Additional file 1: of The pro-metastasis effect of circANKS1B in breast cancer
Creators: name: Zeng, Kaixuan
name: He, Bangshun
name: Yang, Burton
name: Xu, Tao
name: Chen, Xiaoxiang
name: Xu, Mu
name: Liu, Xiangxiang
name: Sun, Huiling
name: Pan, Yuqin
name: Wang, Shukui
Publisher: name: figshare
Year: 2018
Subjects: subject: Biophysics
subject: Biochemistry
subject: Cell Biology
subject: Genetics
subject: FOS: Biological sciences
subject: Molecular Biology
subject: Physiology
subject: Pharmacology
subject: Biological Sciences not elsewhere classified
subject: Developmental Biology
subject: Cancer

description: Tables S1. Correlations between circANKS1B expression and clinical characteristics in breast cancer patients (n = 165). Tables S2. Univariate and multivariate overall survival analysis of prognostic factors for breast cancer patients (n = 165). Tables S3. Primers and RNA sequences used in this study. Figure S1. (A) Genomic origin of circRNAs (n = 69,815) identified in human breast tissues. 87% were derived from exons, and the others were derived from introns, intergenic region and 3′ or 5′ UTR, etc. (B) The length distribution for circRNAs (n = 69,815) identified in human breast tissues. Most of the circRNAs are less than 1,300 nucleotides (nt) in length. Figure S2. (A-D) qRT-PCR analysis of the screened top ten most increased and decreased circRNAs in twenty pairs of TNBC and adjacent normal tissues. Four circRNAs (circ-PGAP3, circ-THSD4, circ-CYP24A1 and circ-ACACB) were validated to be significantly dysregulated. (E-F) qRT–PCR analysis of the abundance of circANKS1B and ANKS1B mRNA in MCF-7 and MDA-MB-231 cells treated with Actinomycin D at the indicated time points. (G) qRT–PCR analysis of circANKS1B and ANKS1B mRNA after treatment with RNase R in MCF-7 and MDA-MB-231 cells. Figure S3. (A) Schematic of two siRNA targeting circANKS1B junction site (left). These two siRNA effectively silenced circANKS1B expression in MDA-MB-231 cells, whereas had no effect on linear ANKS1B expression (right). (B) Schematic of construction of circANKS1B overexpression vector, the sequence of the 5′-flanking intron was copied and inversely inserted the downstream of 3′-flanking intron (left). The overexpression vector effectively increased circANKS1B expression in MCF-7 cells, while did not affect its precursor expression. Figure S4. (A-C) CCK-8 and EdU analysis of the proliferative abilities of MCF-7 cells with circANKS1B overexpression and MDA-MB-231 cells with circANKS1B knockdown. Scale bar = 20 μm. (D) The images of tumor-bearing nude mice from the indicated treatment groups (n = 5 for each group) on the 49th day. (E) Annexin V-PE/7-AAD double staining analysis of apoptosis of MCF-7 cells with circANKS1B overexpression and MDA-MB-231 cells with circANKS1B knockdown. Figure S5. (A) Transwell migration and matrigel invasion assays for T47D cells with circANKS1B overexpression and BT549 cells with circANKS1B knockdown. Scale bar = 20 μm. (B) Immunofluorescence analysis of E-cadherin and Vimentin in circANKS1B-overexpressing MCF-7 cells and circANKS1B knockdown MDA-MB-231 cells. Scale bar = 20 μm. Figure S6. (A) Schematic illustration showing two targeted siRNAs. si-ANKS1B targets the ANKS1B linear transcript, si-both targets both the linear ANKS1B and circANKS1B (left). Their respective inhibitory effects were verified by qRT-PCR (right). (B-C) Wound healing, transwell migration and invasion assays for MDA-MB-231 cells transfected with si-NC, si-both or si-ANKS1B. Scale bar = 20 μm. Figure S7. (A-B) qRT-PCR analysis of miR-148a-3p and miR-152-3p in circANKS1B-overexpressing MCF-7 cells and circANKS1B knockdown MDA-MB-231 cells. (C) qRT-PCR analysis of circANKS1B in MCF-7 and MDA-MB-231 cells transfected with miR-148a/152-3p mimics or control mimics. Figure S8. (A) Schematic of the selection of these 14 metastasis-related genes targeted by miR-148a-3p and miR-152-3p. (B) qRT-PCR analysis of these 14 metastasis-related genes in MCF-7 with miR-148a-3p or miR-152-3p overexpression. Solid red and black circles indicate the genes regulated and non-regulated by miR-148a-3p or miR-152-3p, respectively. Figure S9. (A) IHC analysis of USF1 in normal tissues (n = 40) and breast cancer tissues (n = 165). (B) The expression of USF1 mRNA in breast cancer tissues from TCGA database. (C) The overall survival curves in breast cancer patients with low and high USF1 expression from KM-plotter database ( http://kmplot.com/analysis/ ). (D) The distant metastasis-free survival curves in breast cancer patients with low and high USF1 expression from KM-plotter database. Figure S10. Immunoblot analysis of USF1 and TGF-β1 in MCF-7 (A) or MDA-MB-231 cells (B) in the indicated groups. β-actin was used as a loading control. Figure S11. (A) qRT-PCR analysis of circANKS1B expression in MDA-MB-231 cells transfected with the indicated siRNAs. (B) Transwell invasion assay for MDA-MB-231 cells co-transfected with si-ESRP1 or si-NC and circANKS1B or control vector. (C-D) The overall and distant metastasis-free survival curves in breast cancer patients with low and high ESRP1 expression from KM-plotter database ( http://kmplot.com/analysis/ ). Data were represented as means ± S.D. of at least three independent experiments. Figure S12. (A-B) Transwell migration and invasion assays for circANKS1B silencing MDA-MB-231 cells transfected with miR-148b-3p inhibitors. Data were represented as means ± S.D. of at least three independent experiments. Scale bar = 20 μm. **p &lt; 0.01, n.s = not significant. (DOCX 25384 kb)