| Severe acute pancreatitis(SAP)is a digestive tract disease characterized by local pancreatic damage and subsequent systemic inflammatory response clinically.The condition of patients are severe,and its mortality rate is as high as 30%.A number of studies have demonstrated that several key events participate in the occurrence and development of SAP together simultaneously or consecutively,including the activation and release of premature trypsinogen,pathological calcium signaling,ductal cell dysfunction,the activation of immune cells and cytokines release,endoplasmic reticulum stress,mitochondrial dysfunction and impaired autophagy,ultimately resulting in systemic inflammation response syndrome(SIRS)and multiple organ failure.Multiple organ failure is the main reason that leads to the death of patients.Currently,the pathophysiology of SAP remains unclear,especially the level of gene regulation.Circular RNA(circRNA)is a class of single-stranded covalently closed RNA molecules.Circ RNAs are expressed widely in mammalian tissues and participate in the occurrence and development of many diseases.In these diseases,they function through the following mechanisms mainly: micro RNA(miRNA)sponge,the regulation of mRNA expression,modulating the transcription of parental genes,the templates for protein synthesis and interplays with RNA-binding proteins.Some studies have found that the entire process of circRNA itself from biogenesis to function is precisely regulated,among which N6-methyladenosine(m6A)modification is the most studied.As the most prevalent internal modification of RNA in eukaryotic cells,m6A is regulated by three homologous factors precisely,namely methyltransferase,demethylase and m6A readers.The methyltransferases and demethylases are mainly responsible for catalyzing the formation and oxidative demethylation of m6A.The m6A readers are mainly responsible for recognizing m6A and regulating RNA splicing,localization,degradation and translation.The m6A modification of circRNA was discovered in 2017 and was confirmed to have the same methyltransferase,demethylase and m6A readers as mRNA.At present,the expression of m6A-modified circRNAs has been found to show the cell-type-specific patterns and disease-specific patterns.m6A can participate in many pathophysiological processes in by regulating the metabolism of circRNAs.For example,in the nuclear phase,the nuclear m6A reader protein YTHDC1 can recognize m6A and promote the nuclear export of circRNA.Once exporting to the cytoplasm,it will be recognized by the related reader protein or binds to other proteins,which can stabilize mRNA,affect the micro RNA sponge,or initiate cap-independent translation.In our previous studies,we found that the pancreatic tissue of SAP mice expressed a large number of circRNAs and found that they were involved in SAP progression.Therefore,we speculate that circRNAs are regulated by m6A in SAP pancreatic tissue,and participate in the occurrence and development SAP progression.Based on the above,this study aims to identify transcriptome-wide map of m6A modification in circRNAs,determine their biological significance and explore the potential mechanisms in SAP,in order to further understand the pathological mechanism of SAP and provide the new insights for seeking new potential therapeutic targets.Part I: The identification and analysis of transcriptome-wide m6A-modified circRNA in SAP pancreatic tissuesObjective: To identify and analyze transcriptome-wide m6A-modified circRNAs in SAP pancreatic tissues.Methods: The SAP in C57BL/6 mice was induced using 4% sodium taurocholate salt(0.1ml/100g).To evaluation the SAP model,the pancreatic tissue damage was observed by HE staining and the activities of serum lipase and amylase were determined by biochemical methods.m6A modified RNA immunoprecipitation sequencing(MeRIP-seq)was used to obtain the sequencing raw data,and cutadapt software was used to remove 3’ adaptor-trimming and low-quality reads.The clean reads with high quality of the input library were aligned to the mouse reference genome(UCSCMM10)with STAR software.Circ RNAs were detected and identified using DCC software.The identified circRNAs were annotated using circ Base database and Circ2 Traits database.The methylated sites were identified in each sample using MACS software.Differentially methylated sites were identified using diff Reps software.The expression characteristics of m6A-modified circRNAs were analyzed.Results: The pathological findings and serum lipase and amylase levels showed the successful establishment of