Regulation Of Lnc RNA PCIR On The Growth And Metastasis Of Triple Negative Breast Cancer Cells And Its Molecular Mechanism

Projective Triple negative breast cancer(TNBC)is characterized by the absence of estrogen and progesterone receptors andhuman epidermal growth factor receptor 2.Although significant progression has been achieved in the therapy of breast cancer,chemotherapy remains the primary treatment option for patients with TNBC.Therefore,the clinical outcome of patients with TNBC is relatively worse compared with that of other types of breast cancer due to the inefficiency of endocrine therapy and targeted therapy.In recent years,long non-coding RNAs have been shown to play an important role in most cellular processes.LncRNAs have been implicated to regulate a range of biological functions.LncRNAs are actively involved in the development,apoptosis,metastasis as well as invasion of breast cancer cells.In this study,we sought to identify clinically relevant lncRNAs deregulated specifically in TNBC patients and aim to reveal the functional role and regulatory mechanism of lncRNA in the progress of TNBC.We identified and characterized the lncRNA RP11-214F16.8,renamed Lnc-PCIR(LncRNA Positively Correlated with Inflammatory Responses)by bioinformational analysis of RNA-seq derived from the TCGA database.In this study,the regulatory effect of LNC-PCIR on triple negative breast cancer cell lines will be further investigated through in vivo and in vitro experiments.Methods1.To identify a subset of lncRNAs overexpressed with clinically relevant in the TNBC subtype compared to normal tissue by using RNA-sequencing(RNA-seq)data from 1084 patients available in the TCGA database and differentially expressed gene analysis.Lnc-PCIR showed the significantly prognostic value in TNBC tissue.The RNA levels of Lnc-PCIR were confirmed by quantitative real-time polymerase chain reaction(q-PCR)analysis in 110 paired TNBC tissues and paired tumor-adjacent non-tumor tissues.Compared with matched normal tissues,Lnc PCIR was significantly up-regulated in TNBC tissues.2.To determine the position and size and full length of LNC-PCIR by RACE experiment and Northern blot technology;To determine the subcellular localization of lnc-PCIR in TNBC cells by q PCR.3.To construct overexpressed,knockdown stable cell lines and siRNA-interfered cell lines.The effects of LNC-PCIR on the growth,migration,invasion,subcutaneous tumor formation and distal metastasis of TNBC cells were observed in vitro and in vivo.4.To identify and verify the interaction protein by RNA pulldown technology,mass spectrometry and RIP technology.To identify he binding region or domain of lnc-PCIR with the interacting protein by sequence deletion.To study the effects of lnc-PCIR on the activity of the interacting protein and its mRNA,and to identify its regulatory mechanism.5.To analyze the influence of lnc-PCIR on gene expression profile of TNBC cells,GSEA /GO analysis was used to analyze downstream regulatory genes and signaling pathways,to study the role of interacting proteins,downstream regulatory genes and signaling pathways in playing biological functions,and to clarify the molecular mechanism of Lnc-PCIR.Results1.FromRNA-sequencing data,we identified Lnc-PCIR is a clinically relevant lncRNA displays a remarkable trendof increased expression in TNBC tissue.higher Lnc PCIR l Hevels predicted lower overall survival rates in TNBCpatients.2.Lnc-PCIR located on13q32.3 and has only one transcript with three exons.we performedthe RACE assay(rapid amplification of c DNA ends)and Northern blot assay to confirm the Lnc-PCIR is a 987-bplong intergenic non-protein-coding RNA in breast cancer cells.We examined the subcellular localization of Lnc-PCIR,finding that Lnc-PCIR predominately resides in thenucleus in 231 and BT549 cells by q RT-PCR.Lnc-PCIR showed the strong oncogenic activity by promoting TNBC cell tumor invasion andmetastasis,proliferation,tumorigenicity in vitro and in vivo.3.There are two types of proteins that specifically bind to Lnc-PCIR: TAB3 and PABPC.The 210-507nt(#2)fragment of lnc-PCIR mediates the interaction with the N-terminal CUE domain of TAB3,while the 625-987nt(#3)fragment is the key region binding with the PABP-1234 superfamily domain(11-624aa)of PABPC4.4.LNC-PCIR can regulate the mRNA and protein levels of TAB3,but only the protein level of PABPC.Lnc-PCIR promotes the growth of triple negative breast cancer by regulating the TNF-α/NF-κB signaling pathway through TAB3.Conclusion As an oncogenic gene,lnc-PCIR is up-regulated in triple negative breast cancer and can significantly promote the growth,invasion,tumorigenicity and metastasis of TNBC cells in vivo and in vitro.Tab3 and PABPC4 are oncogenes.Lnc-PCIR inhibits the ubiquitination degradation of PABPC4 protein and promotes its accumulation by binding to Tab3 and PABPC4.Lnc-PCIR promotes the dissociation of Tab2 / Tab3 complex by binding to Tab3 mRNA,and promotes the activation of downstream TNF-NF-κ B signaling pathway by Tab3 to play a regulatory role.Therefore,Lnc-PCIR may be a potential target for the treatment of TNBC.