Function And Mechanism Study Of Long Non-coding RNA LINC00882 In Breast Cancer Progression

Background:Breast cancer(BC)is a highly heterogeneous disease that poses a huge threat to women ’s health.Among them,BC metastasis is the main cause of death in BC patients,and it also causes great difficulties for treatment.Long noncoding RNAs(LncRNAs)are composed of more than 200 nucleotides.Although most LncRNAs do not have the ability to encode proteins,they can exert biological functions by regulating the expression of multiple target genes in cellular processes and diseases.Recently,more and more studies have shown that LncRNAs play an important role in the progression of BC,including apoptosis,autophagy,DNA repair,cell cycle,epithelial-mesenchymal transition(EMT),epigenetic modification and tumor microenvironment(TME)Therefore,exploring the mechanisms of LncRNAs remains essential.Methods:In this study,we used real-time fluorescence quantitative PCR(RT-qPCR)to detect the expression of LINC00882 in BC tissues.At the same time,the receiver operating curve(ROC)was used to analyze whether LINC00882 could be used to distinguish BC tissues with or without lymph node metastasis(LNM)Fluorescence in situ hybridization(FISH)was used to determine the subcellular localization of LINC00882.The upstream modification,promoter region and transcription factors regulating LINC00882 expression were determined by RT-qPCR,chromatin immunoprecipitation(ChIP)and dual luciferase assay.The m6A modification of LINC00882 was predicted on the bioinformatics SRAMP website,and the m6A methylation immunoprecipitation(Me-RIP)experiment confirmed the existence of m6A modification on LINC00882.The effects of LINC00882 on the biological behavior of BC cells were detected by CCK8,EdU,Transwell migration and invasion experiments in vitro.At the same time,we transfected lentivirus LV-LINC00882 or LV-NC into MDA-MB-231 cells to construct LINC00882 stably overexpressed cell lines.Subcutaneous xenograft model and caudal vein metastatic model were constructed to explore the role of LINC00882 in vivo.In terms of mechanism,we revealed the molecular mechanism of LINC00882 promoting BC metastasis by RNA pull-down,RNA immunoprecipitation(RIP),RT-qPCR,FISH combined with immunofluorescence analysis,dual luciferase,ChIP and Western blot experiments.Results:1.The higher expression of LINC00882 in BC tissues with LNM.The results of RT-qPCR showed that LINC00882 was relatively highly expressed in BC tissues with LNM.ROC curve results showed that LINC00882 effectively distinguished LNM from non-metastasis.CPAT website predicted coding potential and RNA pull-down experiments showed that LINC00882 did not have coding ability.In addition,FISH results manifested that LINC00882 was mainly localized in the cytoplasm.2.HDAC1 and CEBPβ synergistically promoted the expression of LINC00882DNA methyltransferase inhibitor(5-Azacytidine,5-AZ).histone deacetylase inhibitor(Trichostatin A,TSA)and histone methyltransferase inhibitor(Deazaneplanocin A,DZNeP)were added to BC cells.The results suggested that TSA up-regulated LINC00882.RT-qPCR results showed that knockdown of histone deacetylase HDAC1 promoted LINC00882 expression.Dual luciferase assay showed that 250 bp upstream of LINC00882 transcription start site was the core promoter region.RT-qPCR,dual luciferase and ChIP assays showed that the transcription factor CEBPβ promoted LINC00882 transcription.In addition,RT-qPCR results suggested that knockdown of HDAC1 and overexpression of CEBPβ synergistically promoted LINC00882 transcription.3.IGF2BP2 recognized METTL14-mediated m6A modification and enhanced the stability of LINC00882Bioinformatics website SRAMP prediction and Me-RIP experimental results showed that m6A modification exists at 264 nt of LINC00882.RT-qPCR assay showed that overexpression of METTL14(N6-adenosine-methyltransferase-like 14)led to up-regulation of LINC00882 expression,and knockdown of METTL3(N6-adenosine-methyltransferase-like 3)or METTL14 led to down-regulation of LINC00882.Actinomycin D(Act D)tracing assay showed that knockdown of METTL14 resulted in a shortened half-life of LINC00882.Knockdown of IGF2BP2(insulin-like growth factor 2 binding protein 2)down-regulated LINC00882 expression.Act D tracing assay showed that knockdown of IGF2BP2 shortened the half-life of LINC00882.4.LINC00882 promoted BC invasion and metastasis in vitro and in vivoTranswell assay showed that LINC00882 promoted BC cells migration and invasion in vitro.CCK8 and EdU assays showed that LINC00882 had no significant effect on BC cells proliferation in vitro.Subcutaneous xenograft model and caudal vein metastatic model showed that LINC00882 promoted BC invasion and metastasis in vivo.5.LINC00882 interacted with PABPC1 in the cytoplasm.RNA pull-down,mass spectrometry,western blot and RIP assay showed that LINC00882 interacted with PABPC1.The 514 nt-615 nt of LINC00882 was the binding region of PABPC1.FISH combined with immunofluorescence analysis demonstrated that LINC00882 and PABPC1 co-localized in the cytoplasm.Nuclear and cytoplasmic extraction and Western blot assays showed that LINC00882 had no significant difference on PABPC1 protein expression and subcellular localization.6.LINC00882 mediated the binding of PABPC1 to ELK3 and activated EMT to promote BC progression.RNA-seq results and RNA pull-down PCR assays showed that LINC00882 bound to the mRNA of DDX3X,ELK3,LIMCH1 and SLC23A2.RT-qPCR results showed that LINC00882 up-regulated the expression of DDX3X,ELK3 and LIMCH1,while knockdown of PABPC1 decreased the expression of DDX3X,ELK3 and LIMCH1.At the same time,RIP assay showed that PABPC1 bound ELK3,and overexpression of LINC00882 significantly increased the enrichment of ELK3.In addition,Act D tracing assay showed that LINC00882 prolonged the mRNA half-life of ELK3,while knockdown of PABPC1 shortened the mRNA half-life of ELK3.Dual luciferase and ChIP assays showed that ELK3 inhibited E-cadherin transcription.Western blot results suggested that LINC00882 promoted the expression of ELK3,N-cadherin and inhibited the expression of E-cadherin.While knockdown of LINC00882 or PABPC1 inhibited the expression of ELK3,N-cadherin and promoted the expression of E-cadherin.Western blot showed that expression of ELK3 and N-cadherin was higher in the LV-LINC00882 group than those in the LV-NC group.Besides,compared with LV-NC group,the expression of E-cadherin was lower.Conclusions:1.LINC00882 is highly expressed in BC tissues with LNM.LINC00882 distinguishes patients with LNM from those without LNM.2.HDAC1 regulate upstream of LINC00882,resulting in the binding of the transcription factor CEBPβ to the core promoter region of LINC00882,thereby promoting the transcription of LINC00882.3.The post-transcriptional m6A modification of LINC00882 promotes its stability.4.LINC00882 acts as a molecular scaffold to pull PABPC1 and ELK3 mRNA closer,prompting PABPC1 to bind to ELK3 mRNA,resulting in enhanced mRNA stability of ELK3.Finally,ELK3 inhibits E-cadherin transcription,leading to the invasion and metastasis of BC.