| Background:Breast cancer(BC)is one of the most frequent malignancies in the female population worldwide and is the primary culprit of tumor-related deaths in women.Triple negative breast cancer(TNBC)is a subtype of breast cancer that lacks expression of estrogen receptor(ER),progesterone receptor(PR)and human epidermal growth factor receptor 2(HER-2).TNBC is most commonly seen in premenopausal women under the age of 50 and accounts for 10-24%of all diagnosed breast cancer patients.In addition,TNBC tumors are poorly differentiated,with a higher recurrence rate and a tendency to metastasize early.Recurrent and metastatic TNBC usually progresses more rapidly and shows strong resistance to chemotherapy and radiotherapy.Alternative therapies do not improve patient prognosis and quality of life due to the lack of combination targeted agents.For patients with advanced TNBC,it is crucial to find the therapeutic target of TNBC.The human genome contains protein-coding and non-coding regions,less than 2%of which are labeled as protein-coding regions and the rest are defined as non-coding regions.As a type of non-coding RNA,long noncoding RNAs(lncRNAs)typically have transcripts longer than 200 nucleotides.Through bioinformatics and high-throughput sequencing approaches,recent studies have revealed that aberrant lncRNA expression is widely involved in tumor pathogenesis processes,including cell proliferation,migration,invasion,epithelialmesenchymal transition(EMT),apoptosis and anti-tumor drug resistance.In general,lncRNAs do not encode proteins or peptides.However,with the availability and accuracy of proteomics and translation technologies,it has been found that many lncRNAs can be translated to produce biologically active proteins or micropeptides.Glutamine(Gln)is one of the most abundant non-essential amino acids in the blood(produced by the body and therefore not an essential part of the diet),and it is involved in almost all biosynthetic pathways in proliferating cells.Metabolic reprogramming is a key marker of tumor progression,and in order to meet the bioenergetic and biosynthetic demands of rapid cell proliferation and to adapt to the tumor microenvironment,the energy metabolic pattern of tumor cells is altered,which also includes the metabolism of glutamine.Although such altered metabolism in tumor cells has been studied,a key question that has not been thoroughly investigated is whether the underlying mechanisms by which glutamine-induced alterations in lncRNA coding properties affect the progression of TNBC are currently unknown.Therefore,it is necessary to explore the molecular mechanisms of triple-negative breast cancer specificity in glutamine metabolism,potentially identifying triple-negative breast cancer-specific prognostic markers and developing new approaches for triple-negative breast cancer targeted therapies.Therefore,it is necessary to explore the molecular mechanisms of TNBC specifically in glutamine metabolism,potentially identifying TNBC-specific prognostic markers and developing new approaches for TNBC targeted therapy.Methods:(1)To analyze whether TNBC and non-TNBC tumor tissues have different glutamine metabolism patterns by bioinformatics,and further to detect whether TNBC cell lines and non-TNBC cell lines are sensitive to the alteration of Gln by cellular assays.(2)Firstly,ribosome profiling sequencing data of four different breast cancer cell lines grown under unrestricted growth conditions or in glutaminedeficient medium were collected through the GEO database.Then,the candidate lncRNAs specific for triple negative breast cancer in response to glutamine stimulation were screened by bioinformatics.(3)The biological properties of the target lncRNA were examined by Northern blot assay,nucleo-plasmic separation assay and fluorescent in situ hybridization(FISH)assay.(4)The coding potential of the target lncRNA was verified by constructing different plasmid expression vectors and using immunoblotting assays and immunofluorescence assays.(5)To explore the effect of glutamine deprivation on the expression level of micropeptide encoded by lncRNA through immunoblotting assay and immunofluorescence assay.(6)To assess the role of target IncRNA and their encoded micropeptide in tumor progression by producing a mouse model of breast cancer xenograft tumors.(7)To analyze the effect of micropeptide expression levels on the prognosis of TNBC and non-TNBC patients using KaplanMeier survival curves.(8)To explore transcription factors regulating target lncRNA expression levels under glutamine deprivation conditions by online analysis of transcription factor prediction tool,chromatin immunoprecipitation(ChIP)assay and dual luciferase reporter gene assay.(9)Probe the target proteins that interact with the micropeptide encoded by the target lncRNA and their mechanisms of interaction in cells by Co-immunoprecipitation(Co-IP),mass spectrometry(MS)and immunoblotting experiments.(10)To investigate the effects of target lncRNA-encoded micropeptide on downstream target proteins and their signaling pathways by immunofluorescence assay,immunoprecipitation assay and immunoblotting assay.(11)To investigate the effects of target lncRNA-encoded micropeptide on angiogenesis and metastasis in TNBC via cell proliferation assay,tube formation assay,cell metastasis assay,wound healing assay,cell sprouting assay,matrix plug angiogenesis assay in vivo,and the model of breast cancer spontaneous tumor mice and transgenic mice.(12)The anti-tumor effects of targeted silencing of lncRNA-encoded micropeptide were explored by TNBC xenograft tumor model.Results:Analysis of metabolic patterns and cellular experimental assays in TNBC and non-TNBC tumor tissues revealed that TNBC were more sensitive to stimulation in response to glutamine deprivation compared to non-TNBC.A candidate lncRNA,MLLT4-AS1,was screened and identified by bioinformatics analysis,immunoblotting assay and immunofluorescence assay.In TNBC cells,both transcript level and translation level of MLLT4-AS1 were significantly increased after glutamine deprivation,and the encoded micropeptide was named XBP1S-binding micropeptide(XBP1SBM).Biological characterization experiments revealed that MLLT4-AS1 was mainly localized in the cytoplasm.Immunoblotting assays and immunofluorescence assays demonstrated that glutamine deprivation promoted the expression of the micropeptide XBP1SBM.Kaplan-Meier survival curve analysis showed that high expression of micropeptide XBP1SBM was significantly associated with poor prognosis of TNBC patients.Upstream chromatin immunoprecipitation(ChIP)assays and dual luciferase reporter gene assays demonstrated that XBP1S bound to the promoter region of MLLT4-AS1 and promoted its transcription under glutamine deprivation conditions.Downstream Co-immunoprecipitation(Co-IP),mass spectrometry(MS),and immunoblotting experiments revealed that the micropeptide XBP1SBM binds to XBP1 s protein,regulates XBP1 s nucleo-plasmic translocation,and enhances its nuclear localization,which in turn promotes the expression of vascular endothelial growth factor(VEGF)transcripts.Cell pHenotyping experiments and in vivo experiments in mouse models demonstrated that the micropeptide XBP 1 SBM promotes angiogenesis and metastasis in TNBC.Meanwhile,TNBC xenograft tumor models demonstrated that targeted silencing target XBP 1 SBM has antitumor effects.Conclusion:Our study shows that TNBC-specific glutamine deficiency leads to a significant increase in the expression of the micropeptide XBP 1 SBM encoded by lncRNA MLLT4-AS1.The micropeptide XBP 1 SBM promotes TNBC growth,angiogenesis and metastasis through the XBP1s/VEGF signaling pathway.This mechanism provides a new potential prognostic biomarker and therapeutic target for TNBC. |
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