCs,and the EXO released from the two re-edited BMMSCs was extracted again separately to obtain BMMSCs/LCC9/EXO and BMMSCs/MCF-7/EXO.The The two BMMSCs/EXO were co-cultured with MCF-7 cells,and MCF-7+PBS was used as a control group.western blot was performed to detect the expression of EMT and CSCs markers.cck8 was used to assess TAM resistance;flow assay was performed to detect the rate of apoptosis and G2/M phase block after TAM dosing;Aldefluor,cell sphere-forming assay was performed to assess the tumor stem cell The tumor stem cell-like properties were assessed by Aldefluor and cell sphere-forming assay;cell migration ability was assessed by Transwell.②After tumor formation,both mice were orally gavaged with tamoxifen citrate and injected with two types of BMMSCs/EXO at multiple points in the tumor.The tumor growth curves of the two groups of mice were plotted,and the expression of CSCs markers and EMT markers of breast cancer in the two groups were detected by WB.2.Screening of key EXO miRNAs and their target genes for TAM resistance induction by BMMSCs/EXO and BCCs/EXO crosstalk using a combination of Chinese medicine network pharmacology,bioinformatics analysis,and EXO miRNA sequencing①The miRNAs of BMMSCs/LCC9/EXO and BMMSCs/MCF-7/EXO were sequenced by high-throughput sequencing,and the differential miRNAs between groups were screened using edgeR package difference analysis;the candidate target genes of the differential miRNAs were obtained by searching the database.②Search the active ingredient and target genes of SGYS and disease genes of HR+BC from the database,respectively.The target genes of SGYS were intersected with HR+BC-related genes to obtain the target genes of SGYS for HR+BC.The obtained target genes were imported into String database to construct the protein-protein interaction network(PPI)of important target genes,imported into Cytoscape for network analysis,and the core target genes of SGYS were screened according to Degree value.③Download mRNA sequencing data of MCF-7 and LCC9 cell lines from the Gene Expression Omnibus(GEO)database,gene chip data of 298 ER+ BC patients treated with TAM for 5 years and clinical information of their distant metastases,and single-cell sequencing data of tumor tissues from 6 ER+BC patients.Differentially expressed genes(DEGs)between MCF-7 and LCC9 cells were screened using the limma R package.Based on distant metastasis information,patients who relapsed within 3 years were categorized as TAM-resistant group,and those who did not relapse after more than 10 years of follow-up were categorized as sensitive group.Weighted gene co-expression network analysis(WGCNA)was performed using the WGCNA R package.Module-trait relationship analysis was performed to explore modules associated with TAM resistance,and modules with correlation coefficients>0.3 and P<0.05 were considered significant.The intersection of DEGs between MCF-7 and LCC9 with the significant module genes of WGCNA was taken as the TAM resistance-related gene set.The final candidate target genes and their upstream EXO miRNAs were obtained by taking the intersection of the above three sets of genes.3.In vitro and in vivo validation of BMMSCs/EXO to induce TAM resistance and stem cell-like properties by delivering target miRNAs to MCF-7 cells and down-regulating their target gene expression①MCF-7 cells were co-cultured with BMMSCs/LCC9/EXO and transfected separately into the following six groups:miR-X mimic group,NC-mimic group,miR-X inhibitor group,NC-inhibitor group,miR-X inhibitor+shRNA-mRNA group,miR-X inhibitor+NC-shRNA group.qRT-PCR to detect miRNA-X and its target gene mRNA levels,Western Blot to detect target gene protein levels,EMT and CSCs marker expression.CCK8 to assess TAM resistance;flow assay to detect apoptosis rate and G2/M phase block rate after TAM dosing.Aldefluor,cell sphere-forming assay to assess tumor stem cell-like properties;Transwell to assess migration ability.