Journal: Oncogene

Article Title: CRISPR interference and activation of the microRNA-3662-HBP1 axis control progression of triple-negative breast cancer.

doi: 10.1038/s41388-021-02089-6

Figure Lengend Snippet: Fig. 4 Identification of HBP1 as a target gene of miR-3662 in MDA-MB-231 cells. A Venn diagram showing the intersection of differentially upregulated genes in RNA-seq and target gene predictions from Targetscan. B Heat-map-based clustering of expression of miR-3662 candidate target genes using our RNA-seq data with TCGA breast cancer dataset. The 26 candidate target genes were divided into three groups according to the expression data from TCGA. The colors of the heat-map represent the expression levels of the 26 candidate genes. The red arrowheads indicate the top candidate 5 genes, for which their expressions were negatively correlated with the expression of miR- 3662, and the Spearman’s correlation coefficient was <−0.1 in TCGA dataset. C qPCR analysis of HBP1 in miR-3662 scrambled control and KO cells. D-F qPCR analysis of HBP1 in scrambled control or KO cells with miR-3662 mimic or inhibitor. Numbers on the x-axis in D indicate treatment time (hours) with the miR-3662 inhibitor. G, H Western blot analyses of HBP1 protein expression in scrambled control and KO cells. I, J Expression of HBP1 protein determined by IHC analysis in scrambled control and KO xenograft tumors from NSG mice. K, L Western blots and quantitative analyses of expression of HBP1 and Wnt signaling proteins in scrambled control, KO, and miR-3662 mimic-transfected KO cells. M, N Western blots and quantitative analyses of expression of HBP1 and Wnt signaling proteins in KO, scrambled control, and HBP1 siRNA-transfected KO cells. Data are presented as means ± SD. P values were determined by a two-tailed t-test or a one-way ANOVA test followed by a Dunnett post hoc test. NC, negative miRNA control; Scr, scrambled control; KO, knockout. All experiments were repeated three times.

Article Snippet: Antibodies used were specific for the following targets: HBP1 (Cat No. 11746-1-AP, 1:5000 for Western blots, 1:100 for IHC staining; Proteintech, Rosemont, IL), β-catenin (Cat No. ab32572, 1:5000 for Western blots, 1:500 for IF staining; Abcam, Cambridge, MA), E-cadherin (Cat No. ab76055, 1:3000 for Western blots; Abcam), N-cadherin (Cat No. ab18203, 1:3000 for Western blots; Abcam), vimentin (Cat No. 5741, 1:200 for IHC staining; Cell Signaling Technology, Danvers, MA), GSK-3β (Cat No. 3915, 1:3000 for Western blots; Cell Signaling Technology), p-GSK-3β (Cat No. 9336, 1:3000 for Western blots; Cell Signaling Technology), c-Myc (Cat No. 904401, 1:3000 for Western blots; BioLegend, San Diego, CA), Ki67 (Cat No. ab15580, 1:200 for IHC staining; Abcam), cyclin D1 (Cat No. ab134175, 1:5000 for Western blots, Abcam), lamin B (Cat No. sc-374015, 1:5000 for Western blots; Santa Cruz Biotechnology, Dallas, TX), GAPDH (Cat No. 5174, 1:5000 for Western blots; Cell Signaling Technology), β-actin (Cat No. sc47778, 1:5000 for Western blots; Santa Cruz Biotechnology), and β-tubulin (Cat No. ab6046, 1:5000 for Western blots; Abcam).

Techniques: RNA Sequencing, Expressing, Control, Western Blot, Transfection, Two Tailed Test, Knock-Out

Journal: Oncogene

Article Title: CRISPR interference and activation of the microRNA-3662-HBP1 axis control progression of triple-negative breast cancer.

doi: 10.1038/s41388-021-02089-6

Figure Lengend Snippet: Fig. 5 Validation of miR-3662-HBP1 axis-mediated regulation of the Wnt/β-catenin signaling pathway in BT-20 cells. A Expression levels and nuclear localization of β-catenin by IF analysis in scrambled control and KO cells. B Quantification of expression levels of nuclear/ cytoplasmic β-catenin and their ratio. C, D Western blot and quantitative analyses of expression of HBP1 and Wnt signaling proteins in miR- 3662 scrambled control and KO cells. E, F Expression of HBP1 protein determined by IHC analysis in scrambled control and KO xenograft tumors from NSG mice. G, H Western blot and quantitative analyses of expression of HBP1 and Wnt signaling proteins in KO, scrambled control, and HBP1 siRNA-transfected KO cells. Data are presented as means ± SD. P values were determined by a two-tailed t-test. Scr, scrambled control; KO, knockout. All experiments were repeated three times.

