| ObjectiveMacrophages are one of the important cells in the innate immune response and play a key role in antigen presentation and immune defense.In the complex network of tumor microenvironment,macrophages,as the most important immune cells,"love and kill"tumor cells,especially in breast cancer.Breast cancer-associated macrophages(BCAMs),including pro-inflammatory and anti-inflammatory macrophages,accounted for 50%of the total tumor microenvironment cells.In the early stage of tumor,pro-inflammatory macrophages infiltrate into tumor tissue in large numbers and play the role in killing tumor,but in the late stage,anti-inflammatory macrophages play the role in promoting tumor.Although the tumor-promoting properties of BCAMs have been widely recognized and have become effective therapeutic targets,the exact mechanisms that regulate their function and maintain their phenotypes in the microenvironment of breast cancer remain unclear.Long non-codingRNA(lncRNA)is an important regulator of macrophage function,it is closely related to the macrophage polarization.Our previous study found that the expression of lncRNA187415.1 was abnormally increased in BCAMs,so this study mainly explored how lncRNA187415.1 regulates the reprogramming of phenotypic function of BCAMs and its potential mechanism,which is of great significance for the treatment of breast cancer.Methods(1)The expression of lncRNA187415.1 in BCAMs and PMs in tumor-bearing mice was detected by RT-qPCR.The specific siRNA was designed to knock down the expression of lncRNA187415.1,and the knockdown efficiency was verified by RT-qPCR.BCAMs transfected with si-lncRNA187415.1 were co-cultured with 4T1cells for 48h.The proliferation,apoptosis,migration and invasion ability of 4T1 cells were detected by Ki-67 antibody immunofluorescence staining,Annexin V-PI assay and Transwell assay.(2)FCM was used to detect the expression of antigen-presenting molecule MHCII,CD206 and CD86 on the surface of BCAMs.RT-qPCR was used to detect the expression of TNFα,IL-1β,IL-4 and IL-10 mRNA in BCAMs.Western-blot was used to detect the expression of Arg1 and i NOS protein in BCAMs.The content of TNFα,IL-1β,IL-10 and IL-4 in BCAMs supernatant was determined by ELISA.(3)The downstream target genes related to lncRNA187415.1 were analyzed by volcano plot,and the CISH gene with the highest differential expression was screened out.The results were verified by RT-qPCR;The CISH protein level in the BCAMs transfected by si-lncRNA187415.1 was detected by Western-blot;The BCAMs knocked down by si-lncRNA187415.1 were treated with Act D using a working concentration of 5μg/m L.The expression of CISH mRNA and protein in BCAMs were detected by RT-qPCR and Western-blot.The expression of MHCII,CD206 and CD86 on BCAMs were detected by FCM after transfection of CISH overexpressing plasmid(CISH EV and empty CISH OE).(4)PROMO and JASPER database were used to predict the combined lncRNA187415.1 promoter regions of transcription factors and binding sites.The C/EBPβsiRNA was designed and its transfection efficiency was verified by RT-qPCR.In order to further verify the regulation of lncRNA187415.1 expression by C/EBPβ,the mRNA expressions of lncRNA187415.1 and CISH in BCAMS with C/EBPβknockdown were detected by RT-qPCR.The combination of C/EBPβand the promoter of lncRNA187415.1 was detected by luciferase reporter assay.In order to determine the approximate binding region,sequence truncation was performed on the lncRNA187415.1 promoter luciferase plasmid.Four luciferase reporter plasmids containing pro-all(including all sites),pro-1500(including 5 sites),pro-800(including 3 sites)and pro-500(including 1 site)were constructed.(5)A mouse model of breast cancer in situ tumor was established(100μL cell suspension containing 1×10~64T1 cells)was absorbed with a 1m L syringe and injected into the fat pad of the fifth mammary gland of the mouse.When the tumor reached about 100mm~3,50μL of the preformulated in vivo-jet PEI?-Man/si-lncRNA187415.1complex was injected into each in situ tumor mouse,once on six days,and four times in total.The growth of the tumor in situ was observed every four days.The PMs were isolated and BCAMs were selected by magnetic beads.The surface of BCAMs was labeled and stained with CD11b and F4/80 flow cytometry antibody,and the purity of BCAMs was detected by FCM.Results(1)RT-qPCR showed that the knockdown effect of siRNA2 of lncRNA187415.1was the best.Ki-67 antibody immunofluorescence staining found the proliferation of4T1 cells was inhibited after 48h co-cultured;Transwell showed that the number of4T1 cells capable of migration and invasion into the lower chamber was reduced in the siRNA2 group;FCM showed that the apoptosis rate of 4T1 cells was significantly increased after co-cultured with siRNA2-transfected BCAMs.(2)FCM showed that the expression of CD206 in BCAMs knocked down by lncRNA187415.1 was lower than that in si-NC group and positive control group,while the expression of CD86 and MHCII were increased;The mRNA expression of TNFαand IL-1βin transfected BCAMs cells were significantly higher than those in si-NC group,while IL-4 and IL-10 were significantly down-regulated;Western-blot showed that the expression of Arg1 in transfected BCAMs was significantly lower than that in si-NC group,while the expression of i NOS was significantly increased;The contents of TNFαand IL-1βin the supernatant of BCAMs transfected by ELISA were significantly higher than those in si-NC group,while the levels of IL-10 and IL-4 were significantly lower.(3)The highest differential CISH was screened by volcano plot.The results of Western-blot and RT-qPCR confirmed that the expression of CISH mRNA and protein in transfected BCAMs was decreased.RT-qPCR showed that down-regulation of lncRNA187415.1 could inhibit the degradation of CISH.Western-blot showed that the expression of CISH in BCAMs transfected with Act D for 24h was significantly lower than that of si-NC.FCM results showed that CD86 and MHCII expression were decreased and CD206 expression was increased on the surface of transfected BCAMs after CISH overexpression.(4)PROMO database was used to predict the combined lncRNA187415.1transcription factor C/EBPβin the promoter region.Then use JASPER database to predict the C/EBPβand lncRNA187415.1 promoter region can be combined with eight sites.RT-qPCR showed that siRNA3#in C/EBPβ-specific siRNA had the best transfection efficiency,and the expression of lncRNA187415.1 and CISH mRNA in BCAMs cells with C/EBPβknockdown were significantly reduced.Dual-luciferase reporter assays showed that the luciferase activity at the plasmid of C/EBPβand the promoter of lncRNA187415.1 was significantly enhanced,which proved that C/EBPβwas the transcription factor of lncRNA187415.1,and only the pro-1500 region was activated.We found that the promoter region of C/EBPβand lncRNA187415.1 was bound between-1.5kb and-2 kb.The results were verified by Ch IP-qPCR.(5)PMs were isolated from model mice and BCAMs were screened by magnetic bead separation.The purity of BCAMs by magnetic bead separation could reach96.61%.RT-qPCR showed that the expression of lncRNA187415.1 in BCAMs was significantly increased compared with that in peritoneal macrophages.At the later stage of the model,compared with the mice in the si-NC group,the tumor of the mice in the siRNA2 group was significantly lighter and the tumor was significantly smaller.Conclusion:(1)LncRNA187415.1 activated by C/EBPβcan inhibit CISH mRNA degradation and maintain the pro-tumor function of BCAMs(2)Targeting lncRNA187415.1 in BCAMs can delay the progression of breast cancer... |
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