②qRT-PCR to detect the expression levels of target miRNAs and their target gene mRNAs in tumor tissues of two groups of tumor-bearing mice in study method 2.WB to detect the expression levels of target gene proteins.4.Clinical study of the effect of SGYS on plasma exosomal miRNA and its target genes in TAM-resistant patientsTen patients with TAM-resistant and TAM-sensitive breast cancer were collected using a non-randomized controlled clinical study.The patients in the TAM-resistant group were given SGYS regularly for 2 weeks after enrollment.At the end of treatment,peripheral blood was collected again from patients in the TAM-resistant group,and the TCM Symptom Score Scale and the Quality of Life Scale were completed.The levels of plasma exosome miR-X and its target plasma protein were measured before and after treatment in the TAM-resistant group and in the TAM-sensitive group.Results:1.Crosstalk between BMMSCs/EXO and TAM-resistant cells LCC9/EXO can convey TAM resistance and stem cell-like properties to sensitive cells MCF-7The IC50 values of TAM were 6.657 μM in MCF-7+PBS group<14.90 μM in MCF-7+BMMSCs/MCF-7/EXO group<21.27 μM in MCF-7+BMMSCs/LCC9/EXO group;the number of cell-forming spheres in MCF-7+PBS group(4.667±0.943)<MCF-7+BMMSCs/MCF-7/EXO group(22±6.481)<MCF-7+BMMSCs/LCC9/EXO group(36.667 ±13.337);breast cancer stem cell ratio MCF-7+BMMSCs/LCC9/EXO group(6.107±0.103)>MCF-7+BMMSCs/MCF-7 group(1.960±0.050)>MCF-7+PBS group(0.853 ± 0.017);apoptosis ratio after TAM dosing MCF-7+PBS group(39.550 ± 0.136)>MCF-7+BMMSCs/MCF-7/EXO group(22.447±0.384)>MCF-7+BMMSCs/LCC9/EXO group(11.697±0.288);the proportion of G2/M phase blocked cells after TAM dosing in MCF-7+PB group(56.933±6.594)>MCF-7+BMMSCs/MCF-7/EXO group(32.883±1.082)>MCF-7+BMMSCs/LCC9/EXO group(8.763± 1.231);cell migration number MCF-7+BMMSCS/LCC9/EXO group(1124± 244.110)>MCF-7+BMMSCs/MCF-7/EXO group(268.667±43.622)>MCF-7+PBS group(85.333± 32.149);Western blot The results showed that the expression of E-Cadherin,ERa and CD24 in MCF-7+BMMSCs/MCF-7/EXO group and MCF-7+BMMSCs/LCC9/EXO group showed an overall decreasing trend,while the expression of N-Cadherin,CD44 and Snail showed an overall increasing trend compared with the control MCF-7+PBS group.The differences between the above experimental groups were statistically significant(P<0.05).The expression of E-Cadherin,ERa and CD24 were lower in the MCF-7+BMMSCs/LCC9/EXO group,while the expression of N-Cadherin,CD44 and Snail were higher in the MCF-7+BMMSCs/LCC9/EXO group.expression was higher;the differences between the two groups in the above experiments were statistically significant(P<0.05)2.The target EXO miR-27a-3p and its target gene BAK1 were obtained through a combination of EXO miRNA sequencing,Chinese medicine network pharmacology and bioinformatics analysis.The differential miRNAs between BMMSCs/LCC9/EXO and BMMSCs/MCF-7/EXO were screened by EXO miRNA sequencing and their target genes were predicted,and a total of 37 miRNAs with more than 4-fold expression difference and their target genes 2624 were obtained;through the network pharmacology study of SGYS,127 core target genes of SGYS for the treatment of ER+/PR+BC core target genes;244 TAM resistance-related gene sets were obtained by bioinformatics analysis based on public databases;a total of 3 genes,BAK1,EGFR,F3 and all 3 core target genes were target genes of miR-27a-3p were obtained by taking the intersection of the three,and BAK1 was finally identified as a downstream molecule of miR-27a-3p through pre-experiments.for subsequent studies.3.BMMSCs/EXO can induce TAM resistance and stem cell-like properties in MCF-7 by down-regulating BAK1 expression through delivery of miRNA-27a-3p to MCF-7 cells①Dual luciferase assay suggested that miR-27a-3p mimic had a significant inhibitory effect on luciferase in BAK1-WT,but in the presence of BAK1-MUT,miR-27a-3p mimic had no significant inhibition on luciferase activity,therefore,it can be assumed that miR-27a-3p has a direct binding to BAK1.qRT-PCR The results showed that miR-27a-3p was negatively regulated with BAK1 in both cells and tumor tissues;Western blot results showed that miR-27a-3p was negatively correlated with BAK1 expression in mouse tumor tissues.