Fig. 4

The hnRNPA1 protein mediates miR-196a packaging into CAF-derived exosomes. a A specific interaction between the miR-196a sequence and RBP motifs was predicted through RBPDB analysis (threshold 0.7). b Western blot and real-time PCR results showing ZRANB2, hnRNPA1, and ELAVL1 expression levels in CAFs at 48 h after transfection with specific siRNAs. c miR-196a expression in exosomes from CAFs transfected with specific siRNAs targeting ZRANB2, hnRNPA1, or ELAVL1 was measured using real-time PCR. d Real-time PCR analysis showing miR-196a expression in CAFs with hnRNPA1 silenced. e Western blot analysis of hnRNPA1 expression in samples derived by miRNA pulldowns performed with nuclear, cytoplasmic, or exosomal CAFs lysates and the indicated biotinylated miR-196a or mutated miR-196a; biotinylated poly(G) was used as a negative control. f RIP assays with anti-hnRNPA1 antibody (or IgG as control) were performed on the cell or exosomal lysates from CAFs. miR-196a levels in immunoprecipitated samples were determined by real-time PCR and were reported as percentages in respect to the input sample (% input). g RIP assay to determine hnRNPA1 enrichment on miR-196a relative to IgG in cytoplasmic or exosomal lysates of CAFs treated with or without cisplatin. h CAL 27 and HN4 cells were co-cultured with CAFs concurrently transfected with Cy3-miR-196a and specific siRNAs targeting hnRNPA1 for 48 h. Fluorescence microscopy was used to detect red fluorescent signals in HNC cells (scale bar, 10 μm). i Nude mice were subcutaneously xenografted with a mixture of CAL 27 cells plus CAFs transfected with hnRNPA1, sh-NC, or sh-hnRNPA1, and the tumor growth curve and tumor volumes are shown. j The distribution of miR-196a in xenograft tumors was detected using FISH assay, and miR-196a expression in tumor cells was assessed. k The hnRNPA1 mRNA expression level in 108 pairs of HNC samples and adjacent normal tissues. l Correlation analysis was performed between miR-196a expression and hnRNPA1 expression in HNC tissues (n = 108) (NT, without cisplatin treatment; CT, cisplatin treatment; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001)