Article Snippet: Antibodies used were specific for the following targets: HBP1 (Cat No. 11746-1-AP, 1:5000 for Western blots, 1:100 for IHC staining; Proteintech, Rosemont, IL), β-catenin (Cat No. ab32572, 1:5000 for Western blots, 1:500 for IF staining; Abcam, Cambridge, MA), E-cadherin (Cat No. ab76055, 1:3000 for Western blots; Abcam), N-cadherin (Cat No. ab18203, 1:3000 for Western blots; Abcam), vimentin (Cat No. 5741, 1:200 for IHC staining; Cell Signaling Technology, Danvers, MA), GSK-3β (Cat No. 3915, 1:3000 for Western blots; Cell Signaling Technology), p-GSK-3β (Cat No. 9336, 1:3000 for Western blots; Cell Signaling Technology), c-Myc (Cat No. 904401, 1:3000 for Western blots; BioLegend, San Diego, CA), Ki67 (Cat No. ab15580, 1:200 for IHC staining; Abcam), cyclin D1 (Cat No. ab134175, 1:5000 for Western blots, Abcam), lamin B (Cat No. sc-374015, 1:5000 for Western blots; Santa Cruz Biotechnology, Dallas, TX), GAPDH (Cat No. 5174, 1:5000 for Western blots; Cell Signaling Technology), β-actin (Cat No. sc47778, 1:5000 for Western blots; Santa Cruz Biotechnology), and β-tubulin (Cat No. ab6046, 1:5000 for Western blots; Abcam).

Techniques: Biomarker Discovery, Expressing, Control, Western Blot, Transfection, Two Tailed Test, Knock-Out

Journal: Oncogene

Article Title: CRISPR interference and activation of the microRNA-3662-HBP1 axis control progression of triple-negative breast cancer.

doi: 10.1038/s41388-021-02089-6

Figure Lengend Snippet: Fig. 6 miR-3662-mediated functional role and post-transcriptional regulation of HBP1. MDA-MB-231 miR3662 KO cells were transfected with either scrambled control siRNA or HBP1-specific siRNA. A Western blot analysis of HBP1 protein expression. B qPCR analysis of HBP1 expression. C Cell proliferation measured by cell counts for 6 days. D, E Clonogenic cell assay. After 2 weeks, the cloning formation was analyzed with crystal violet staining. F, G Transwell invasion assays. H Luciferase activity of HEK293T cells transfected with the miR-3662 mimic or inhibitor. The dual-luciferase reporter assay was performed on aliquots of the samples, and firefly luciferase activity, normalized to Renilla luciferase, was plotted. I Specificity of the miRNA-HBP1 interaction validated by directed mutagenesis of miRNA-binding sites in HBP1 3′-UTR. J Interaction analysis of miR-3662 with the 3′-UTRs of HBP1 mRNA by miRNA/mRNA IP assay in MDA-MB-231 cells. Data are presented as means ± SD. P values were determined by a two-tailed t-test or a two-way ANOVA test. siRNA, small interfering RNA; KO, knockout. All experiments were repeated three times.

Article Snippet: Antibodies used were specific for the following targets: HBP1 (Cat No. 11746-1-AP, 1:5000 for Western blots, 1:100 for IHC staining; Proteintech, Rosemont, IL), β-catenin (Cat No. ab32572, 1:5000 for Western blots, 1:500 for IF staining; Abcam, Cambridge, MA), E-cadherin (Cat No. ab76055, 1:3000 for Western blots; Abcam), N-cadherin (Cat No. ab18203, 1:3000 for Western blots; Abcam), vimentin (Cat No. 5741, 1:200 for IHC staining; Cell Signaling Technology, Danvers, MA), GSK-3β (Cat No. 3915, 1:3000 for Western blots; Cell Signaling Technology), p-GSK-3β (Cat No. 9336, 1:3000 for Western blots; Cell Signaling Technology), c-Myc (Cat No. 904401, 1:3000 for Western blots; BioLegend, San Diego, CA), Ki67 (Cat No. ab15580, 1:200 for IHC staining; Abcam), cyclin D1 (Cat No. ab134175, 1:5000 for Western blots, Abcam), lamin B (Cat No. sc-374015, 1:5000 for Western blots; Santa Cruz Biotechnology, Dallas, TX), GAPDH (Cat No. 5174, 1:5000 for Western blots; Cell Signaling Technology), β-actin (Cat No. sc47778, 1:5000 for Western blots; Santa Cruz Biotechnology), and β-tubulin (Cat No. ab6046, 1:5000 for Western blots; Abcam).

