Fig. 4.

IL7Rα as a target gene of miR-3614-5p in CRC. (A) The volcano plot showed that there were 127 differentially expressed genes including 31 upregulated and 96 downregulated genes. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis showed miR-3614-5p down-regulated genes enriched in JAK/STAT3, TNFα, and inflammatory response pathways. (C) Venn diagram showed the overlapping genes among RNA-seq data, miR-3614-5p target genes, and genes enriched in JAK/STAT3, TNFα, and inflammatory response pathways. (D) Schematic diagram of the RNA pull-down assay. (E) The RNA pulldown results showed the enrichment of miR-3614-5p and IL7Rα mRNA by the miR-3614-5p probe. (F) Schematic diagram of the Ago2 RIP assay. (G) The Ago2-RIP results showed the effect the enrichment of IL7Rα mRNA in Ago2-containing miRNPs. (H) Schematic diagram of the binding sites between IL7R mRNA and miR-3614-5p. (I) Dual-luciferase reporter assay was performed to evaluate the effect of miR-3614-5p mimics or inhibitors transfection on the luciferase activity of IL7Rα wild type (Wt) and IL7Rα mutant type (Mut) in 293T and HCT116 cells. (J) RT-qPCR detected the effects of upregulation of miR-3614-5p expression on IL7Rα expression in HCT116 and HT29 cells. (K) Western blot analysis showing reduced IL7Rα and CD132 protein levels following miR-3614-5p overexpression. (L-M) Representative flow cytometry plots (L) and quantification (M) of IL7Rα-positive and IL7Rα-negative cells in HCT116 and HT29 cells, showing decreased IL7Rα expression after miR-3614-5p overexpression.*p < 0.05