LncRNA-Hh Strengthen Cancer Stem Cells Generation In Twist-Positive Breast Cancer Via Activating Hedgehog Signaling Pathway

Objective To understand the effects of lncRNAs relatedTwist induce EMT on CSC characteristics in Twist-positive breast cancerMethods (1) Human breast cancer cells MCF-7 were infected with the retrovirus carrying Twist gene. The expression of EMT markers was determined by qRT-PCR and immunofluorescence. (2) Mammosphere formation efficiency was determined by mammosphere assay in MCF-7/Twist cells and MCF-7/Vector cells. CSC characteristics were measured using the changes of CSC markers by qRT-PCR, Western blot and immunofluorescence assay, the capacity of self-renewal, proliferation and migration were determined by each mammosphere-generated cell numbers and the tanswell assay. (3) LncRNAs and mRNA expression profiles were analyzed between MCF-7/Twist mammosphere cells and MCF-7/Vector mammosphere cells by using Agilent Human lncRNA/mRNA microarrays. The chip results were accurately reproduced by qRT-PCR. (4) Bioimformatic analysis (pthway) of dysregulated genes was performed via DAVID v6.7 using KEGG pathway database. The excitation of pathway was verified by western blot. (5) Mammosphere formation efficiency, the expression of CSC markers and the excitation of Hedgehog pathway were measured by mammosphere assay and western blot via the knockout of Twist gene and blocking the activation of Hedgehog pathway in Twist-positive breast cancer. (6) The potential lncRNAs associated with Hedgehog signaling pathway were determined by bioinformatics and qRT-PCR. (7) The relationship between lncRNA-Hh and Twist was analyzed by qRT-PCR. The transcriptional activity of lncRNA-Hh was measured by luciferase reporter assay. (8) Mammosphere formation efficiency, the expression of CSC markers, lncRNA-Hh potential-target GAS1 and the excitation of Hedgehog pathway were measured by mammosphere assay and western blot via the over-expression of lncRNA-Hh in MCF-7 and the knockout of lncRNA-Hh in Twist-positive breast cancer. (9) The functional role of lncRNA-Hh in tumorigenesis was determined in vivo. The expression levels of lncRNA-Hh, and the target GAS1, GLI1 and CSC-related proteins in xenografts were confirmed by qRT-PCR, Western blotting assay and Irnmunohistochemistry assay.Results (1) Over-experssion of Twist results a typical EMT phenotype in MCF-7. (2) Twist-induced EMT endows cells with cancer stem cell-like characteristics and more capacity of self-renewal, proliferation and migration. The larger size of mammospheres and more MFEs were acquired. (3) Compared with MCF-7/Vector mammosphere cells,270 up-regulated lncRNAs,192 down-regulated lncRNAs (Fold change>5) and 2065 differentially expressed mRNAs (Fold change>1.5) were identified by microarray. The potential targets neighboring genes of 462 differentially expressed lncRNAs (Fold change>5) via cis-or trans-regulatory effects were predicted. (4) Several well-known canonical pathways which are related with CSC were identified in the lncRNAs predicted gene group and the different mRNA gene group. The Hedgehog signaling, which was indicated to be significantly changed by lncRNA/mRNA array assay, has a dramatically enhanced. (5) Twist-knockdown and blocking the activation of Hedgehog pathway obviously reduced the mammosphere formation efficiency and the expression of CSC markers. The excitation of Hedgehog pathway was weakened. (6) Hedgehog signaling pathway associated-LncRNA (lncRNA (chr9,89563614-89616948,+), LncRNA-Hh) was ascertained. (7) Twist was involved in lncRNA-Hh transcription. (8) lncRNA-Hh promoted the the mammosphere formation efficiency, the expression of CSC markers, the excitation of Hedgehog pathway and the expression of potential-target GAS1. (9) lncRNA-Hh promotes breast cancer tumorigenesis in vivo and the expression levels of lncRNA-Hh, GAS1, GLI1 and CSC-related proteins in xenografts.Conclusion our results show that lncRNA (e.g. lncRNA-Hh) plays a key role in endowing Twist-induced EMT cells to gain CSC-like signature via egulating the corresponding signaling pathways (e.g. Hedgehog signaling). Hopefully, this study could provide an impetus for a novel insight into the CSC pathology genesis in breast cancer.