Exosomal LncRNA SNHG16 Derived From TAMs Promoted The Metastasis Of Triple Negative Breast Cancer In Tumor Hypoxia Microenvironment

Background and objectivesBreast cancer is the largest malignant tumor in the world and has the highest incidence in women which seriously threatens women health and life safety.Among different types of breast cancer,triple negative breast cancer(TNBC)lacks targeted and specific treatment methods due to the lack of ER receptor and HER-2 receptor.In addition,TNBC progresses rapidly with a high degree of malignancy,and is easy to lymph node metastasis and distant metastasis,which seriously harms the survival and prognosis of breast cancer patients.Tumor-associated macrophages(TAMs)are the most abundant immune cells in the tumor microenvironment,and are currently one of the most promising therapeutic targets.A large number of studies have proved that TAMs can significantly affect the recurrence and metastasis of breast cancer.TAMs secrete a variety of cytokines that promote invasion and metastasis,induces the activation of a variety of matrix metalloproteinases,and binds with angiotensin to promote angiogenesis,extensively participating in various processes of tumor metastasis.Hypoxia is the basic feature of the tumor microenvironment of most solid tumors,which is also a powerful driving force to promote tumor metastasis.Moreover,tumor cells secrete various chemokines to recruit TAMs to the hypoxia region in the hypoxia microenvironment,which induces tumor metastasis further.Exosomes,which have been a hot topic of interest for researchers in recent years,are mediators of biogenic intercellular communication and are widely involved in the progression of tumor malignancy.Tumor cells usually release more exosomes to communicate between proximity and distance in the tumor microenvironment.Exosomes are involved in almost all tumor features,including sustained proliferation,angiogenesis,immune evasion,genomic instability,resistance to apoptosis,dysregulated energy metabolism,invasion and metastasis.And hypoxic microenvironment induces the secretion of exosomes as well as the loading of their contents.However,the effect of exosomes derived from TAMs in hypoxic microenvironment on TNBC has not been studied.Based on high metastasis rate ofTNBC,our project focused on the features of the tumor microenvironment,i.e.the pro-metastatic effect of TAMs and the hypoxic tumor microenvironment.Exosomes derived from hypoxic TAMs were investigated to identify targets that effectively inhibit the metastasis of triple negative breast cancer and provide new theoretical basis and strategies for the prevention and treatment of TNBC.Methods:1.Differentiation of THP-1 cells to TAMs and identification.Human bone marrow mononuclear cell line THP-1 cells were differentiated to TAMs with the culture medium of MDA-MB-231 cells(conditioned medium)in vitro experiment.The expression of M2 phenotype markers CD68 and CD206 in TAMs induced by conditioned medium were detected by flow cytometry.2.Establishment and identification of hypoxic model.The hypoxia incubator(1%O2,5%CO2,37℃)was used as a hypoxia model.The expression and distribution of HIF-la in TAMs under normoxic and hypoxic condition was determined by immunofluorescence.3.Extraction and identification of exosomes secreted by TAMs.TAMs were incubated with medium containing exosome-free serum under normoxic or hypoxic condition for 48h.Exosomes were extracted from the supernatant using ultra-high speed centrifugation and purified by the commercial kit.The extracts were identified as exosomes using transmission electron microscopy(TEM),zeta potential and nanoparticle size analyzer(DLS)and Western blot method to observe the morphology,particle size,and the expression of protein markers CD9 and CD63,respectively.4.Detection of the effect of exosomes derived from hypoxic TAMs on the survival,migration,invasion and vascular mimicry formation of MDA-MB-231 cells.DiR staining was applied to the exosomes and after co-incubation with MDAMB-231 cells for 4h,the uptake of exosomes by tumor cells was observed by fluorescence microscopy.Exosomes from normoxic and hypoxic TAMs were quantified by BC A protein quantification and co-incubated with MDA-MB-231 cells for 24h.The viability of the tumor cells was examined by MTT assay.Exosomes extracted from normoxic and hypoxic TAMs with the same cell number were incubated with MDA-MB-231 cells for 24h.The effects of exosomes extracted from normoxic and hypoxic TAMs on the migration,invasion and vascular mimicry formation of MDA-MB-231 cells were detected by scratch assay,transwell migration assay,transwell invasion assay and vascular mimicry formation assay respectively.5.Detection of the effect of LncRNA SNHG16 in exosomes derived from hypoxic TAMs on the migration,invasion and vascular mimicry formation of MDA-MB-231 cells.The expression levels of SNHG16 in invasive breast cancer or in different breast cancer types,and the correlation with survival prognosis were analyzed by the UALCAN platform combined with the TCGA