The Mechanism Of RAB5A Regulating Exosomal Transport Of MiR-21 Affecting Phenotypic Reprogramming Of Macrophages And Malignant Features Of Triple-negative Breast Cancer

Objective: Triple negative cancer(TNBC)lacks effective therapeutic targets due to the loss of ER,PR and HER-2 expression,and patients often have poor prognosis.Tumor cell-derived exosomes act on recipient macrophages by transmitting bioactive molecules such as miRNA,which form M2-phenotype-tumor-associated macrophages with immunosuppressive function,playing an important role in tumor development and the formation of immunosuppressive micoenvironment.Studies have shown that RAB5 A protein can regulate intracellular vesicle foration and exosomesecretion.Therefore,this study preliminarily clarifies the mechanism of RAB5 A regulating exosomesecretion and transmisson of miR-21 mediating TNBC-TAMs interaction affecting macrophage phenotype reprogramming and triple negative breast cancer malignant characteristics through the following three parts of experiments,providing a theoretical basis for finding potential therapeutic targets for TNBC.Chapter One: Regulation of TNBC Cell-Derived Exosome Secretion by RAB5 A Protein and Its Impact on Macrophage PolarizationMethods: Firstly,RAB5 A knockdown MDA-MB-231 and MDA-MB-468 cell lines were constructed using cell transfection technology,and their evaluation was conducted through RT-qPCR and Western blot experiments.Secondly,TNBC-derived exosomes were isolated and purified from MDA-MB-231 and MDA-MB-468 cells,and exosome morphology,particle size,and exosome numbers were observed and quantified using transmission electron microscopy and nanoparticle tracking analysis(NTA).Further investigation was undertaken regarding the impact of RAB5 A knockdown on TNBC-derived exosomesecretion.Subsequently,the uptake of TNBC-derived exosomes by THP-1 cells was observed through PKH26 staining.Finally,the effect of RAB5 A knockdown on macrophage polarization was assessed by examining the mRNA expression of M1(i NOS and IL-1β)and M2(CD163,Arg1,and IL-10)macrophage markers via RT-qPCR,in conjunction with treatment with the exosome inhibitor GW4869 to observe whether macrophage polarization could be influenced by RAB5 A knockdown through the regulation of TNBC-derived exosomesecretion.Results: This study successfully established RAB5 A knockdown TNBC cells.TEM observation revealed that exosomes exhibited typical circular or elliptical structures with diameters ranging from 30 to 150 nm,consistent with exosomal features.NTA results indicated a lower quantity of TNBC-derived exosomes in the RAB5 A knockdown group compared to the control and si NC groups(F: 16.79,P<0.05).PKH26 staining experiment showed uptake of TNBC-derived exosomes by THP-1 cells.RT-qPCR results demonstrated a significant decrease in CD63 and CD9 expression in the RAB5 A knockdown cell culture medium,particularly for CD9 in MDA-MB-468 cell culture medium,suggesting that RAB5 A knockdown could inhibit TNBC-derived exosomesecretion(F: 45.44,P < 0.05).Analysis of macrophage polarization markers showed that RAB5 A knockdown suppressed M2 macrophage marker CD163(F: 161.01,P<0.05),Arg1(F: 39.71,P<0.05),and IL-10(F: 44.59,P<0.05)mRNA expression by inhibiting TNBC-derived exosomesecretion,thereby inducing macrophage polarization.Chapter Two: Regulation of TNBC Exosomal miR-21 Transfer by RAB5A Protein Modulates PELI1 Gene Expression to Promote Macrophage Polarization Towards the M2 PhenotypeMethods: The miRNA differentially expressed in TNBC cells was screened by RT-qPCR.Subsequently,based on the miRNA screening results(miR-21),the impact of RAB5 A knockdown on thesecretion of miR-21 in MDA-MB-231 and MDA-MB-468 cells was detected by RT-qPCR,and the regulatory mechanism of RAB5 A knockdown on miR-21 secretion was explored in conjunction with exosome inhibitor GW4869.Further,the expression of miR-21 in TNBC-derived exosomes was examined through RT-qPCR to investigate its effect and mechanism on macrophage polarization.To elucidate the regulatory mechanism of miR-21 on macrophage polarization,downstream target genes of miR-21 were screened based on the miRDB,Targetscan,and miRTar Base databases,and validation was performed using RT-qPCR and Western blot experiments,with a luciferase reporter assay to detect the interaction between miR-21 and its target gene(PELI1).To investigate whether RAB5 A knockdown could inhibit PELI1 expression by suppressing TNBC-derived exosomesecretion and thus affecting miR-21 delivery,Western blot experiments were conducted to assess the impact of RAB5 A knockdown on PELI1 protein expression.Finally,this study examined the mediation role of PELI1 on miR-21-induced macrophage polarization through RT-qPCR experiments on macrophage biomarkers.Results: Firstly,screening of differentially expressed miRNAs revealed a significant increase in miR-21 expression in MDA-MB-231 and