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. 2019 Apr 23;10(1):1858.
doi: 10.1038/s41467-019-09712-x.

Excessive miR-25-3p Maturation via N 6-methyladenosine Stimulated by Cigarette Smoke Promotes Pancreatic Cancer Progression

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Free PMC article

Excessive miR-25-3p Maturation via N 6-methyladenosine Stimulated by Cigarette Smoke Promotes Pancreatic Cancer Progression

Jialiang Zhang et al. Nat Commun. .
Free PMC article

Abstract

N6-methyladenosine (m6A) modification is an important mechanism in miRNA processing and maturation, but the role of its aberrant regulation in human diseases remained unclear. Here, we demonstrate that oncogenic primary microRNA-25 (miR-25) in pancreatic duct epithelial cells can be excessively maturated by cigarette smoke condensate (CSC) via enhanced m6A modification that is mediated by NF-κB associated protein (NKAP). This modification is catalyzed by overexpressed methyltransferase-like 3 (METTL3) due to hypomethylation of the METTL3 promoter also caused by CSC. Mature miR-25, miR-25-3p, suppresses PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2), resulting in the activation of oncogenic AKT-p70S6K signaling, which provokes malignant phenotypes of pancreatic cancer cells. High levels of miR-25-3p are detected in smokers and in pancreatic cancers tissues that are correlated with poor prognosis of pancreatic cancer patients. These results collectively indicate that cigarette smoke-induced miR-25-3p excessive maturation via m6A modification promotes the development and progression of pancreatic cancer.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
MiR-25-3p induction by CSC and its effect on survival time of individuals with PDAC. a Heatmap of significantly different expression of microRNAs (P < 0.05, fold change > 2 or < 0.5) detected with microRNA array in HPDE6-C7 cells exposed to CSC (100 µg/ml) or equal amount of DMSO as solvent control. The red (higher expression) or green (lower expression) color represents the normalized expression value of indicated microRNAs. b Expression levels of indicated miRNAs in HPDE6-C7 cell exposed to CSC (100 μg/ml) or equal amount of DMSO as vehicle control. Results represent means ± standard derivation (S.D.) from three independent measurements. c The induction of miR-25-3p overexpression in HPDE6-C7, PANC-1, and BXPC-3 cells was in a CSC dose (0, 0.1, 1.0, 10, and 100 µg/ml) dependent manner. Data represent mean ± S.D. from three independent experiments. d Expression levels of indicated miRNAs in non-tumor tissue samples of smokers (N = 67) or nonsmokers (N = 108). Results represent means ± S.D. from three independent measurements. e Aberrant overexpression of miR-25-3p in surgically removed PDAC samples compared with their paired non-tumor tissue samples in two patient groups and combined sample. fh Kaplan–Meier estimates of survival time in two groups of patients with PDAC and combined sample by different miR-25-3p levels in tumor. The median survival time for individuals with high miR-25-3p levels (≥median) in Guangzhou, Beijing and combined sample was 10.1, 9.0, and 10.1 months compared with 16.6, 16.0, and 17.8 months in those with low miR-25-3p levels (<median), with the hazard ratio and 95% confidence interval (CI) of 2.20 (95% CI, 1.53–3.63), 1.82 (95% CI, 1.15–3.36), and 2.07 (95% CI, 1.50–3.09). i Significant difference in serum miR-25-3p levels among smokers (N = 22) and nonsmoker (N = 23). Data of (e) and (i) 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. Student t tests were used in (a), (b), and (c) (NS, non-significant, *P< 0.05, **P< 0.01, and ***P< 0.001) and Wilcoxon rank-sum tests were used in (d), (e), and (i) (NS, non-significant, **P< 0.01, and ***P< 0.001)
Fig. 2
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
Fig. 3
Fig. 3
m6A modification is a major mechanism for METTL3-promoted pri-miR-25 maturation. a Left: Identification of m6A by m6A individual-nucleotide resolution cross-linking and immunoprecipitation sequencing (miCLIP-seq). The m6A residues were detected by cross-linking induced mutation sites (CIMS) and cross-linking induced truncation sites (CITS) in pri-miR-25. Red tracks of miCLIP-seq are unique tag coverage and blue tracks represent C>T transition or truncation, respectively. Filled purple circles denote miCLIP-called m6A and the horizontal blue bars indicate transcript models of pre-miR-25. Right: The sequences of pre-miR-25 and miR-25-3p are highlighted, respectively, by different colors, and the m6A motif (GGACU) is located at the putative splicing site. b PDAC had significantly higher levels of [m6A]pri-miR-25 than paired non-tumor tissues. [m6A]pri-miR-25 was detected by immunoprecipitation followed by qRT-PCR analysis. ***P< 0.001 by Wilcoxon rank-sum test. c, d Effects of CSC exposure (100 µg/ml) and overexpression or knockdown of METTL3 on the levels of [m6A]pri-miR-25 in PDAC cells. Data represent mean ± S.D. from