Fig. 2

CSC promotes pri-miR-25 maturation by upregulating METTL3 expression. a Relative expression levels of pri-miR-25, pre-miR-25, and miR-25-3p in HPDE6-C7, PANC-1, and BXPC-3 cells exposed to CSC (100 µg/ml). b Effects of CSC on the expression of miR-25-3p and METTL3 mRNA (upper panel) and protein (lower panel) in HPDE6-C7, PANC-1, and BXPC-3 cells in a dose (0, 0.1, 1.0, 10, and 100 µg/ml) dependent manner. c METTL3 overexpression or knockdown significantly affected the expression of pri-miR-25, pre-miR-25, and miR-25-3p in PDAC cells. OE, overexpression. d METTL3 knockdown significantly affected the expression of miR-25-3p in HPDE6-C7, PANC-1, and BXPC-3 cells exposed to CSC. e CSC (100 µg/ml) induced hypomethylation of CpG island in the METTL3 promoter region (between –284 and –564 bp from transcription start site) detected by bisulfate sequencing (upper panel) and methylation-specific PCR (lower panel) in HPDE6-C7 and PDAC cells. f Representative methylation-specific PCR (MSP) of non-tumor tissues from smokers or nonsmokers showing the METTL3 CpG islands hypomethylation in smokers. Ratio (M/M + U) represents the methylated rate in indicated samples. g ChIP-qPCR assays showing decreased bindings of DNMT1 and DNMT3a but not DNMT3b within the METTL3 CpG islands caused by CSC. h Quantitative ChIP analysis of DNMT1, DNMT3a, and DNMT3b levels within the METTL3 regulatory region in non-tumor tissues from 36 patients (18 smokers and 18 nonsmokers). i NFIC knockdown significantly affected the expression levels of METTL3 proteins (left panel) and RNA and miR-25-3p (right panel). Results of RNA represent means ± S.D. from three independent experiments. j ChIP-qPCR assays showing significantly increased direct binding of NFIC to METTL3 promoter in HPDE6-C7 and PDAC cells exposed to CSC. k Quantitative ChIP analysis showing significant difference in the NFIC levels within the METTL3 regulatory region between smokers’ and nonsmokers’ non-tumor pancreatic tissues (both N = 18). Data of (h) and (k) are displayed in min to max boxplot. The line in the middle of the box is plotted at the median while the upper and lower hinges indicated 25th and 75th percentiles. Values are the mean ± S.D. from three independent experiments, and all statistic analyses in this figure are Student t test. *P < 0.05, **P < 0.01, ***P < 0.001 and NS, not significant as compared with the corresponding control