Pancreatic cancer is a malignant tumor of the digestive tract with a high degree of malignancy and is difficult to diagnose and treat. About 90% of ductal adenocarcinomas originate from the ductal epithelium. Its morbidity and mortality have increased significantly in recent years. The 5-year survival rate is <1%, which is one of the worst malignant tumors. m6A methylation modification is an important mechanism for pri-miRNA processing and maturation, but its role in abnormal regulation in human diseases remains unclear. The authors found that cigarette smoke condensate (CSC) produced by smoking can induce mR-25-3p precursor maturation through m6A modification, thereby promoting the development of pancreatic cancer.
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Experimental methods: miRNA sequencing , m6A pri-miRNA sequencing , ChIP qPCR , RNA pull down
Experimental materials: CSC (cigarette smoke condensate), DMSO (general chemical dissolver), pancreatic ductal adenocarcinoma, pancreatic cells, etc.
CSC-treated pancreatic duct epithelial cells and untreated groups were subjected to miRNA sequencing (the cloud sequence can provide this service) , and the differentially expressed miRNA----miR-25-3p in the experimental group was screened. Moreover, the expression level of miRNA is positively correlated with the CSC concentration. The front is in the cell, and then the authors examined the expression levels of miRNAs in pancreatic tissue in smoked and non-smokers and found that miR-25-3p is also highly expressed in smokers. In normal and pancreatic cancer cells, miR-25-3p was also found to be significantly up-regulated in cancer cells. The above results indicate that smoking and PDAC diseases cause a significant up-regulation of miR-25-3p expression. Then the author combined with clinical indicators, found that patients with high expression of miR-25-3p PDAC had shorter survival time. At the same time, miR-25-3p is associated with cell proliferation, migration and invasion phenotypes.
To further elucidate the molecular mechanism of CSC-induced miR-25-3p, the authors first examined the expression of pri-miR-25, pre-miR-25 and miR-25-3p in CSC-treated PDAC cells, and found that CSC treatment can reduce pri -miR-25 expression increases both pre-miR-25 and miR-25-3p expression, suggesting that CSC may induce high expression of miR-25-3p by regulating the processing and maturation of pri-miR-25. Since m6A methylation plays an important regulatory role in miRNA precursor processing and maturation, the authors examined several important m6A methyltransferases to see if these enzymes affect CSC-induced miR-25-3p The expression showed that CSC induced high expression of METTL3 and showed concentration-dependent, but had no effect on METTL14 and WTAP expression. Subsequently, the authors further verified that overexpression of METTL3 enzyme in PDAC cells significantly reduced pri-miR-25 levels and increased pre-miR-25 and miR-25-3p levels, and vice versa. At the same time, knockdown of METTL3 also significantly reduced CSC-induced miR-25-3p expression. The above data suggest that CSC may promote high expression of miR-25-3p by increasing the expression of METTL3.
First, the authors demonstrated that CSC can inhibit the methylation of the promoter region of the MeTTL3 gene by DNA methylation low-throughput verification technology (the cloud sequence provides this service) . The results of ChIP-qPCR (the cloud sequence can provide this service) showed that CSC treatment can reduce the binding of DNA methyltransferases DNMT1 and DNMT3a to the METTL3 promoter. This condition also exists in the pancreatic tissue of the smoker. The authors then identified four potential transcription factors that regulate METTL3 by biosignal analysis, but only NFIC, a transcription factor, is positively correlated with METTL3 mRNA expression levels in both tumor and non-tumor pancreatic tissues. For further validation, the authors knocked down NFIC in PDAC cells and found that the expression levels of METTL3 and miR-25-3p were significantly reduced. In addition, the binding of NFIC to the METTL3 promoter was also significantly increased in non-tumor pancreatic tissues of smokers. The above results indicate that CSC promotes the expression of METTTL3 by reducing the DNA methylation of the METTL3 promoter region and binding it to the transcription factor NFIC.
To further explore whether METTL3 affects miR-25-3p maturation, the authors first analyzed the m6A site on pri-miR-25 by m6Apri-miRNA methylation sequencing (the cloud sequence provides this service) and found that m6A does exist in pri-miR-25 cleavage site. Analysis of MeRIP-qPCR (which is available in the cloud format ) showed that the m6A modification of pri-miR-25 in PDAC samples was much higher than that of non-PDAC samples. Similarly, CSC treatment increased the m6A modification of pri-miR-25 in cells; overexpression of METTL3 also increased the m6A modification of pri-miR-25, whereas knockdown of METTL3 was reversed. These results suggest that METTL3 plays an important role in the formation of m6A and the maturation of miR-25-3p. To test whether m6A directly regulates miR-25 maturation, the authors performed in vitro RNA maturation using in vitro transcribed m6A-containing pri-miR-25 ([m6A]pri-miR-25) and m6A-modified pri-miR-25. The results showed that the [m6A]pri-miR-25 was significantly increased by the sheared pre-miR-25; when the METTL3 binding region of pri-miR-25 was mutated, pri-miR-25 was cleaved Pre-miR-25 was significantly reduced. These in vitro results were consistent with intracellular phase, suggesting that high expression of miR-25-3p in smokers and PDAC patients may be due to high stimulation of METTL3 due to cigarette stimulation and increased m6A modification on pri-miR-25.
To find the reader enzyme of pri-miRNA, the authors found 22 proteins that bind to pri-miRNA m6A by RNA Pull Down (this cloud provides this service) combined with mass spectrometry. DGCR8 is an important enzyme in the process of pri-miR-25 processing. After coIP (the cloud sequence can provide this service) , 9 proteins were found to interact with DGCR8. NKAP was found to be the recognition protein of m6A in pri-miR-25.
Next, the authors studied the mechanism of action of miRNAs and their target genes, and predicted the potential target gene PHLPP2 of miR-25-3p by means of biosignal analysis. It was confirmed by luciferase and overexpression and knockout experiments that PHLPP2 is indeed a potential target gene for miR-25-3p. Finally, the article was sublimed and confirmed that the target gene is involved in the AKT signaling pathway and affects the phosphorylation level of p70S6K.
to sum up
In this study, the authors first screened the highest expression of miR-25-3p in the CSC treatment group by miRNA high-throughput sequencing, and was clinically and functionally significant.
Subsequently, the authors were divided into two ways to explore the causes and functions of miR-25-3p differential expression.
On the one hand: it was confirmed that CSC induced METTL3 promoter hypomethylation and increased its expression by transcription factor NFIC, thereby increasing the m6A methylation degree of pri-miR-25; on the one hand: finding m6A of pri-miR-25 The methylation recognition protein is NKAP, which promotes the maturation of pri-miR-25 to miR-25-3p by forming a complex with miRNA mature processing proteins DGCR8 and Drosha. Finally, the target gene PHLPP2 of miR-25-3p was found by biosignal. It was confirmed that it promotes the occurrence and development of PDAC by activating the AKT cancer signaling pathway.
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