Fig 1: miR-31-5p targets EGLN3 and MBOAT2 expression. (A) Venn diagram showing the intersection between 86 genes that were the most derepressed after antimiR-31 treatment and predicted miR-31 target genes from TargetScan human/rat software. (B) Transfection experiments were performed in HEK-293FT cells using constructs for 3 predicted miR-31 targets, MBOAT2, EGLN3, and CTNND2. Renilla luciferase activity was normalized to firefly luciferase activity. The normalized luciferase activity in 293FT cells transfected with empty psiCHECK2 vector and scrambled negative control 1 (premiR-N.C.1) and relative luciferase activities of 293FT cells transfected with all other combinations of psiCHECK2 constructs and premiR oligos are shown. Experiments were repeated thrice in duplicate and data are presented as mean ± SD. Luciferase reporter assays indicate direct interactions between miR-31-5p and MBOAT2 (P < 0.005) and between miR-31-5p and EGLN3 (P < 0.001), two-tailed Student’s t test. (C) mRNA expression of Egln3 and Mboat2 in esophageal mucosa-derived RNA from ZD:CTRL, ZS:CTRL, and ZD:miR-31-/- rats (Psmb6 as normalizer; error bars represent mean ± SD; n = 7 to 11 rats). P values represent the significant difference between each individual group and ZD:CTRL rats; two-tailed, one-way ANOVA and Tukey post hoc t tests. (D) Protein expression of miR-31 targets EGLN3, MBOAT2, and STK40 (arrowheads emphasize the abundant/intense miR-31 overexpression in ZD:CTRL vs. ZS:CTRL esophagus). Representative photos of ZD:CTRL, ZS:CTRL, ZD:miR-31-/-, and ZS:miR-31-/- esophagus showing miR-31 ISH signal (blue, NBT/BCIP) (EGLN3, MBOAT2, and STK40 protein expression; brown, DAB; n = 10 rats per cohort). (E) Connection between miR-31 expression, protein expression of EGLN3 and STK40, and inflammation markers in human ESCC tissue. Representative photos of human ESCC tissue (FFPE sections): Case 1, case 2, and normal esophageal mucosa in case 2 showing miR-31 ISH signal (blue, NBT/BCIP) (EGLN3, STK40, COX-2, S100A8, S100A9, and NF-?B p65 protein expression; brown, DAB; n = 10 patient cases).