database.The expression levels of SNHG16 in normoxic and hypoxic TAMs,their secreted exosomes and co-incubated tumor cells were detected by qPCR.The expression levels of SNHG16 in TAMs as well as in MDA-MB-231 cells after siRNA-SNHG16 knockdown were detected by qPCR.Exosomes secreted from hypoxic TAMs which were transfected with siRNASNHG16 in advance were co-incubated with MDA-MB-231 cells(SNHG16 normal expression)or with MDA-MB-231 cells transfected with siRNA-SNHG16.Transwell migration assay,Transwell invasion assay,and vascular mimetic formation were used to detect the effect of down-regulation of SNHG16 expression in TAMs or/and tumor cells on the metastasis and vascular mimicry formation of MDA-MB-231 cells.6.Detection of the downstream molecular mechanism of LncRNA SNHG16 on the promotion of the migration,invasion and vascular mimicry formation of MDA-MB231 cells.The LncRNA subcellular localization information platform(LncATLAS platform https://lncatlas.crg.eu/)was used to analyze the localization information of SNHG16 in 15 human cell lines.Nucleus-cytoplasmic fractionation assay was used to analyze the localization information of SNHG16 in MDA-MB-231 cells.miRNAs with binding sites to SNHG16 and expression levels negatively correlated with SNHG16 in breast cancer were searched by Starbase database,TCGA database and bioinformatics literatures.After downregulating SNHG16 in MDA-MB-231 cells,the expression levels of the screened miRNAs were detected by qRT-PCR.The expression levels of miR-205-5p in invasive breast cancer,and in patients with different lymph node metastasis status were analyzed by UALCAN database and TCGA database.Dual luciferase reporter gene assay was performed to detect whether SNHG16 directly binds to miR-205-5p.The miR-205-5p inhibitor or NC was co-transfected with siRNA SNHG16 to observe the metastatic ability of MDAMB-231 cells using scratch assay,transwell invasion assay,and vascular mimicry assay.Starbase database was used to analyze the co-expression analysis between SNHG16 and the screened target proteins in breast cancer.Then we performed transfection siRNA-SNHG16 or miR-205-5p mimics to verify their regulatory effect on the target protein.Rescue experiment was used to verify the upregulation of SNHG16 on the expression of the target protein by inhibiting miR-205-5p.7.Detection of the promotion of exosomes derived from hypoxic TAMs on the lung metastasis of MDA-MB-231 cells in nude mice.The exosomes secreted by TAMs under normoxic and hypoxic environments were collected and co-incubated with MDA-MB-231-Luc cells.And then MDA-MB-231Luc cells were injected into nude mice through the tail vein.The pulmonary metastasis of MDA-MB-231-Luc cells in vivo was observed by IVIS.Lung tissues of nude mice were isolated to observe the pulmonary metastatic nodules.Fresh lung tissues were made into frozen sections for H&E staining,and the expression of Ncadherin and E-cadherin in lung tissue was observed by immunofluorescence assay.The expression levels of SNHG16 in lung tissues of each group were detected by qRT-PCR.8.Detcection of the effects of exosomal LncRNA SNHG16 derived from hypoxic TAMs and endogenous SNHG16 in tumor cells on the lung metastasis of MDA-MB23 1 cells in nude mice in vivo.The exosomes secreted by TAMs under hypoxic environments transfected with siRNA-SNHG16 or si-NC were collected and co-incubated with MDA-MB-231-Luc cells.And then MDA-MB-231-Luc cells were injected into nude mice through the tail vein.The lung metastasis of MDA-MB-231-Luc cells in vivo was observed by IVIS.Lung tissues of nude mice were isolated to observe the tumor metastasis.Results:1.The identification of M2 phenotype of TAMs,establishment of hypoxia model and identification of exosomes secreted by TAMTAMs induced by tumor conditioned medium was close to M2 phenotype.The expression of M2 macrophages markers CD68 and CD206 in TAMs induced by MDA-MB-231 cell conditioned medium was significantly higher than that in THP cells,which was similar to M2 macrophages induced by PMA/IL-4/IL-13,indicating that TAMs was a pro-tumor phenotype(M2),HIF-1α expression was increased and activated into the nucleus of hypoxic TAMs,indicating that the physical hypoxia model was successfully established using hypoxic incubater.The extract obtained from the supernatant of TAMs was isolated by ultra-high speed centrifugation and purified,with a typical double-concave disc-shaped structure under electron microscope,and with a particle size of 150 nm and high expression of CD9 and CD63 markers,which was identified as exosome.2.Exosomes secreted by TAMs in hypoxia promoted the migration,invasion and vascular mimicry formation of MDA-MB-231 cells.3.Exosomal LncRNA SNHG16 derived from TAMs under hypoxic condition facilitated MDA-MB-231 cells metastasis.(1)TCGA database analysis showed that LncRNA SNHG16 was highly expressed in TNBC,and breast cancer patients with higher SNHG16 levels had shorter survival.(2)LncRNA SNHG16 was highly expressed in hypoxic TAMs,exosomes secreted by hypoxic TAMs and MDA-MB-231 tumor cells.