MDA-MB-468 cells after RAB5 A knockdown(t: 9.79,P<0.05).RT-qPCR results showed reduced miR-21 expression in the culture medium of RAB5 A knockdown MDA-MB-231 and MDA-MB-468 cell lines(t:8.70,P<0.05);however,pri-miR-21 and pre-miR-21 levels remained unchanged(t: 1.74,P > 0.05).After blocking exosome synthesis andsecretion with GW4869,miR-21 expression levels unchanged(t: 0.94,P > 0.05),indicating that RAB5 A knockdown reduces miR-21 transport between cells by blocking exosome synthesis andsecretion.Investigation into the effect of miR-21 expression on macrophage polarization revealed that co-culture of THP-1 cells pre-treated with PMA with exosomes overexpressing miR-21 led to a significant upregulation of IL-10(t: 12.61,P<0.05)and CD163(t: 13.01,P<0.05)expression and a suppression of i NOS(t: 7.59,P<0.05)and IL-1β(t: 8.88,P<0.05)expression in THP-1 cells.This suggests that miR-21 overexpression can induce macrophage polarization and depends on TNBC-derived exosomes.Bioinformatics analysis combined with molecular biology experiments showed that PELI1 is a target gene of miR-21 and its expression is inhibited through interaction with PELI1(F: 50.89,P<0.05).Treatment of miR-21 overexpressing THP-1 cells with TNBC-conditioned medium resulted in a significant increase in M2 marker expression and a decrease in M1 marker expression,while PELI1 reversed the regulatory effect of miR-21 on macrophage polarization: IL-10(F: 85.62,P<0.05)and CD163(F: 42..94,P<0.05),i NOS(F: 381.95,P < 0.05)and IL-1β(F: 82.72,P < 0.05).These results indicate that miR-21-PELI1 regulates macrophage polarization.Further research revealed that exosomes derived fromRAB5 A knockdown MDA-MB-231 and MDA-MB-468 cells increased the expression of PELI1 protein in TPH-1 cells(F: 50.89,P<0.05).This suggests that RAB5 A knockdown may promote PELI1 expression by inhibiting TNBC-derived exosomesecretion,thereby mediating macrophage polarization by suppressing miR-21 transport between cells.Chapter Three: The Impact of TAMs Formed through the RAB5A-miR-21-PELI1 Axis on the Biological Characteristics of TNBCMethods: Based on the results,thissection of the study employed CCK-8 assays,scratch assays,Transwell chamber assays,flow cytometry,and immunohistochemistry to investigate the effects of RAB5 A knockdown and miR-21 inhibition on TNBC-derived exosome-treated macrophages regarding the activity,migration,invasion,and apoptosis of MDA-MB-231 and MDA-MB-468 cells.Furthermore,the impact of RAB5 A knockdown on tumor size and weight in mouse TNBC xenografts was observed by injecting RAB5 A knockdown MDA-MB-231 cells.Additionally,immunohistochemistry experiments were conducted to assess the in situ expression of the M2 macrophage marker CD163 in mouse TNBC tumor tissues,validating the mechanism of RAB5A-mediated exosome-transferred miR-21 affecting macrophage phenotype reprogramming and malignant characteristics of triple-negative breast cancer at an in vivo level.Results: Following treatment with TNBC-derived exosomes,the activity,migration,and invasion capabilities of MDA-MB-231 and MDA-MB-468 cells were significantly upregulated,while apoptosis levels were suppressed.In the co-culture system of macrophages treated with TNBC-derived exosomes with RAB5 A knockdown,the activity,migration,and invasion of MDA-MB-231 and MDA-MB-468 cells were inhibited,and apoptosis levels were induced,consistent with the results from the miR-21inhibitor-treated group: activity(t: 5.78,P<0.05),apoptosis(t: 4.81,P<0.05).In vivo experimental results demonstrated that the injection of RAB5 A knockdown MDA-MB-231 cells led to a significant decrease in tumor size and weight.Furthermore,the injection of MDA-MB-231 cells and THP-1 cells resulted in a significant increase in tumor size and weight,while the injection of RAB5 A knockdown MDA-MB-231 cells and THP-1 cells led to a significant decrease in tumor size and weight(F: 408.62,P<0.05).Immunohistochemistry results revealed high expression of CD163 in tumor tissues injected with sh NC-transfected TNBC cells and THP-1 cells,whereas tumor tissues injected with sh RAB5A-transfected TNBC cells and THP-1 cells showed reduced positive staining for CD163(F: 112.82,P<0.05).Conclusion:1.RAB5 A not only acts as an oncogene in TNBC cells,but also plays an important role in promoting exosomessecretion and macrophage polarization;2.The effect of exosmal transport miRNA induce macrophage polarization on tumor microenvironment.By revealing that knocking down RAB5 A inhibits the biosynthesis and delivery of exosomes and reduces the release of miR-21,thus affecting the immune regulatory function of macrophages.This study expanded our understanding of the relationship between exosomes,miRNAs and the formation of tumor immune microenvironment.3.The results of this study can provide theoretical basis for further research and development of TNBC therapy targeting RAB5 A,exosomes and TAMs.