②The IC50 values of each group on TAM were miR-27a-3p mimic group 30.49μM>miR-27a-3p inhibitor+sh-BAK1 group 22.77μM>NC-inhibitor group 20.11μM>NC-mimic group 19.88μM>miR-27a-3p inhibitor group 16.88μM>miR-27a-3p inhibitor+sh-NC group 16.58μM.cell sphere-forming ability of the six groups miR-27a-3p mimic group(78.92±17.88)>NC-inhibitor group(39.67± 12.56)>NC-mimic group(35.67±9.13)>miR-27a-3p inhibitor ± sh-BAK1 group(9.33±3.25)>miR-27a-3p inhibitor group(4.75±1.79)>miR-27a-3p inhibitor+sh-NC group(4.25±1.42).Proportion of ALDH+cells in each group(%)miR-27a-3p mimic group(7.803±0.539)>NC-inhibitor group(5.547±0.318)>NC-mimic group(5.26±0.301)>miR-27a-3p inhibitor+sh-BAK1 group(3.65± 0.119)>miR-27a-3p inhibitor+sh-NC group(2.05±0.110)>miR-27a-3p inhibitor group(1.967±0.301).Proportion of cells in each group that developed G2/M phase organization after TAM dosing(%)miR-27a-3p mimic group(5.75±0.44)<NC-inhibitor group(9.90±2.28)<NC-mimic group(10.38±2.26)<miR-27a-3p inhibitor +sh-BAK1 group(15.80±0.64)<miR-27a-3p inhibitor+ sh-NC group(18.83±1.53)>miR-27a-3p inhibitor group(20.663±5.48).The proportion of cells that underwent apoptosis after TAM dosing in each group(%)miR-27a-3p mimic group(4.75±0.273)<NC-mimic group(8.307±0.132)<NC-inhibitor group(8.577±0.366)<miR-27a-3p inhibitor+sh-BAK1 group(15.57±0.123)<miR-27a-3p inhibitor group(24.373±0.506)<miR-27a-3p inhibitor+sh-NC group(25.123 ±0.260).transwell cell migration number miR-27a-3p mimic group fine(957.67±97.41)>NC-mimic group 97.41)>NC-mimic group(345.67±40.75)>miR-27a-3p inhibitor+sh-BAK1 group(324.67±73.92)>NC-inhibitor group(281 ±28.08)>miR-27a-3p inhibitor group(70.33± 14.38)>miR-27a-3p inhibitor+sh-NC group(59±14.51).Some of the above experiments did not produce statistical differences between the groups,but the overall trend of TAM resistance and tumor stem cell-like properties were enhanced with the increase of miR-27a-3p level;inhibition of miR-27a-3p could inhibit TAM resistance and stem cell-like properties caused by the uptake of exosomal miR-27a-3p,and this inhibition could be achieved by This inhibition could be reverted to some extent by silencing of BAK1.4.The expression of plasma exosomal miR-27a-3p was relatively downregulated in the TAM-resistant group before treatment compared with the TAM-sensitive group,and the expression of plasma exosomal miR-27a-3p was downregulated in the TAM-resistant group after treatment compared with that before treatment,but the differences were not statistically significant./mL)was significantly lower than that of patients in the TAM-sensitive group(34.901±4.04μg/mL),P<0.0001;and after treatment with the Shuganyishen formula,BAK1 protein in the TAM-resistant group decreased to 21.67 ± 4.13μg/mL,and the difference was non-significant compared with that before treatment(P>0.05).And the TCM symptom score and quality of life were significantly improved(P<0.05).Conclusion:1.In the tumor microenvironment,crosstalk of BMMSCs/EXO and BCCs/EXO can induce TAM resistance by delivering EXO miR-27a-3p to sensitive MCF-7 cells,down-regulating the expression level of its target gene BAK1,delivering TAM resistance information,and regulating the homeostasis of BCSCs in the microenvironment.2.The Shuganyishen formula can improve the quality of life and TCM symptom scores of TAM-resistant patients and may have a regulatory effect on the levels of plasma EXO miR-27a-3p and its target gene BAK1 in TAM-resistant patients. |
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