Fig. 6

CDKN1B and ING5 are direct targets of exosomal miR-196a in HNC cells. a A diagram showing the predicted candidate target genes of miR-196a by Gene Ontology analysis. b CDKN1B and ING5 mRNA expression in CAL 27 and HN4 cells transfected with miR-196a or anti-miR-196a at 48 h after transfection. c CDKN1B and ING5 mRNA levels in CAL 27 and HN4 cells at 48 h after incubation with exosomes (25 μg/mL) from HNC cells (Ctrl Exos) and CAFs. d, e Correlation analysis was performed between miR-196a expression and CDKN1B or ING5 expression in HNC tissues (n = 108). f Predicted miR-196a target sequences in the 3′ UTRs of CDKN1B and ING5 genes. g Relative CDKN1B or ING5 reporter activities in 293T cells co-transfected with miR-196a and luciferase reporters. h The effects of anti-miR-196a on CDKN1B or ING5 reporter luciferase activity in 293T cells. i The effects of CAF-derived exosomes (25 μg/mL) on CDKN1B or ING5 reporter luciferase activity in 293T cells. j A diagram showing the program for the immunoprecipitation of miRNA targets. k Interaction of target transcripts with miR-196a. CAL 27 or HN4 cells were transfected with biotinylated miR-NC or miR-196a for 48 h. Levels of CDKN1B and ING5 mRNA in the materials pulled down by biotin-miR-196a were analyzed by real-time PCR and normalized to β-actin. l CAL 27 and HN4 cells were transfected with miR-196a or anti-miR-196a or were incubated with CAF-derived exosomes (25 μg/mL) for 48 h. p27 and ING5 expression in the indicated cells was detected by western blot analysis. m The results from the cell cycle and cisplatin-induced cell apoptosis analyses in HNC cells transfected with miR-NC, miR-196a plus control vector, miR-196a plus CDKN1B plasmid, or miR-196a plus ING5 plasmid at 48 h after transfection. (ns, no significant difference; *p < 0.05; **p < 0.01; ***p < 0.001)