Figure 4

MiR-296-3p directly regulates ICAM-1 in PCa cells. (a) Screening of candidate miRNAs (in target cells) involved in the resistance to NK cells were performed using calcein release assay. Data are the mean±S.E.M. of three independent experiments. (b) The expression level of miR-296-3p and transcription level of ICAM-1 in P69 and M12 were analyzed by miRNA transcriptome sequencing and DGE sequencing, respectively. TPM means transcripts per million. (c) Real-time PCR in ΔΔCt method was performed to identify the expression levels of miR-296-3p in P69 and M12. One representative experiment out of three was shown. (d) The protein levels of ICAM-1 in P69 and M12 were determined by western blotting. One representative experiment out of three was shown. (e) The predicted miR-296-3p binding site (red) and their mutant (blue) in the 3′-UTR of ICAM-1 mRNA are indicated. Number shows position of nucleotides in the 3′-UTR of ICAM-1 mRNA. (f) HEK-293T cells were transfected with psiCHECK-2 containing WT or mutant ICAM-1 3′-UTR and miR-296-3p or mock control vector. The Renilla luciferase activity was normalized on the constitutive activity of firefly luciferase. Data are the mean±S.E.M. of three independent experiments. (g) The expression of membrane-bound or total ICAM-1 in P69 and M12 were analyzed by flow cytometry (upper panel) and western blotting (bottom panel), respectively. One representative experiment out of three was shown