Fig. 4

CRLF1 is a downstream target of miR-3065-3p in colorectal cancer cells. a Venn diagrams showing the number of genes identified as potential targets of miR-3065-3p using three databases. b RT-qPCR analysis of TMEM47, CRLF1 and CLDN11 expression in miR-3065-3p-overexpressing HCT116 or RKO cells. c Western blot analysis of CRLF1 expression in HCT116 or RKO cells ectopically expressing miR-3065-3p and vector control (MCS) cells. The right panel shows the statistical results. d Western blot analysis and statistical results of CRLF1 expression in tumor tissues from the xenograft mouse model. ‘1–4’ represented the different four mice in the same group. e Representative images of immunohistochemical staining with antibodies against CRLF1 in tumor tissues. The right panel shows the statistical results. f Predicted miR-3065-3p binding sites in the 3ʹUTR of CRLF1 and the seed sequences of miR-3065-3p of different species. g The wild-type (WT) or mutant (Mut) construct with miR-3065-3p (accession number: MIMAT0015378) binding sites in the 3ʹUTR of CRLF1. h HEK293T cells were cotransfected with a pmirGLO reporter harboring the 3ʹUTR of CRLF1 with wild-type (WT) or mutated (Mut) miR-3065-3p binding sites and miR-3065-3p mimics or NC. Luciferase activity was analyzed 40 h after transfection. i Analysis of CRLF1 expression in colorectal cancer and normal tissues according to data from TCGA database using UALCAN website ( http://ualcan.path.uab.edu/index.html). j, k RT-qPCR (j) and western blot (k) analysis of CRLF1 expression in colorectal cancer and adjacent normal tissues. l Correlation analysis between CRLF1 and miR-3065-3p expression in colorectal cancer. The results are shown as the mean values ± SEM of three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001