three independent experiments. e, f m6A modification substantially enhanced pri-miR-25 processing and maturation in an in vitro reaction system containing starting materials of pri-miR-25 or [m6A]pri-miR-25 and the whole cellular lysates of 293T cells transfected with plasmids carrying DROSHA and DGCR8. Pri-miR-1-1 which has no methylation site was included as a control. e Northern blot detection of starting materials (left panel) and the levels of resultants pre-miR-25 and pre-miR-1-1 in the reaction. f Quantification of pre-miR-25, miR-25-3p, and pri-miR-25 in the reaction mixture. Data represent fold change ± S.D. relative to pri-miR-25 as starting material. g, h Mutation of [m6A]pri-miR-25 at METTL3-recognizing site abolished pri-miR-25 processing and maturation in the in vitro reaction system. g Northern blot detection of starting materials (left panel) and the levels of resultants pre-miR-25 and pre-miR-1-1 in the reaction. h Quantification of pre-miR-25, miR-25-3p, and pri-miR-25 in the reaction mixture. Data represent fold change ± S.D. relative to [m6A]pri-miR-25 WT as starting material from three experiments. All statistic analyses in this figure are Student t test unless specified. *P< 0.05, **P< 0.01, and ***P< 0.001 compared with the corresponding control
Fig. 4
Fig. 4
Identification of NKAP as a mediator of m6A in pri-miR-25 maturation. a Schematic of RNA pulldown and immunoprecipitation experiment and proteomic screening by mass spectrometry (MS) for the identification of [m6A]pri-miR-25 reader. b Western blot analysis of potential m6A readers obtained from proteomic screening as indicated in (a) shows specific association of NKAP with both [m6A]pri-miR-25 and DGCR8. c RNA immunoprecipitation assays with m6A antibody show association of NKAP (FLAG-tagged) with pri-miR-25 in PDAC cells. Data from three independent experiments represent RNA levels associated with NKAP relative to input. Antibody against DGCR8 or DROSHA was included as positive control and IgG served as negative control. d Electrophoretic mobility shift assays of recombinant NKAP with unmethylated or methylated pri-miR-25 probes. The probes were maintained constantly while a gradient of 0–10 μM recombinant NKAP was added to the reactions. e, f Reciprocal immunoprecipitation assays show interaction among DGCR8, DROSHA, and NKAP (FLAG-tagged) in PANC-1 cells in the presence of RNase A. g Confirmation of binding of DGCR8 to NKAP by truncation mapping test. Upper panel shown are the schematic of the domain structures of DGCR8 and NKAP protein. Lower panel: Western blot analysis of constructs for His-tagged DGCR8 (wild-type versus domain truncation mutants) and FLAG-tagged NKAP or for FLAG-tagged NKAP (wild-type versus domain truncation mutants) and His-tagged DGCR8 that were co-transfected into PANC-1 cells, respectively. h Knockdown of NKAP expression in PDAC cells substantially decreased pre-miR-25 and miR-25-3p levels but substantially increased the level of pri-miR-25. i RNA immunoprecipitation assays show significantly decreased enrichment of pri-miR-25 with NKAP (left panel) or DGCR8 (right panel) in PDAC cells with METTL3 knockdown. j RNA immunoprecipitation assays show significantly decreased enrichment of pri-miR-25 with DGCR8 in PDAC cells with NKAP knockdown. k NKAP knockdown affected the expression of miR-25-3p in PDAC cells exposed to CSC. Values of (hk) are the mean ± S.D. from three independent experiments. All statistic analyses in this figure are Student t test. *P< 0.05, **P< 0.01, and ***P< 0.001 compared with the corresponding control
Fig. 5
Fig. 5
NKAP is a reader of m6A on pri-miR-25. a The NKAP-binding sites on pri-miR-25 detected by CIMS and CITS miCLIP-seq. Orange tracks of NKAP-iCLIP-seq are unique tag coverage and blue tracks represent C>T transition and truncation, respectively. Filled purple circles denote iCLIP-called NKAP-binding site and the horizontal blue bars indicate the transcript model of pre-miR-25. b The DGCR8-binding sites on pri-miR-25 detected by DGCR8 HITS-CLIP. Filled purple circles denote iCLIP-called NKAP-binding site and the horizontal blue and red bars represent the transcript model of pre-miR-25 and the DGCR8-binding site, respectively. c In vivo binding of NKAP-FLAG to pri-miR-25 in PDAC cells exposed to CSC or DMSO detected by CLIP-qPCR. Values are the mean ± S.D. from three independent experiments. d The levels of NKAP-FLAG bound to pri-miR-25 were significantly elevated by overexpression of METTL3, but significantly decreased by knockdown of METTL3 in both PANC-1 (left panel) and BXPC-3 (right panel) cells. Values are the mean ± S.D. from three independent experiments. e Data of m6A residues and NKAP-binding sites identified by the analysis of cross-linking-induced mutation sites (CIMS) or cross-linking-induced truncation sites (CITS) in iCLIP-seq data. f Venn diagram showing overlap between m6A residues and NKAP-binding sites. g Significant enrichment of RGAC motif is in NKAP footprints (CIMS or CITS along with 5-nucleotide flanking sequences) analyzed by MEME SUIT with default parameters. h Shown is the intensity of NKAP binding centered at m6A residues. i Shown is the intensity of m6A CLIP signal centered at NKAP-binding sites. j Shown is the distribution of NKAP iCLIP tag density around the splicing factor binding sites. k Shown is the binding intensity of NKAP at splicing sites near or not near m6A residues. All statistic examinations in this figure are Student t test unless specific. *P< 0.05, **P< 0.01, and ***P< 0.001 compared with the corresponding control
