Epigenetic Regulation Of NAMPT By NAMPT-AS Drives Metastatic Progression In Triple-Negative Breast Cancer

BACKGROUND AND AIMSBreast cancer is one of the most common female malignancies in the world.The incidence and mortality rate of breast cancer in China have shown an increasing trend over the past ten years,which seriously endangers the physical and mental health of women in China.Breast cancer is highly heterogeneous from the aspect of either morphology or genetics.The inter-and intra-heterogeneity of breast cancers still remain as obstacles in assessment and treatment,which promotes the tailored individualized therapy based on molecular classification.Based on comprehensive gene expression profiling analysis,breast cancer can be categorized into three major subtypes,namely hormone receptor positive/ERBB2 negative,ERBB2 positive and basal-like.TNBC is characterized by negative estrogen receptor(ER),negative progesterone receptor(PR)and negative ERBB2 status.TNBC was repetitively reported as a predisposition to worse prognosis.TNBC occurs frequently in younger adults,presented with aggressive phenotype,higher rates of lymph node involvement,metastasis and poor response to treatment.Since patients with TNBC lack the expression of the above-mentioned receptors,the survival of TNBC patients could not be improved by endocrine therapy and HER2-targeted therapy.Chemotherapy is currently the main adjuvant treatment for patients with TNBC,but only 20%of TNBC patients are sensitive to standard chemotherapy regimens,and a significant proportion of patients will develop chemotherapy resistance.Distant metastasis is more likely to occur in patients developing chemotherapy resistance and associated with worse prognosis.Elucidating the underlying molecular mechanism of breast cancer metastasis will have important clinical significance.Progression and metastasis of TNBC are complex biological processes involving multiple genes and epigenetic regulation.A large number of studies have shown that the abnormal expression of non-coding RNA plays a vital role in the epigenetic regulation of malignant tumors.Long non-coding RNA(LncRNA)is a general term for a class of RNA transcripts longer than 200 nt that does not have protein encoding capabilities;it occupies a large proportion in the transcriptome and includes enhancer RNA,snoRNA host,intergenic transcripts and overlaps with other genes transcripts,sense or antisense transcripts and so on.From the perspective of cellular localization,IncRNAs localized in cytoplasm primarily function as endogenous miRNA sponge to competitively bind target genes,whereas those localized in nucleus act as scaffolds for protein-protein interaction,guiding for protein-DNA interaction and chromatin modifiers.From the perspective of genomic position of IncRNAs and their target genes,lncRNAs may be involved in regulating either the expression of their neighboring genes in cis or more distant genes in trans through various mechanisms.Less than a decade ago,strand-specific high-throughput sequencing was developed and proved that some IncRNAs were natural divergent antisense transcriptions of protein-coding genes.Because antisense lncRNAs(aslncRNA)coincide with 50%of the protein-coding genes,it is frequently observed that the aslncRNAs positively regulate the sense coding gene in cis or in trans.Herein,silencing aslncRNAs can be a potential cut point for intervention on the oncogenic driver genes.In order to find lncRNAs that are closely related to the progression and metastasis of TNBC,we conducted in-depth mining of The Human Genome Atlas(TCGA)and Gene Expression Omnibus(GEO)database.We selected the overexpressed NAMPT-AS(NAMPT-antisense,NAMPT-AS)as the target LncRNA in TNBC.Combined with the bioinformatics analysis from both the Encyclopedia of DNA Elements database(ENCODE)and FANTOM5(Function Annotation of The Mammalian Genome,FATOM)projects,we studied the function and underlying mechanism of NAMPT-AS through in vivo and in vitro experiments and validated in clinical samples.METHODS AND RESULTS:In this project,we intend to further study the role of