the SAP mouse model.We identified 409 m6A-modified circRNAs totally using MeRIP-seq.Among them,178 were specifically expressed in the SAP group,107 were specifically expressed in the control group,and 124 were shared in both groups.Moreover,57 differentially expressed m6A-modified circRNAs were identified between the SAP group and the control group.These results suggested that the m6A modified circRNA in the SAP group was altered and displayed a disease-specific pattern.The total m6A level of circRNA in the SAP group was lower than that in the control group.In addition,compared with non-m6A-modified circRNAs,the percentage of circRNAs derived from protein-coding genes in m6A-modified circRNAs was increased,suggesting that circRNA m6A modification may play an important role in SAP.Part II: Preliminary exploration of the biological significance of m6A-modified circRNAs in SAPObjective: To explore the biological significance of m6A-modified circRNAs in SAP preliminarily.Methods: The parental genes of circRNAs with differential m6A modification were selected to analyze their underlying biological roles by Gene Ontology(GO)analysis and Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis.According to the level of m6A,the top 10 up-regulated and top 10 down-regulated circRNAs were select to construct a circRNA-miRNA network using Target Scan software and miRanda software.The relationship between m6A modification and the expression of circRNA was analyzed by cumulative distribution.Conservation analysis with human circRNAs,the sequences of human circRNAs were downloaded from the circ Base database,and each selected m6A circRNA sequence was aligned with the human circRNA sequence by the blastn function of the Blast software.Results: m6A modification did not affect the expression of circRNA,suggesting that m6A modification in circRNA function through other mechanisms in SAP progression.The GO and KEGG pathway analysis revealed that the function of differentially m6A-modified circRNAs were associated with protein digestion and autophagy regulation,which were two important events in the development of SAP.In the m6A circRNA-miRNA networks,we found that several important miRNAs that have been confirmed to be involved in the pathological process of SAP bind to these differentially m6A-modified circRNAs,such as miR-24-3p,miR-26 a,miR-92 b,miR-216 b,miR-324-5p and miR-762,suggesting that m6A may affect the interaction between circRNA and miRNA.Furthermore,conservation analysis with human circRNAs suggests that these most differentially m6A-modified circRNAs may have similar roles in human SAP.Part III: Exploration of the regulatory mechanism of m6A modification in circRNA of SAPObjective: To preliminarily explore the regulatory mechanism of modification in circRNA of SAPMethods: Based on the decrease of total m6A level in SAP pancreatic tissue,the expression of demethylase was detected by Western blotting and real-time quantitative PCR.The effect of screened demethylase to pancreatic tissue of SAP was evaluated by specific knockdown through adeno-associated virus.Results: The expression of demethylase ALKBH5 in the pancreatic tissue of SAP mice was significantly increased,while the expression of FTO was decreased,which was consistent with the decrease of total m6A level in the SAP group.Therefore,it is speculated that ALKBH5 is involved in the dynamic regulation of m6A and reduce the m6A level.After specific knockdown of ALKBH5 in pancreatic tissue,pancreatic tissue damage was alleviated in SAP.ConclusionIn this study,we identified the transcriptome-wide map of m6A modification in circRNAs of pancreatic tissue between normal and SAP mice,and explored their biological significance and potential mechanisms.Differentially m6A-modified circRNAs were involved in the key pathological processes of SAP,and m6A modification may influence the interaction between circRNA and miRNA.Some differentially m6A-modified circRNAs are highly homologous to human circRNAs,suggesting that these circRNAs may have similar roles in human SAP.Total m6A level was elevated in SAP,while the expression of the demethylase ALKBH5 was increased,and knockdown of ALKBH5 expression in pancreatic tissue using adeno-associated virus can reduce the damage of pancreatic tissue in SAP,suggesting that ALKBH5 may be the key regulator of regulating m6A modification in SAP.Our results provide new insights for further exploring the pathological mechanisms of SAP and searching for potential therapeutic targets. |
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