Techniques: Functional Assay, Transfection, Control, Western Blot, Expressing, Cloning, Staining, Luciferase, Activity Assay, Reporter Assay, Mutagenesis, Binding Assay, Two Tailed Test, Small Interfering RNA, Knock-Out

Journal: Oncogene

Article Title: CRISPR interference and activation of the microRNA-3662-HBP1 axis control progression of triple-negative breast cancer.

doi: 10.1038/s41388-021-02089-6

Figure Lengend Snippet: Fig. 7 Regulation of the miR-3662-HBP1 axis and its effect on growth and the Wnt signaling pathway. A, B Schematic illustration of the CRISPRi/a system. C Design and sequences for CRISPRi/a sgRNAs. D, E Relative expression levels of mi-R3662 measured by qPCR in CRISPRi/a HEK293T cells after Dox induction (days 0–3). F, G Expression of dCas9-KRAB, dCas9-VPR, and sgRNA determined by flow cytometric and IF analyses with GFP and mIFP in CRISPRi/a MDA-MB-231 cells. H, M Expression changes of mi-R3662 in CRISPRi/a MDA-MB-231 cells after Dox induction (days 0–7) measured by qPCR. I, N Expression changes of HBP1 in CRISPRi/a MDA-MB-231 cells after Dox induction (days 0–7) measured by qPCR. J, O. Changes of proliferation of CRISPRi/a MDA-MB-231 cells after Dox induction (days 1–7) assayed by MTT. K, L. Western blot and quantitative analyses of expression of HBP1 and Wnt signaling proteins in CRISPRi MDA-MB-231 cells before and after Dox induction (days 0 and 4). P, Q Western blot and quantitative analyses of expression of HBP1 and Wnt signaling proteins in CRISPRa MDA-MB-231 cells before and after Dox induction (days 0 and 4). Data are presented as means ± SD. P values were determined by a two-tailed t-test or a one- or two-way ANOVA test. All experiments were repeated three times.

Article Snippet: Antibodies used were specific for the following targets: HBP1 (Cat No. 11746-1-AP, 1:5000 for Western blots, 1:100 for IHC staining; Proteintech, Rosemont, IL), β-catenin (Cat No. ab32572, 1:5000 for Western blots, 1:500 for IF staining; Abcam, Cambridge, MA), E-cadherin (Cat No. ab76055, 1:3000 for Western blots; Abcam), N-cadherin (Cat No. ab18203, 1:3000 for Western blots; Abcam), vimentin (Cat No. 5741, 1:200 for IHC staining; Cell Signaling Technology, Danvers, MA), GSK-3β (Cat No. 3915, 1:3000 for Western blots; Cell Signaling Technology), p-GSK-3β (Cat No. 9336, 1:3000 for Western blots; Cell Signaling Technology), c-Myc (Cat No. 904401, 1:3000 for Western blots; BioLegend, San Diego, CA), Ki67 (Cat No. ab15580, 1:200 for IHC staining; Abcam), cyclin D1 (Cat No. ab134175, 1:5000 for Western blots, Abcam), lamin B (Cat No. sc-374015, 1:5000 for Western blots; Santa Cruz Biotechnology, Dallas, TX), GAPDH (Cat No. 5174, 1:5000 for Western blots; Cell Signaling Technology), β-actin (Cat No. sc47778, 1:5000 for Western blots; Santa Cruz Biotechnology), and β-tubulin (Cat No. ab6046, 1:5000 for Western blots; Abcam).

Techniques: Expressing, Western Blot, Two Tailed Test