NAMPT-AS in the progression and metastasis of TNBC.The specific content includes the following four parts:PART ? NAMPT-AS was identified as triple negative breast cancer metastastic progression associated LncRNAMETHODS1.The identification of NAMPT-AS in TNBC progression and metastasis1.1 The level of NAMPT-AS in TNBC and non-TNBC based on TCGA database,GEO database and breast cancer Tissue Pathology Bank of Qilu Hospital1.2 The level of NAMPT-AS in TNBC and normal breast tissues based on GEO database and breast cell linesRESULTS1.NAMPT-AS is expressed higher in TNBC tissues and cell lines1.1 The expression of NAMPT-AS is higher in TNBC than non-TNBC based on TCGA,GEO and Qilu database;1.2 The level of NAMPT-AS is higher in TNBC tissues than normal tissues,higher in TNBC cell lines than others.PART ? The underlying molecular mechanism of reciprocal regulation between NAMPT-AS and NAMPTMETHODS1.Phenotype and bioinformatic analysis of NAMPT-AS1.1 RNA-Seq analysis and GSEA after knockdown of NAMPT-AS in MDA-MB-4681.2 The detection of autophagy by MDC staining and LC3 immunofluorescence1.3 The ultrastructure of MDA-MB-231 and MDA-MB-468 cells after knockdown of NAMPT-AS by transmission electron microscopy(TEM)1.4 The expression of key molecules in mTOR signaling pathway by western blot2.The detection of localization of NAMPT-AS through bioinformatic analysis,qPCR after partition of nuclear and cytoplasmic RNA,and RNA-FISH3.Nuclear NAMPT-AS recruits POU2F2 to the promoter of NAMPT to enhance the transcriptional activity of NAMPT3.1 The transcription activity of NAMPT when NAMPT-AS overexpressed by dual luciferase assay3.2 Using bioinformatic analysis to identify a potential binding protein of NAMPT-AS;detecting the level of NAMPT-AS and NAMPT under the knockdown of the target binding factor by qPCR;co-transfection of the target binding factor and the NAMPT promoter region in sense and antisense direction,and detecting the luciferase activity by dual luciferase assay;confirmation of binding between target factor and NAMPT-AS by RNA immunoprecipitation(RIP),and the recruitment of factor to the promoter region3.3 Analysis of ChIP-Seq data in ENCODE database to predict the bidirectional transcription potential of NAMPT promoter region;and demonstrated by dual luciferase assay and ChIP assay4.The prediction and confirmation of the downstream microRNA of NAMPT-AS4.1 The prediction of microRNAs downstream of NAMPT-AS,and upstream of NAMPT by Microcosm,Targetscan,RegRNA and SegalLab database4.2 The secondary structure of NAMPT-AS and the binding site of predicted microRNA4.3 Detection of the level of predicted microRNA under knockdown of NAMPT-AS4.4 Detection of the level of NAMPT-AS under overexpression of predicted microRNA5.Confirmation of the binding between microRNA and NAMPT-AS We applied dual luciferase and RIP assay to confirm the binding between target microRNA and NAMPT-AS6.The prediction and confirmation of the downstream gene of microRNA6.1 The prediction of the downstream gene by DIANA,Targetscan,miRWalk,miRDB database6.2 Western blot to detect the protein level of the gene under microRNA overexpression6.3 We applied dual luciferase and RIP assay to confirm the binding between microRNA and target geneRESULTS1.NAMPT-AS influences autophagy via mTOR signaling pathway1.1 GSEA analysis showed that NAMPT-AS knockdown was associated with mTOR signaling pathway,and western blot showed key molecules in mTOR signaling was depressed,such as phospho-mTOR,p70s6k,4E-BP11.2 Knockdown of NAMPT-AS induced autophagy phenotype,including the autolysosomes and acid vesicles in MDA-MB-231 and MDA-MB-468 cells by MDC staining,LC3 immunofluorescence and TEM2.NAMPT-AS recruits transcription factor POU2F2 to the promoter of NAMPT2.1 Bioinformatic prediction,qPCR based on partition of nuclear and cytoplasmic RNA,RNA-FISH assay confirmed that NAMPT-AS localized in both nucleus and cytoplasm2.2 Nuclear NAMPT-AS recruited POU2F2 to the promoter of NAMPT to enhance the transcriptional activity of NAMPT2.2.1 Co-transfection of NAMPT-AS could augment the transcription of NAMPT by dual luciferase