Acute Pancreatitis In The Blood Circulation MicRNA Spectrum Analysis And Its Influence On Prognosis And Therapeutic Targets

Background: Acute pancreatitis(AP)is a reversible inflammatory process of the pancreas with a wide range of clinical variations.Annual incidence of AP is about 40/100000 in the western countries,and incidence increases year by year,the totall of fatality rate is 5% ~ 10%.Mild Acute pancreatitis(MAP)is often self-limited and has a very low mortality rate(<1%).However,severe Acute pancreatitis(SAP)can progress rapidly,leading to multiple organ failure and becoming life threatening.The death rate for SAP has been reported to be as high as 25%-30%.The clinical management of AP depends largely on disease severity.Treatment for MAP patients is relatively straightforward and requires only brief hospitalization,whereas treatment for SAP patients often involves intensive care.Therefore,identifying the serverity ofacute pancreatitispatients during an early stage of the disease isvery important.Current clinical classification method is widely accepted in 1992 and 2012,Atlanta meeting standards,the 1992 meeting set diagnostic criteria for severe acute pancreatitis in organ failure(including shock,pulmonary dysfunction,kidney failure and hemorrhage of upper gastrointestinal tract)and/or local complications(pancreatic necrosis and abscess or pseudocyst).2012 will come up for severe acute pancreatitis diagnosed as organ failure time lasts for more than 48 hours.In recent decades a large number of single factor and multiple factors scoring system was used to predict early SAP such as hematocrit,c-reactive protein,inflammatory response syndrome,Ranson score and APACHE II score and computed tomography(CT)and CTSI scores,BISAP score,etc.,they have been as advantages as disadvantages,but physicians is not satisfied with these predict factors.miRNAs are 22-nucleotide,non-coding RNA molecules that are involved in post-transcriptional regulation either by promoting m RNA degradation or by repressing m RNA translation into proteins.miRNAs play important roles in embryonic development,cell proliferation,apoptosis,and differentiation.Recently,miRNAs were shown to regulate various pancreatic biological processes including development,regeneration,beta cell proliferation,and apoptosis.Kong XY,through research of two kinds of AP rats model,found that serum micrornas-216-a significantly elevated in 24 hours after building,maintain to 48 hours,they think that serum micrornas in AP-216-a,may be a new kind of specific biomarkers of pancreatic injury.Micrornas may become the new mark of diagnosis and estimate the prognosis of acute pancreatitis.There is no special treatment of acute pancreatitis.At early stage,the major therapies inchudes removing the cause and maintaining organ function,At late stage,nutrition and treatment of infection is very important.As the emergence of micrornas,especially the emergence of high throughput gene sequencing,can you provide us with the treatment of acute pancreatitis is extremely complications provide therapeutic targets.Recent studies have shown that micrornas in serum may be associated with acute pancreatitis and sepsis.Martinez-Nunez,RT,and other studies have shown that in macrophages in the miRNA-155 direct effects on IL13 R alpha 1,reduce IL13 R alpha 1 protein levels,inhibit STAT6 pathway,and dependency by IL13 R alpha 1 gene(SOCS1,DC-SIGN,CCL18,CD23,and SERPINE),to influence the immune response.At present a large number of studies have found that serum micrornas can be used as diagnosis of tumor and judge the prognosis of clinical indicators.Whether serum micrornas as the severity of acute pancreatitis diagnosis or judgment of a target? Whether serum micrornas as therapeutic targets to provide basis for treatment of acute pancreatitis is extremely complications? According to gene chip screening mild acute pancreatitis and severe acute pancreatitis serum micrornas spectrum,we will choose the part of micrornas with clinical data statistical analysis,to investigate the relationship between the serum micrornas and prognosis of acute pancreatitis.At the same time analyze the acute pancreatitis serum micrornas provide targets for extremely complications for the treatment of acute pancreatitis.Materials and methods 一.serum micrornasspectrum of acute pancreatitis detection and analysis 1.Patient information Collect three healthy volunteers,six within 48 hours of onset of mild acute pancreatitis(three biliary pancreatitis,three hyperlipidemia pancreatitis)and six within 48 hours of onset of severe acute pancreatitis(three biliary pancreatitis,three hyperlipidemia pancreatitis)of plasma and diagnostic criteria in 1992 Atlanta meeting standards,plasma samples wascollected in 24 hours after admission.2.miRNA microarray Peripheral blood(4 ml)was collected from each donor and supplemented with EDTA to avoid coagulation.Plasma was isolated