Clinical Implications Of Long Non-coding RNA LUCAT1 In Breast Cancer And The Mechanism Of LUCAT1 In Regulating Breast Cancer Stemness

Objective:On a global scale,breast cancer is the most frequent malignancy and the leading cause of cancer death in women.Heterogeneous clusters of tumor cells exist in solid tumors,a special subgroup of which is called cancer stem cells(CSCs),characterized by self-renewal and pluripotency.Breast cancer stem cells(BCSCs)are regarded as the source of tumor development,differentiation,invasion and metastasis,drug resistance and recurrence in breast cancer.Therefore,studies on stemness regulation of BCSCs are significant in theory and clinical practice.Long non-coding RNAs(lncRNAs)recruit transcription factors to regulate gene expression,or interact with microRNAs(miRNAs)and influence the stability of mRNAs.LncRNAs participate in epigenetic regulation in pathophysiology.LncRNAs are involved in the tumorigenesis and progression and considered to be a potential biomarker for cancer diagnosis,prognosis and therapy.The competing endogenous RNA(ceRNA)hypothesis is a classical mode of gene expression regulation.LncRNAs exert a?miRNA Sponge‘by competitively binding miRNAs to antagonize the function of miRNAs in inhibiting specific target mRNAs.Several studies reported that lncRNAs,as ceRNA,affected tumorigenesis and progression,reflecting a new level of post-transcriptional regulation of genes and providing new insights for the molecular mechanism of CSCs.In the present study,large scale screening for the differentially expressed lncRNAs between BCCs and BCSCs,is performed by lncRNAs microarray.After filtration by bioinformatics analysis and consultation based on previous studies,LUCAT1(lung cancer associated transcript 1)is selected as the target.LUCAT1 is located at 5q14.3,firstly found in the airway epithelium of smokers.LUCAT1 is significantly more-expressed in non-small cell lung cancer(NSCLC)tissues than normal tissues.Its high expression is correlated with high TNM staging,positive lymph node metastasis and poor prognosis.LUCAT1 is related to cisplatin resistance in ovarian cancer and tumorigenesis in colorectal cancer.Nonetheless,little is known regarding the expression of LUCAT1 in breast cancer and the stemness regulation of LUCAT1 in BCSCs.Herein,we assessed LUCAT1 expression in breast cancer specimens and firstly reported the association of LUCAT1 with clinical pathology factors and prognosis in breast cancer.The mechanism of LUCAT1 in regulating BCSCs characteristics might provide theoretical support for finding new diagnostic markers and therapeutic targets of breast cancer.Methods:1.Induction and identification of breast cancer stem cells:Breast cancer cells MCF-7 and T47D were cultured into MCF-7 CSCs and T47D CSCs in serum free medium.The characteristics of breast cancer stem cells were identified by morphological observation,qRT-PCR,western blot and flow cytometry.2.Indicator selection based on genomics and bioinformatics:LncRNA LUCAT1,which was significantly differentially expressed in MCF-7 CSCs and MCF-7,was screened by cluster analysis of lncRNAs microarray.RNA-seq data were extracted from TCGA to analyze the differential expression of LUCAT1 in CD44~+CD24~-and non-CD44~+CD24~-breast cancer patients and its correlation with stemness markers.3.Analysis of LUCAT1 expression and clinical significance in breast cancer tissue samples:In 26 pairs of fresh tissue samples,the expression of LUCAT1 in breast cancer and adjacent tissues was detected by qRT-PCR,and the correlation between LUCAT1expression and SOX2 expression was analyzed.The expression of LUCAT1(In situ hybridization),SOX2 and TCF7L2(Immunohistochemistry)were detected in 151 breast cancer tissues.The correlation between LUCAT1 and clinical pathological factors was analyzed by Pearson chi-square test,Fisher exact test,univariate and multivariate logistic regression.The correlation between LUCAT1 expression and prognosis of breast cancer patients was evaluated by kaplan-meier survival analysis.4.Cytological experiments:Mi R-5582 mimic and inhibitorwere transfected into MCF-7 and T47D.MCF-7 CSCs and T47D CSCs were transfected with sh-Ctrl or sh-LUCAT1 lentiviral transduction particles.MCF7 and T47D were transfected with NC-cDNA or LUCAT1-c DNA.Sphere-formation assay and colony formation assay were used to detect cell self-renewal and proliferation,respectively.Stemness markers OCT4,Nanog and SOX2,as well as TCF7L2 and Wnt1 were detected by qRT-PCR and western blot,in mRNA and protein levels.The expression of CD24 and CD44 were detected by flow cytometry.Intracellularβ-catenin-mediated transcriptional activity was detected by TOP/FOP-Flash reporter assays.5.Target gene prediction based on bioinformatics and molecular mechanism experiments:Target genes were predicted by mirDB,RNA hybrid,DIANA and