Fig. 6
Fig. 6
MiR-25-3p targets PHLPP2 and evokes oncogenic AKT-p70S6K signaling. a Relative reporter gene activity of psiCHECK2 vector bearing PHLPP2 3′-UTR fraction 1 (left panel) or fraction 2 (right panel) in PDAC cells co-transfected with increasing amounts (1, 5, and 10 pmol) of miR-25-3p mimic. b Relative reporter gene activity of constructs bearing PHLPP2 3′-UTR fraction 1 (left panel) or fraction 2 (right panel) in PDAC cells co-transfected with 5 pmol of miR-25-3p mimic or its inhibitor. c Reporter gene activity of psiCHECK2 vector bearing PHLPP2 3′-UTR fraction 1 (left panel) or fraction 2 (right panel) or their mutant counterparts in PDAC cells in the presence of 5 pmol miR-25-3p mimic. Results in (a), (b), and (c) represent means ± S.D. from three experiments and each had six replicates. d Stable overexpression or knockdown of miR-25-3p on the levels of PHLPP2 RNA (left panel) and protein (right panel). Results of mRNA represent means ± S.D. from three independent experiments. e RNA immunoprecipitation assays show significantly increased association with Ago2 of both miR-25-3p (left panel) and PHLPP2 mRNA (right panel) in PDAC cells overexpressing miR-25-3p. Results represent means ± S.D. from three independent experiments. f MiR-25-3p evokes AKT-p70S6K signaling via PHLPP2. Knockdown of PHLPP2 substantially enhanced AKT and p70S6K phosphorylation activation in PDAC cells. g Overexpression or knockdown of miR-25-3p had substantial effects on PHLPP2 expression and phosphorylation of its downstream signaling modules. h Knockdown of PHLPP2 expression substantially rescued the activation of AKT and p70S6K suppressed by miR-25-3p knockdown in PDAC cells. Ctrl control, KD knockdown. i Knockdown of PHLPP2 expression significantly reversed the effects of miR-25-3p knockdown on PDAC cell proliferation. Results represent mean ± S.D. from three independent experiments. j Knockdown of PHLPP2 expression significantly reversed the effects of miR-25-3p knockdown on PDAC cell migration and invasion. Left panel shows representative images of transwell assays and right panel shows quantitative statistics. Results are mean ± S.D. from three random fields. All statistic analyses in this figure are Student t test. *P< 0.05, **P< 0.01, and ***P< 0.001 compared with the corresponding control
Fig. 7
Fig. 7
Cigarette smoking evokes the activation of AKT-p70S6K signaling via miR-25-3p in PDAC. a CSC evokes the activation of AKT-p70S6K signaling via miR-25-3p in PDAC cells. b, c Exposure to CSC significantly enhanced the ability of proliferation (b) and migration and invasion (c) in PDAC cells. Scale bars, 200 μm. Right panel shows quantitative statistics. Results are mean ± S.D. from three random fields. d Correlations between miR-25-3p and METTL3 RNA levels and between PHLPP2 RNA and miR-25-3p or METTL3 RNA levels in smokers’ non-tumor pancreatic tissues (N = 67, left panel) and PDAC (N = 67, right panel) by Pearson’s test. e Correlations between miR-25-3p and METTL3 RNA levels and between PHLPP2 RNA and miR-25-3p or METTL3 RNA levels in nonsmokers’ non-tumor pancreatic tissues (N = 108, left panel) and PDAC (N = 108, right panel) by Pearson’s test. f Western blot analysis of protein expression levels of indicated genes in non-tumor tissues from nonsmokers (N = 18, upper panel) or smokers (N = 18, lower panel). R, the same positive reference sample for loading adjustment on each gel. Each protein band was semi-quantified by gray density and the value for each band is relative to density of both β-ACTIN and the corresponding band of R. g Significant difference in the expression levels of indicated proteins in non-tumor tissues from nonsmokers or smokers (both N = 18). Data 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. *P< 0.05, **P< 0.01, and ***P< 0.001 by Student t test. h Significant Pearson correlations among the expression levels of METTL3, PHLPP2, p-AKT, and p-p70S6K proteins in smokers’ non-tumor tissues (N = 18). i Shown are Pearson correlations between METTL3, PHLPP2, p-AKT, and p-p70S6K protein expression levels in non-tumor tissue specimens from nonsmokers (N = 18). Data of (gi) were from western blot analyses showing in (f). j A proposed action model for excessive miR-25-3p maturation via m6A modification stimulated by cigarette smoking in the development and progression of PDAC

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