Fig 2: Correlations among RCAN1.4 and RUNX3 expression in breast cancer tissues. a,b Three pairs of breast cancer tissues and matched normal breast tissues were subjected to ChIP analysis using antibody against H3K27ac. The association with the supper-enhancer of RCAN1.4 (a) and promoter region of RCAN1.4 (b) were quantified by qPCR. c The mRNA expression of RCAN1.4 (left) and RUNX3 (right) in three pairs of breast cancer tissues and matched normal tissues. d Kaplan-Meier analyses of OS, RFS and DMFS based on RUNX3 (204197_at) mRNA levels using the KM-plotter breast cancer database (http://kmplot.com/analysis). Auto select best cutoff was chosen in the analysis. e Kaplan-Meier plots of the overall survival of patients, stratified by both mRNA expression of RUNX3 and RCAN1.4. The data were retrieved from breast cancer patients of TCGA database. f The representative images of strong RUNX3 staining in the matched adjacent normal cells (N) and weak staining in tumor cells (T) (left). Quantitative IHC analysis of RUNX3 staining in primary breast tumors and adjacent normal breast tissues was shown (n = 99, right). g The representative images for low and high expression of RUNX3 staining in 258 primary breast cancer tissues were shown (left). Kaplan-Meier plots of the overall survival of patients, stratified by protein expression of RUNX3(right). h The Spearman correlation of RUNX3 with RCAN1.4 expression in breast cancer patient tumors. i Kaplan-Meier plots of the overall survival of patients, stratified by protein expression of RUNX3 and RCAN1.4. j Proposed model of RUNX3 deficiency-mediated disruption of SE-driven RCAN1.4 expression to promote the malignancy of breast carcinoma. During the mammary epithelial cell differentiation, the tumor suppressor gene RCAN1.4 expression is driven by a ~ 23 kb-long super-enhancer region, which is located ~ 266 kb away with the epigenetic signature of active enhancers H3K4me1 and H3K27ac. This SE is occupied by the key transcription factor RUNX3 and physically interacts with the RCAN1.4 locus via DNA looping. In breast cancer cells, the loss of RUNX3 results in disruption of super-enhancer-driven RCAN1.4 expression, which promotes the malignancy of breast carcinoma. The error bars represent mean ± SD, n = 3 biological independent samples. **P < 0.01. The P value in a, b was determined by one-way ANOVA with Tukey’s multiple comparisons test, no adjustments were made for multiple comparisons. The P value in c was determined by a two-tailed unpaired Student’s t test. The P value in f was determined by Wilcoxon matched-pairs signed rank test (two-sided), and the error bars represented lower hinge - 1.5 * IQR to upper hinge + 1.5 * IQR (where IQR is the inter-quartile range, or distance between the first and third quartiles). Data in a, b, c were representative of three independent experiments