Targetscan databases;RNA pull down and luciferase reporter assays were used to identify LUCAT1 and TCF7L2 as the direct target of miR-5582-3p.6.In vivo experiments:The mice were randomized into four groups(n=3 per group):NC-cDNA mice injected with agomir NC;LUCAT1-cDNA mice injected with agomir NC;NC-cDNA mice injected with miR-5582-3p agomir and LUCAT1-cDNA mice injected with miR-5582-3p agomir.In vivo experiments was performed to confirm the regulatory mechanism of the whole LUCAT1/miR-5582-3p/TCF7L2 axis and Wnt/β-catenin pathwayResults:1.Induction and culture of breast cancer stem cells:The morphology of MCF-7 and T47D changed obviously and size of mammospheres rapidly increased 7-8 days later.MCF-7 CSCs and T47D CSCs possessed typical properties,such as high CD44~+CD24~-phenotype,high expression of CSCs markers(OCT4,SOX2 and Nanog)in mRNA and protein levels.2.LUCAT1 is over-expressed in the BCSCs than BCCs and related to breast cancer stemness:LncRNAs microarray chips indicated that LUCAT1 expression in MCF-7CSCs was significantly higher than that in MCF-7 cells.In 26 fresh specimens,LUCAT1mRNA expression was positively correlated with SOX2 protein expression(r=0.6701,p<0.001).TCGA analysis indicated that LUCAT1 expression was distinctly higher in patients with CD44~+CD24~-phenotype(n=236)than others(n=868)(p=0.0274).The stemness markers including OCT4(p<0.0001),ABCC1(p<0.0001),Wnt1(p=0.0209)and HIF-1α(p<0.0001)expressed higher in LUCAT1-high patients than LUCAT1-low patients.3.There was a significant correlation between LUCAT1 expression and tumor size(p=0.015),lymph node metastasis(p=0.002)and TNM staging(p<0.001).TNM staging was an independent factor of LUCAT1 expression.LUCAT1-high patients had significantly lower OS(p=0.006)and DFS(p=0.011)than LUCAT1-low patients.4.LUCAT1 contributes to stem-like properties of BCCs and stemness of BCSCs:SOX2,Nanog,OCT4 expression and the rate of CD44~+CD24~-cells were upregulated in oe-LUCAT1 MCF-7 and T47D cells in mRNA and protein levels.Compared with oe-NC group,oe-LUCAT1 cells formed more and bigger colonies.SOX2,Nanog,OCT4expression and rate of CD44~+CD24~-cells were down-regulated in sh-LUCAT1 MCF-7CSCs and T47D CSCs.LUCAT1 silencing reduced mammosphere diameter and quantity.5.LUCAT1 competitively binds mi R-5582-3p with TCF7L2 and enhances Wnt/β-catenin pathway:mirDB and RNAhybrid databases were used to predict the combination of mir-5582-3p with LUCAT1;Dual luciferase gene reporter assay indicated that miR-5582-3p mimic could inhibit the luciferase activity in LUCAT1-WT group,with no effect in LUCAT1-Mut group(p<0.01).The RNA pull-down experiment manifested that miR-5582-3p was more enriched in the wild-type LUCAT1 compared with that in the mutant-type LUCAT1.RNAhybrid,DIANA and Targetscan were used to predict the binding of mir-5582-3p with TCF7L2.Analysis of luciferase reports indicated miR-5582-3p decreased luciferase activity in the WT vector compared with that in MUT type(p<0.01).The up-regulation of LUCAT1 in MCF-7 cells could reverse the decline in the transcriptional activity of TOP/FOP by miR-5582-3p mimic.The up-regulation of LUCAT1 remarkably increased protein expression ofβ-catenin in the nucleus,TCF7L2,Wnt1 and SOX2 in total,while miR-5582-3p mimic induced opposite results which could be restored by the co-transfection of oe-LUCAT1.The down-regulation of LUCAT1 expression in MCF-7 CSCs could reverse the enhancement of the transcriptional activity of TOP/FOP by miR-5582-3p inhibitor.The similar results of western blot were also observed in MCF-7 CSCs transfected with miR-5582-5p inhibitor and/or sh-LUCAT1.6.LUCAT1 regulates breast cancer stemness in vivo:The effect of LUCAT1 on increasing tumor size and weight could be inhibited by miR-5582-3p,while the effect of miR-5582-3p on decreasing tumor size and weight could be rescued when LUCAT1 was overexpressed in the nude mouse model.Consistent with in vitro findings,the up-regulation of LUCAT1 expression in MCF-7 cells could reverse the decreased expression ofβ-catenin in nucleus,SOX2,TCF7L2 and Wnt1 in total in xenograft tumors,caused by miR-5582-3p agomir.The down-regulation of LUCAT1 expression increased miR-5582-3p expression,and decreased TCF7L2 and Wnt pathway-related proteins expression in MCF-7 CSCs-derived tumors.Conclusion:1.High expression of LUCAT1 in breast cancer indicates poor prognosis.LUCAT1 might be a significant biomarker to evaluate prognosis in breast cancer.2.LUCAT1 increased stem-like properties of BCCs and stemness of BCSCs.3.LUCAT1,as ceRNA,competitively combined with mir-5582-3p,to reduce the inhibitory effect of mir-5582-3p on TCF7L2,and then activate the Wnt/β-catenin signal pathway to regulate the stem cell stemness of breast cancer.The LUCAT1/miR-5582-3p/TCF7L2 axis provides insights for regulatory mechanism of stemness,and new strategies for clinical practice.