(3)Downregulation of SNHG16 expression in MDA-MB-231 cells inhibited the migration of MDA-MB-231 cells.But following by exosomes derived from hypoxic TAMs to promoted the migration of MDA-MB-231 cells significantly.After downregulating SNHG16 in MDA-MB-231 cells,SNHG16 from hypoxic TAMs still significantly promoted the migratory ability of MDA-MB-231 cells.Simultaneous downregulation of SNHG16 expression levels in hypoxic TAMs as well as in MDA-MB-231 cells more significantly attenuated the migratory ability of MDA-MB-231 cells.The results of invasion assay,vascular mimicry assay and migration assay were consistent.These indicated that LncRNA SNHG16 in TAMs-derived exosomes under hypoxic environment significantly promoted MDA-MB-231 cell metastasis.4.SNHG16 promoted migration,invasion,VM formation of MDA-MB-231 cells byupregulating the expression of HMGB3 by inhibiting miR-205-5p.(1)LncRNA SNHG16 was localized in the cytoplasm of MDA-MB-231 cells.(2)The miRNAs that were differentially expressed and significantlydownregulated in breast cancer in the TCGA database,negatively correlated with SNHG16 expression in breast cancer in the Starbase database,and had predicted binding sites to SNHG16 in the starbase database included hasmiR-205-5p,has-miR-30a-5p,and has-miR-195.After SNHG16 knockdown in MDA-MB-231 cells,only the expression level of miR-205-5p was significantly elevated in MDA-MB-231 cells which was detected by qRTPCR among the three miRNAs.(3)MiR-205-5p levels were significantly downregulated in breast cancer patients compared to healthy people,and miR-205-5p levels were significantly downregulated in breast cancer patients who developed lymph node metastasis and were lowest in breast cancer patients with stage N3 lymph node metastasis in the TCGA database.(4)The dual luciferase reporter gene assay validated the direct binding of miR205-5p to SNHG16.(5)After suppression of SNHG16 expression and transfection with miR-205-5pinhibitor,the inhibitory effect of downregulation of SNHG16 expression on MDA-MB-231 cell migration,invasion and vascular mimicry formation was reversed by miR-205-5p inhibitor.These results indicated that LncRNA SNHG16 promoted the migration,invasion and vascular mimicry formation of MDA-MB-231 cells mediated by miR-205-5p.(6)The expression of HMGB3 was positively corelated with the expression of SNHG16 in breast cancer with starbase database.The expression of HMGB3 and EMT-related proteins were decreaed after transfection with siRNASNHG16 or miR-205-5p mimics in MDA-MB-231 cells.Transfection with miR-205-5p inhibitor reversed the inhibition on the expression of HMGB3 induced by suppresion of SNHG16 expression in MDA-MB-231 cells.5.Exosomes derived from hypoxic TAMs promoted the lung metastasis of MDA-MB231 cells in nude mice in vivo.In vivo experiment,the fluorescence signal intensity of metastasis in the lungs of nude mice in the hypoxic exosome group was significantly higher than that in the control group and the normoxic exosome group.There were significantly more tumor metastases in the lung tissues of nude mice in the hypoxic exosome group than the control group and the normoxic exosome group.Increased tumor cells were observed in the lung tissues of the normoxic exosome group as well as the hypoxic exosome group in comparison to control group,and metastatic nodules were significantly higher in the hypoxic exosome group than in the other two groups in H&E staining sections.The expression level of SNHG16 was significantly higher in the lung metastases of the hypoxic exosome group.The expression of N-cadherin expression was increased and E-cadherin expression was decreased in the hypoxic exosome group compared to the other groups,which meant exosomes derived from hypoxic TAMs promoted EMT in triple negative breast cancer cells.6.Dual inhibition of exosomal LncRNA SNHG16 derived from hypoxic TAMs and endogenous SNHG16 in tumor cells significantly reduced the lung metastasis of MDA-MB-231 cells in nude mice in vivo.In vivo experiment,the fluorescence signal intensity of metastases in the lungs of nude mice was observed by IVIS.Downregulation of SNHG16 expression in MDAMB-231-Luc inhibited the lung metastasis of MDA-MB-231-Luc in nude mice.After downregulating SNHG16 in MDA-MB-231-Luc cells,SNHG16 from hypoxic TAMs exosomes still significantly promoted the lung metastasis of MDA-MB-231Luc in nude mice.Simultaneous downregulation of SNHG16 expression levels in hypoxic TAMs exosomes as well as that in MDA-MB-231-Luc cells dramatically attenuated the lung metastasis of breast cancer cells in nude mice.Conclusion:Exosomes derived from TAMs under hypoxic condition significantly promoted the metastasis of TNBC,and this effect was mediated by SNHG16 highly expressed in exosomes derived from hypoxic TAMs.SNHG16 promoted the metastasis of breast cancer through ceRNA mechanism by binding and inhibiting miR-205-5p and subsequently upregulating the expression of HMGB3.Dual inhibiting SNHG16 of TAMs exosomes and breast cancer cells can effectively inhibited triple negative breast cancer metastasis.