assay2.2.2 Bioinformatic prediction with Annolnc and RPISeq based on the nucleotide sequence of NAMPT-AS suggested that POU2F2 could not only function as binding protein of NAMPT-AS,but also functioned as transcription factor by binding to the promoter region of NAMPT;the level of NAMPT-AS and NAMPT were decreased after knockdown of POU2F2;The dual luciferase reporter gene experiment proved that POU2F2 could bind to the promoter region of NAMPT,and promoted the transcription of NAMPT-AS and NAMPT;the binding capability between NAMPT-AS and POU2F2 was demonstrated by RIP assay in MDA-MB-231 and MDA-MB-468 cell lines2.3 The bi-directional transcriptional capability of NAMPT promoter region2.3.1 The detection of H3K4me3 and H3K27Ac chromatin modification and RNA polymerase ? distribution in this region by the ENCODE project,which was demonstrated by ChIP assay in MDA-MB-231 and MDA-MB-468 cell lines;The counts of CAGE read provided by FANTOM5 database suggested that this promoter region was capable of bidirectional transcription2.3.2 We applied dual luciferase assay to confirm the bidirectional transcription of the promoter.The luciferase activity in the direction of NAMPT promoter was almost three-fold higher than that in the direction of NAMPT-AS in 293T,MDA-MB-231 and MDA-MB-468 cell lines3.The underlying signaling network of NAMPT-AS/miR-548b-3p/NAMPT axis3.1 miR-548b-3p is downstream of NAMPT-AS3.1.1 miR-548b-3p was predicted as the common microRNA downstream of NAMPT-AS,and upstream of NAMPT by Microcosm,Targetscan,RegRNA and SegalLab database3.1.2 miR-548b-3p increased under knockdown of NAMPT-AS;NAMPT-AS decreased under miR-548b-3p overexpression3.1.3 miR-548b-3p could bind NAMPT-AS;Dual luciferase assay proved that miR-548b-3p could bind to NAMPT-AS;RIP assay confirmed both miR-548b-3p and NAMPT-AS could bind AG023.2 NAMPT is downstream of miR-548b-3p3.2.1 NAMPT was the common target gene by DIANA,Targetscan,miRWalk,miRDB database3.2.2 Western blot showed decreased NAMPT under miR-548b-3p overexpression3.2.3 Dual luciferase assay proved that miR-548b-3p can bind to NAMPT 3'UTR;RIP assay confirmed both miR-548b-3p and NAMPT could bind AG02PART ? The function of NAMPT-AS/miR-548b-3p/NAMPTMETHODS1.The function of NAMPT-AS in vitro1.1 The effect of knockdown or overexpression of NAMPT-AS on the proliferation,apoptosis and metastasis in MDA-MB-231 and MDA-MB-468 by MTT,clone formation,flow cytometry and transwell assay1.2 The expression of apoptosis and proliferation-related genes by western blot2.The relationship between the level of microRNA and overall survival3.The function of microRNA in vitro3.1 The effect of microRNA on the proliferation,apoptosis and metastasis by MTT,clone formation,flow cytometry,transwell assay and western blot3.2 The detection of autophagy by MDC staining,LC3 immunofluorescence and western blot4.The function of NAMPT in vitro4.1 The effect of NAMPT on the proliferation,apoptosis and metastasis by MTT,clone formation,flow cytometry,transwell assay and western blot4.2 The detection of autophagy by MDC staining,LC3 immunofluorescence and western blot4.3 Rescue experiment:Western blot to detect the level of mTOR signaling and autophagy related genes under the treatment of rapamycin,with NAMPT overexpression or NAMPT knockdown5.The effect of NAMPT-AS on proliferation and metastasis by in vivo assay5.1 Construct stable NAMPT-AS overexpressing cell line and inject them subcutaneously,and record changes in tumor volume every week and tumor weight at the endpoint5.2 Confirm the overexpression of NAMPT-AS and the level of NAMPT by qPCR5.3 Detect the protein level of mTOR signaling pathway and autophagy-related proteins in tumor tissue5.4 Inject the stable NAMPT-AS overexpression cells through tail vein and observe the incidence of lung metastasis in nude mice6.The effect of NAMPT on proliferation in vivo Construct stable NAMPT knockdown cell line and inject them subcutaneously,and record changes in tumor volume every four days and tumor weight at the endpointRESULTS1.The function of NAMPT-AS/miR-548b-3p/NAMPT by in vitro assay1.1 NAMPT-AS