TGF-β Promotes EMT In Breast Cancer Through LncRNA-mediated Multilevel Regulation Of SNAI2

BACKGROUND Breast cancer is the most common female malignant tumor,with concomitantly high incidence and mortality.In China,the number of new cases of breast cancer is up to 273,000 per year,which is still increasing with a youth oriented tendency.Metastasis is the most important cause of death in breast cancer patients,and 20-30% of breast cancer patients eventually die from tumor recurrence and metastasis.Epithelial-mesenchymal transition(EMT)is among the core cellular behaviors that underlie the metastasis of primary tumors.Multiple signaling pathways,growth factors and tumor microenvironment factors are involved in the EMT process.TGF-β signaling pathway is widely accepted as a key signaling pathway that promotes EMT via upregulation of transcriptional factors Snail,Slug and ZEB1 through Smad-dependent and-independent pathways.Long non-coding RNAs are a large heterogeneous class of transcripts longer than 200 nucleotides without a protein-coding potential.They can regulate gene expression in cis or in trans via epigenetic mechanisms or in transcriptional and post transcriptional levels.Recent studies have shown that lnc RNAs play important roles in metastasis.Given the critical involvement of TGF-β–elicited EMT in metastasis,it is of great significance to explore the role of lnc RNAs in fine-tuning the TGF-β signal pathway.PURPOSES The purpose of this study was to identify lnc RNAs that are regulated by TGF-β and play key roles in mediating TGF-β-induced EMT of breast cancer cells.In particular,the regulation of two candidate lnc RNAs UCA1 and AC026904.1,and their regulatory roles in EMT were to be characterizedMETHODS AND RESULTS 1.TGF-β1 was used to induce EMT of a breast cancer cell line,MCF-7 and microarray analysis was performed to screen for the differentially expressed lnc RNAs.Quantitative RT-PCR assay confirmed that two of these candidate RNAs,UCA1 and AC026904.1,were upregulated by TGF-β.FISH experiment showed that UCA1 was mainly distributed in the cytoplasm and AC026904.1 was mainly located in the nucleus.A variety of inhibitors were used to pretreat breast cancer cells,in which we found that the Smad3 inhibitor SB431542 and the MEK/ERK signaling inhibitor U0126 can abolish TGF-β induction of UCA1 and AC026904.1,respectively.CHIP-q PCR verified that the binding ability of Smad2/3 to UCA1 promoter was significantly enhanced after TGF-βinduction,while the binding capacity of Smad2/3 to the AC026904.1 promoter region showed no significant change upon treatment with TGF-β.2.In situ hybridization showed that UCA1 was positively associated with the malignancy of breast cancer,while IHC showed that E-cadherin was negatively correlated with the malignancy of breast cancer.q RT-PCR results showed that AC026904.1 and UCA1 had higher expression levels in metastatic breast cancer tissues.Kaplan-Meier’s analysis revealed that breast cancer patients with lower expression of AC026904.1 or UCA1 displayed longer progression-free survival(PFS).3.q RT-PCR was used to detect the efficiency of si RNAs targeting AC026904.1 and UCA1.Western blot and ICC resluts indicated that AC026904.1 and UCA1 can upregulate SNAI2 and ZEB1 positively and downregulate E-cadherin.q RT-PCR results showed that in clinical samples of breast cancer,AC026904.1 and UCA1 were positively correlated with the level of Slug m RNA.4.Bioinformatics analysis showed that the transcript of UCA1 contains potential binding sites of mi R-1 and mi R-203 a.Western Blot experiments showed that mi R-1 and mi R-203 a inhibitors could significantly restore the Slug protein levels in stable UCA1 knockdown MDA-MB-231 cells.RIP and double luciferase reporter gene experiments showed that UCA1 could directly bind to mi R-1 and mi R-203 a,and UCA1 derepressed the expression of SNAI2 through sequestering mi R-1 and mi R-203 a.5.We interrogated a 500 kb window around the AC026904.1 gene locus and found that only two protein-coding genes,SNAI2/SLUG and EFCAB1,were contained within this genomic region.The 3C experiment and the double luciferase reporter gene experiment confirmed that AC026904.1 can function as an enhancer RNA(e RNA)for SNAI2.Chromatin immunoprecipatation(Ch IP)and q PCR experiment confirmed that AC026904.1 has the characteristics of an e RNA.Chromatin RNA immunoprecipitation(Ch IRP)experiment confirmed that AC026904.1 can be enriched on the promoter region of Slug.Transwell experiment showed that the migration of breast cancer cells were significantly reduced after stable knockdown of AC026904.1 or UCA1,which was restored by Slug overexpression.6.Parental and AC026904.1/UCA1 knockdown breast cancer cells were inoculated into the mammary fat pads of nude mice to establish xenograft tumor model.We found that knockdown of either lnc RNA in breast cancer cells suppressed the development and metastasis of in vivo tumors,and significantly prolonged the survival of tumor-bearing mice.CONCLUSION 1.The expression of the lnc RNAs,UCA1 and AC026904.1,was regulated in breast cancer cells via canonical and non-canonical TGF-β signal pathway.2.UCA1 and AC026904.1 were highly expressed in metastatic breast cancer,and were correlated with a poor prognosis of breast cancer patients.3.UCA1 and AC026904.1 cooperate to upregulate the transcription factor Slug in breast cancer cells.4.UCA1 functions as a competing endogenous RNA(ce RNA)to derepress the expression of Slug in breast cancer cells.5.AC026904.1 promotes SNAI2/SLUG transcription in cis by acting as an enhancer RNA(e RNA).6.Silencing of UCA1 or AC026904.1 efficiently impairs the metastasis of breast cancer.