within 30 min after blood collection by centrifugation at 820 g for 10 min at room temperature,followed by another 10-min centrifugation at 10,000 g to further eliminate the cellular components in the plasma.Total serum miRNA was isolated using Trizol in combination with the miRNeasy mini kit(QIAGEN)according to the manufacturer’s instructions.A Nanodrop spectrophotometer(ND-2000,Nanodrop Technologies)was used to determine the quality and quantity of the isolated RNA.Total serum miRNAs including six SAP,six MAP,and three control cases were used for miRNA profiling.The miRCURYHy3/Hy5 Power labeling kit(Exiqon,Vedbaek,Denmark)was used for miRNA labeling according to the manufacturer’s instructions.The labeled miRNA samples were hybridized onto the miRCURY LNA Array(v.16.0,Exqo).The complete procedures of hybridization,incubation,and washing were conducted according to the manufacturer’s instructions.The resulting signals were scanned using an Axon Gene Pix 4000 B microarray scanner.3.Statistical analysis The miRNA array results were extracted and analyzed using Gene Pix Pro 6.0 software(Axon).The miRNA expression signals were normalized using median normalization.miRNAs with signal values higher than 50 in all groups were included in further analyses.The signals from multiple probes for each miRNA wereaveraged to represent the expression of the miRNA.Two-tailed Manne Whitney unpaired test was used to determine the significance of differences in miRNA expression between groups.The selection criteria for differentially expressed miRNAs included:(1)fold change > 2,and(2)p value < 0.01.Hierarchical clustering of differentially expressed miRNAs was analyzed as a heat map,using MEV(version 4.6).二.the effect of different micrornas to diagnosis and judgment the prognosis of acute pancreatitis 1.Patient information The clinical characteristics of patients,including levels of serum calcium,C reactive protein(CRP),and procalcitonin(PCT),APACHE II scores,the status of complications,and disease progression for all patients involved in this study were recorded by two designated clinicians.The clinical diagnosis of AP severity for all the patients was retrospectively determined by the same clinicians,according to following criteria: A)patients were admitted to the hospital within 48 h of initial disease presentation;B)patients demonstrated two of the three AP clinical features: i)AP-characteristic abdominal pain,ii)serum amylase and/or lipase levels at least 3-fold higher than the upper limit of the normal range,and iii)AP-characteristic CT scan results;C)MAP refers to AP with clinical and biochemical features but no organ failure or complications;D)SAP refers to AP with clinical and biochemical features as well as organ failure andor complications.Based these criteria,74 AP patients including 43 MAP and 31 SAP patients were recruited for participation in this study.Twenty-one healthy volunteers were also recruited to participate in the study.2.miRNA quantification by RT-q PCR Peripheral blood(4 ml)was collected from each donor and supplemented with EDTA to avoid coagulation.Plasma was isolated within 30 min after blood collection by centrifugation at 820 g for 10 min at room temperature,followed by another 10-min centrifugation at 10,000 g to further eliminate the cellular components in the plasma.Total serum miRNA was isolated using Trizol in combination with the miRNeasy mini kit(QIAGEN)according to the manufacturer’s instructions.A Nanodrop spectrophotometer(ND-2000,Nanodrop Technologies)was used to determine the quality and quantity of the isolated RNA.Total serum miRNAs(15 ng)isolated from the validation cohort(18 controls and 62 patients)were reverse transcribed using the One Step Prime Script miRNA c DNA Synthesis Kit(Takara Biotechnology,D350A)with a universal adaptor primer.The RT reaction was performed at 37 C for 60 min,followed by 85 C for 5 s.The synthesized c DNA was then amplified by quantitative PCR,using SYBR Premix EX Taq II(Takara Biotechnology,DRR081A),Uni-miRQ PCR Primer(Takara Biotechnology),and primers specific to each individual miRNA.The reactions were conducted using PRISM 7500 System(Applied Biosystems,USA).Reactions with no template were used as negative controls,and has-miR-16 was used as internal control.The reaction conditions were 95 C for 30 s followed by 40 cycles of 95 C for 15 s and 60 C for 60 s(ramping was set to 1.6 C/sec).3.Statistical analysis RT-q PCR data were analyzed usingthe comparative CT method.DCT for each target miRNA was defined as CTtarget miRNA e CT miR-16.Two-sided,two-tailed Student t-tests were performed to determine the statistical significance(p < 0.05)of differences between groups.ROC curve analysis was performed using Graph Pad Prism version 5.