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Fig. 4 | Journal of Experimental & Clinical Cancer Research

Fig. 4

From: Loss of exosomal miR-3188 in cancer-associated fibroblasts contributes to HNC progression

Fig. 4

miR-3188 directly targets BCL2 in HNC cells. a The target genes of miR-3188 were predicted by bioinformatics analysis and then subjected to gene ontology analysis. Six candidate genes related to proliferation, cell cycle and apoptosis were screened for further verification. b, c After a 24 h transfection with miR-3188 mimics in HN4 and HN30 cells, the expression levels of potential targets for miR-3188 were analyzed using real-time PCR. d After a 24 h transfection with miR-3188 inhibitor in HN4 and HN30 cells, the BCL2 expression levels were analyzed using real-time PCR. e, f After a 48 h transfection with miR-3188 inhibitor in HN4 and HN30 cells, the BCL2 expression levels were analyzed using western blotting. g CAFs were transfected with miR-3188 mimics and CM was collected 48 h later. After treatment with CM of miR-3188 overexpressed CAFs for 48 h, BCL2 protein levels in HN4 and HN30 were analyzed using western blotting. h, i miR-3188 and BCL2 expression in 102 normal epithelial tissues and 115 HNC tissues were detected by real-time PCR. j Pearson analysis was performed in the above cohort and an inverse correlation was observed between the miR-3188 and BCL2 expression levels. k Predicted miR-3188 binding site in the 3’UTR region of BCL2. A mutated miR-3188 binding site was generated in the complementary site for the seed region of miR-3188. l, m An miR-3188 mimics or inhibitor and a luciferase vector encoding the wild-type or mutant BCL2 3′-UTR region were co-transfected into HN4 and HN30 cells, after another 24-h incubation the relative luciferase activity was measured. n HN4 cells was transfected with a luciferase vector encoding the wild-type or mutant BCL2 3′-UTR region, and treated with CM of CAFs overexpressed miR-3188 or CM of NFs silenced miR-3188, then the luciferase activity was measured. (NC, negative control. ns, no significant difference, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001)

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