overexpression promotes cell proliferation and metastasis,and inhibits apoptosis1.1.1 Through MTT,clone formation,transwell and flow cytometry in MDA-MB-231 and MDA-MB-468 cell lines,NAMPT-AS overexpression promoted proliferation and metastasis,and inhibited apoptosis while NAMPT-AS knockdown has an opposite direction1.1.2 Western blot showed that apoptosis-related molecules Bax,cleaved-Caspase3,cleaved-Caspase8 upregulated while BCL2 downregulated under the knockdown of NAMPT-AS in MDA-MB-231 and MDA-MB-4681.2 The function of miR-548b-3p in vitro1.2.1 Higher miR-548b-3p predicts better overall survival1.2.2 The function of miR-548b-3p in vitro1.2.2.I miR-548b-3p inhibited proliferation and metastasis,promoted apoptosis by MTT,clone formation,flow cytometry,transwell assay and western blot1.2.2.2 Overexpression of miR-548b-3p promoted autophagy by MDC staining,LC3 immunofluorescence and western blot1.3 The function of NAMPT in vitro1.3.1 NAMPT promoted proliferation and metastasis,inhibited apoptosis by MTT,clone formation,flow cytometry,transwell assay and western blot1.3.2 Knockdown of NAMPT stimulated autophagy by MDC staining,LC3 immunofluorescence and western blot1.3.3 Rescue experiment:Western blot showed NAMPT overexpression could partially reverse the inactivated mTOR signaling and stimulated autophagy related genes under the treatment of rapamycin,while NAMPT knockdown intensified the phenotype of autophagy2.The function of NAMPT-AS/NAMPT by in vivo assay2.1 NAMPT-AS promotes proliferation and metastasis in vivo2.1.1 The tumor volume and tumor weight were significantly larger in NAMPT-AS overexpression group than the control group2.1.2 The mRNA levels of NAMPT-AS and NAMPT were higher in NAMPT-AS overexpression group2.1.3 The mTOR signaling pathway was inactivated and autophagy-related proteins elevated in NAMPT-AS overexpression tumor tissue2.1.4 The incidence of lung metastasis was higher in NAMPT-AS overexpression nude mice2.2 The effect of NAMPT on proliferation in vivo and survival outcomes The tumor volume and tumor weight were significantly smaller in NAMPT knockdown group than the control group PART ? The role of NAMPT-AS/NAMPT in clinical prognosisMETHODS1.The relationship between NAMPT-AS and survival outcomes1.1 The relationship between the expression level of NAMPT-AS and clinical prognosis in all types of breast cancer based on TCGA database,and breast cancer samples collected from Qilu Hospital Tissue Pathology Bank1.2 The association between the level of NAMPT-AS and clinical prognosis in TNBC patients based on TCGA database2.The effect of NAMPT on survival outcomes Immunohistochemical staining of NAMPT in a tissue microarray and analysis of the association between NAMPT and OS and breast specific survivalRESULTS1.The role of NAMPT-AS in clinical prognosis by TCGA database1.1 Higher expression of NAMPT-AS was associated with poor overall survival(OS)and relapse-free survival(RFS)in all types of breast cancer based on TCGA database,and breast cancer samples collected from Qilu Hospital Tissue Pathology Bank1.2 NAMPT-AS was associated with poor OS and RFS outcomes in TNBC patients2.The role of NAMPT in clinical prognosis by tissue array Higher level of NAMPT was correlated with worse OS and breast specific survival in all breast cancer patients and patients with TNBCCONCLUSIONS:1.The expression level of LncRNA NAMPT-AS/miR-548b-3p/NAMPT is closely related to the progression and metastasis of TNBC2.The high expression of NAMPT-AS/NAMPT is closely related to the poor prognosis of patients with all types of breast cancer and TNBC,which can be regarded as potential prognostic factor3.Both in vivo and in vitro experiments have shown that NAMPT-AS/NAMPT affects autophagy by regulating mTOR signaling pathway and thus promotes the progression and metastasis of TNBC4.In vitro experiments show that NAMPT-AS regulates NAMPT expression from both transcriptional and post-transcriptional level,recruiting the transcription factor POU2F2 to promote the transcription of NAMPT,and also competitively binding miR-548b-3p to reduce the degradation of NAMPT...