Fig. 4

MTDH is a direct target of miR-224-3p. a Venn diagram showing the putative target genes of miR-224-3p computationally predicted by 4 algorithms (miRmap, TargetScan, miRWalk and DIANA microT). b The TOP20 upregulated genes in chemoradiotherapy-resistant ESCC, compared to chemoradiotherapy-sensitive ESCC, in public GSE45670 dataset. c Representative images of positive and negative IHC staining of MTDH in ESCC tissues (× 200). MTDH predominantly locates in cytoplasm. d The expression of MTDH is higher in CDDP-resistant ESCC cells than in matched parental cells. e Predicted binding sites on 3′-UTR of MTDH to sponge miR-224-3p and matched established mutant sequences. f Luciferase reporter assay shows the luciferase activity of MTDH-3′-UTR-WT, MTDH-3′-UTR-Mut#1, MTDH-3′-UTR-Mut#2, MTDH-3′-UTR-Mut#3 and MTDH-3′-UTR-Mut#4 in KYSE30/CDDP-R cells co-transfected with miR-224-3p mimic. g The effects of miR-224-3p mimic on the expression of MTDH in KYSE30/CDDP-R cells transfected with MTDH-3′-UTR-WT, MTDH-3′-UTR-Mut#1, MTDH-3′-UTR-Mut#2, MTDH-3′-UTR-Mut#3 or MTDH-3′-UTR-Mut#4. h NORAD knockdown downregulates MTDH expression in KYSE30/CDDP-R cells and miR-224-3p mimic rescues this downregulation. i Overexpression of NORAD upregulates MTDH expression in KYSE30 cells and miR-224-3p inhibitor rescues this downregulation. j Three-dimensional scatter plot of NORAD, miR-224-3p and NORAD expressions in 11 CDDP-resistant and 82 CDDP-sensitive ESCC tissues from patients. g-i Error bars denote SD of triplicates. ***P < 0.001