三.miR-551b-5p effect the intracellular concentration of calcium ion 1.The ICC isolation and cultivation Geting intestine from the SD neonatal rat,stripping the mesentery、serosal layer and blood vessel used the microscope,further more,stripping the mucous membrane of small intestine and submucous layer,geting the ICC from the smooth muscle layer and culture the ICC.2.According to the research objectives to divide the cultured ICC into five groups : A:Normal comparison group.B:miR-551b-5p overexpression group(Electransfection the miR-551b-5p mimic).C: miR-551b-5p mimic negative control group.D:miR-551b-5p down-regulated expression group(Electransfection the miR-551b-5p inhibitor).E:miR-551b-5p inhibitor negative control group.3.Testing the specificity miR-551b-5p expression in ICC which raised and lowered it expression or before it expressed.Using the RT-PCR technique to test the miR-551b-5p m RNA expression in ICC,Using the Laser Scanning Confocal Microscope Testing the morphologic characteristics of apoptosis of ICC and the concentration change of calcium ion in ICC.using the Fluo-3AM technique to test the concentration change of calcium ion.4.Data analysis: All the experimental results of SPSS data are used l8.0 statistical software for processing,all data is represented in mean±s,data for normal distribution and homogeneity of variance;ANOVA,variance,were compared by SNK test;homogeneity of variance,comparison between groups using Dunnett s T3;P < 0.05 was statistically significant.Result: 一、There has 895 micrornasin the chip,We found that patients with acute pancreatitis plasma and normal plasma micrornas have 205 differentially expressed micrornas,severe acute pancreatitis with mild acute pancreatitis plasma micrornas nine differentially expressed。In acute biliary pancreatitis,there has 128 micrornas differentially expressed than normal.In acute hyperlipidemia type pancreatitis,there has 13 micrornas differentially expressed than normal,severe acute pancreatitis has 10 micrornas differentially expressed than mild acute pancreatitist.二.Target genes data based on information from three databases: mirbase,Miranda and target Scan.Prediction results in three overlapping part of the database.Selection of 62 a total of 1064 target genes differentially expressed micrornas prediction.In KEGG pathway analysis,inflammation,autophagy,barriers and gastrointestinal miRNA target genes expressed in different signaling pathways are involved in concentration.These pathways may be associated with the development of acute pancreatitis.The GO signal path analysis,the release of calcium channel activity are obvious enrichment of miRNA target genes.Calcium signaling pathways through SPHK1 and Fc gamma R mediated phagocytosis pathway is associated,by ITPR2 ITPR3 and Gap junctions,associated with EGFR adhesive connection,Endocytosis associated with it.三.In plasma miRNA expression based on the analysis of the spectrum,we further screening the most likely to be diagnostic markers of 11 micrornas.Mi R-92-b,ebv-miR-BART6-3 p,miR-146-b-5 p,miR-7,miR-10.A,miR-132 *,miR-30 c,miR-514-b-3 p,miR-214 is found in differences in the AP compared to normal plasma;Mi R-551-b *,miR-890 in SAP contrast MAP with differentially expressed in plasma,miR-92-b,miR-10.A,miR-7 in plasma in acute pancreatitis patients decreased,miR-551-b-5 p in severe acute pancreatitis in plasma increased.四.Comparing with the normal comparison group,the miR-551b-5p expressing increased remarkably in miR-551b-5p mimic group(P<0.01);the miR-551b-5p expressing decreased remarkably in miR-551b-5p inhibitor group(P<0.01);Comparing with the Mi R-551b-5p mimic negative control group,the miR-551b-5p expressing increased remarkably in miR-551b-5p mimic group(P<0.01);Comparing with the Mi R-551b-5p inhibitor negative control group,the miR-551b-5p expressing decreased remarkably in miR-551b-5p inhibitor group.Comparing with the normal comparison group,between the miR-551b-5p mimic group and miR-551b-5p inhibitor group,Comparing with the normal comparison group,the calcium ion concentration of ICC increased remarkably in miR-551b-5p mimic group(P<0.05);the calcium ion concentration of ICC decreased remarkably in miR-551b-5p inhibitor group(P<0.05).Conclusion: 一.Compared acute pancreatitis patientsplasma with normal plasma micrornas have 205 differentially expressed micrornas,severe acute pancreatitis with mild acute pancreatitis plasma have nine differentially expressed micrornas.二.Target genes data information and the signal pathway analysis revealed inflammation,autophagy,barrier,gastrointestinal pathways are involved in the development or the occurrence of acute pancreatitis,which release calcium channel activity(calcium-release channel activity)are obvious enrichment of miRNA target genes.三.Micrornas.miR-92-b,miR-10.A,miR-7 May as supplementary indexes of early diagnosis of acute pancreatitis,miR-551-b-3 p may be the early important indicator for the severity of acute pancreatitis.四.The expression of SAP relevancy miR-551b-5p had positive correlation with the calcium ion concentration of ICC.