LncRNA Expression Signatures Of Twist-induced Epithelial-to-mesenchymal Transition In MCF10A Cells

Objective：The aim of this study was to research a signal network andlncRNAs in twist-induced epithelial-to-mesenchymal transition in MCF10Acells.Methods：The immunohistochemistry was used to stain normal breasttissue samples, ductal carcinoma in situ samples, and various breastcarcinoma samples (grade>2). A pBABE-Puro-based vector andpBABE-Puro-based vectors expressing c-Myc-tagged Twist cDNA weredescribed and stable cell lines were created via infection of target cells usingretrovirus. The expressions of Twist、E-cadherin、N-cadherin、Vimentin andFibronectin associated with EMT were analyzed by Western blotting、qRT-PCR and immunofluorescent staining. To construst a network regulatedby Twist through lncRNAs in contributing to EMT process, we performedlncRNA and mRNA array analysis in MCF10A/Twist and MCF10A/Vectorcells. The differentially expressed lncRNA and mRNA profiles were analyzedusing paired Significance Analysis in Microarray (SAM). The chip results were verified by qRT-PCR. These differentially expressed mRNAs were thengrouped using hierarchical clustering of heatmap. The target genes oflncRNAs were predicted through UCSC genome browser and RNAplexsoftware. Bioinformatic analysis (gene ontology, pathway and networkanalysis) of the integrated genes was performed through the GeneCodis webtool and Integrated Discovery (DAVID) v6.7. The activation of β-catenin(total、nuclear and p-β-catenin) and the downstream target products (Vimentin,Fibronectin, Snail and Slug) of the high-enrichment WNT signaling wereanalyzed by Western blotting、qRT-PCR and immunofluorescent stainingunder treatment with or without special inhibitor. The invasive potential ofMCF10A/Vector, MCF10A/Twist and MCF10A/Twist under blockage ofWNT signaling were examined by Transwell array.Results：The expression of Twist in high-grade breast tumors was inincreasing levels comparable to normal breast tissues. Over-expression ofTwist led to decreasing classic epithelial markers (E-cadherin) and increasingmesenchymal markers, N-cadherin, Vimentin and Fibronectin. The lncRNAmicroarray reveals differentially expressed lncRNAs betweenMCF10A/Twist and MCF10A/Vector cells. In the MCF10A/Twist group,30lncRNAs were upregulated and69were downregulated (fold change≥5.0or≤0.2). There was3,164mRNAs differentially expressed. The qRT-PCR dataaccurately was in accordance with the chip results. Target gene-relatedpathway analysis and mRNA pathway enrichment analyses showed significant changes associated with EMT, including the WNT, MAPK,JAK/STAT, TGF-β, mTOR, Hedgehog and P53signaling pathway.Interestingly, we showed that β-catenin was activated in MCF10A/Twistcells. Nuclear β-catenin was greatly increased in MCF10A/Twist checked,while the total β-catenin was slightly decreased. Moreover, thephosphorylation of β-catenin was significantly reduced in MCF10A/Twistgroup. A complex series of downstream target genes of WNT pathway,including Vimentin, Fibronectin, Snail and Slug were up-regulated. And theinvasion ability of MCF10A/Twist cell was enhanced.Conclusions: The lncRNAs expression profile was marklydysregulated in MCF10A/Twist and MCF10A/Vector cells. A networkregulated by Twist through lncRNAs function independently or coordinatelyto activate the EMT program were constructed. More interestingly, lncRNA(chr17,44833874–44834830,+), lncRNA (chr17,21142183–21156578,),lncRNA (chr6,26124411–26139312,+) and lncRNA (chr19,438420–2083745,) may be involved in regulation or activation of WNTsignaling pathway in the Twist-induced EMT process. These findings firstdetermine that Twist contributes to invasion and metastasis by inducingwide-ranging transcriptional and functional changes of lncRNAs and signalpathways in our study.