The Expression Level Of Plasma MiRNAs In Acute Pancreatitis And The Role Of MiR-494 In Pancreatic Acinar Cells Apoptosis And Its Mechanisms

Acute pancreatitis(AP) is a kind of acute inflammatory disease of the pancreas. AP could be caused by hyperlipidemia, excessive drinking and biliary diseases. According to whether the patients showing symptoms of transient or persist organ failure, AP patient could be classified into mild acute pancreatitis(MAP), moderate severe acute pancreatitis(MSAP) and severe acute pancreatitis(SAP). The prognosis of AP patients varies greatly. MAP patients have a better prognosis with a fatality rate close to 0%. But the prognosis of SAP patients is poor. The characteristic of SAP is persist organ failure over 48 hours. Of all AP patients, 10% to 20% could show persist organ failure, and the fatality rate of these patients could reach 30% to 50%.Micro RNAs(miRNAs) are a class of 17 to 25 nucleotide long, highly conservative short non-coding RNAs that regulate the expression of target genes at post-transcription level. miRNAs bind to the 3’ untranslated region of protein coding m RNAs by complementary base pairing. By doing so, miRNAs negatively regulate the expression of their target genes. It is estimated that miRNAs could regulated over 30% of the protein coding genes of mammalian. Since miRNAs play such an important role in gene expression regulation, the expression level of miRNAs change dramatically in many physiological or pathological conditions. In the progression of many diseases, the expression level of miRNAs in tissue or body fluid such as circulation blood changes.The aim of present study is to evaluating the changes of the expression level of miRNAs in the circulation blood plasma of AP patients, finding miRNAs as new biomarkers to stratify the severity of AP at an early stage, and finding the role certain miRNAs play in AP progression.1. Study the abnormal expression of miRNAs in the plasma of AP patients Objectives: To analyze the abnormal expression of miRNAs in the plasma of AP patients. To compare the difference of miRNAs expression level in the plasma of MAP patients and SAP patients.Methods: Collect the blood samples of healthy control, MAP patients and SAP patients. Extract total RNAs from the plasma samples and test the quality of the RNA samples. Analyze the differences of the expression level of miRNAs in plasma samples using the method of microarray. Results: The expression level of miRNAs in the plasma of AP patients changed greatly. Compared to healthy controls, the expression level of 75 miRNAs changed significantly in the plasma of AP patients(over 2 folds, P<0.05), of which 55 miRNAs was up-regulated and 20 miRNAs were down-regulated. Of all the AP patients, miRNAs expression level in the plasma of MAP patients changed slightly. Only 19 miRNAs in the plasma of MAP patients were significantly different from that of the health control, of which 14 miRNAs was up-regulated and 5 miRNAs were down-regulated. The expression level of miRNAs in the plasma of SAP patients changed dramatically. A total of 177 miRNAs in the plasma of SAP patients were significantly different from that of the health control, of which 135 miRNAs was up-regulated and 42 miRNAs were down-regulated. Furthermore, we found that the plasma miRNAs expression pattern of SAP patients was differ from MAP patients. A total of 80 miRNAs in the plasma of SAP patients were significantly different from that of the MAP patients, of which 57 miRNAs was up-regulated and 23 miRNAs were downregulated. Conclusions: The expression level of plasma miRNAs in AP patients was significantly different form healthy control, this suggests that miRNAs may play important regulative role in AP. The miRNAs expression pattern of SAP patients and MAP patients showed great differences. Further study may focus on the regulative role of miRNAs in AP and on finding novel biomarker for stratifying the severity of AP at early stages.2. Stratify the severity of AP using circulation blood miRNAs biomarkers Objectives: To analyze the difference of the expression level of plasma miRNAs between SAP, MAP and MSAP patients. To find new biomarkers to diagnose SAP. Methods: Prospectively collect the blood samples of AP patients. Extract the total RNAs from the plasma of AP patients. Analyze the expression level of certain miRNAs by the method of RT-q PCR. Using ROC curve to evaluated the sensitivity and specificity of miRNAs markers for diagnosing SAP. Results: In this study, we found 12 miRNAs(miR-1208, miR-3127-5p, miR-3180-3p, miR-3187-3p, miR-4265, miR-4294, miR-4513, miR-4725-3p, miR-4776-5p, miR-516a-3p, miR-6083 and miR-770-5p) were significantly up-regulated compared with MAP and MSAP patients(P<0.05). ROC analysis showed that all these 12 miRNAs could be used as biomarker for diagnosing SAP. Of all these 12 miRNAs, miR-1208(AUC 0.88,sensitivity 80.95%,specificity 84.88%,P<0.001)、miR-3180-3p(AUC 0.914,sensitivity 80.95%,specificity 88.37%,P<0.001)、miR-4265(AUC 0.881,sensitivity 80.95%,specificity 86.05%,P<0.001)和 miR-4776-5p(AUC 0.896,sensitivity 90.48%,specificity 76.74%,P<0.001)showed high sensitivity and specificity. Conclusions: The expression level of miRNAs in the plasma of SAP patients was different from that of MAP and MASP patients. miRNAs in circulation blood plasma could be used as biomarkers to diagnose SAP, with high sensitivity and specificity.3. Expression of miR-494 in AP and its function on pancreatic acinar cells apoptosis Objectives: To evaluate the expression level of miR-494 in AP and study the function of miR-494 on acinar cells apoptosis and its mechanisms. Methods: In this study, we built a cerulein induced AP mouse model and a cerulein induced AR42 J cell model. We verified the model by the method of pathology and by evaluating the expression level of amylase, TNF-α, IL-1 and IL-6. The expression of miR-494 were determined by RT-q PCR. The influence of miR-494 on pancreatic acinar cells apoptosis was determined using the method of flow cytometry. The expression levels of miR-494’s potential target apoptosis regulative proteins such as ROCK1, PTEN and CAMKIIδ were analyzed by Western blot. Results: Pancreas of mice from the AP group showed AP characteristics such as pancreas injury and inflammation. Amylase and inflammatory cytokines were up-regulated in AP groups. The expression level of miR-494 was up-regulated in AP. miR-494 could suppress pancreatic acinar cells apoptosis. The results of Western blot showed that miR-494 could down-regulate the expression of pro-apoptotic proteins ROCK1, PTEN and CAMKIIδ. Conclusions: miR-494 was over-expressed in AP. miR-494 could suppress pancreatic acinar cells apoptosis by targeting pro-apoptotic proteins ROCK1, PTEN and CAMKIIδ.4. TGF-β/Smad3 signal pathway transregulates miR-494 expression Objectives: To study the mechanisms of miR-494 over expression in AP. Methods: Determine the activity of TGF-β signal pathway by analyze the expression of p Smad3 using the method of Western blot. Observe the effect of TGF-β on the expression level of miR-494 in pancreatic acinar cells. Finding binding sites of Smad proteins on the promoter of miR-494 by gene sequence analysis. Built a luciferase reporter gene plasmid vector that contains the promoter of miR-494. Study the effect of TGF-β on the activity of miR-494 promoter by dual-luciferase reporter gene assay. Results: The expression level of p Smad3 was up-regulated in AP, indicating the activation of TGF-β signal in AP. TGF-β could up-regulate the expression of miR-494 in pancreatic acinar cells. Multiple Smad3 binding site were found on miR-494 promoter. TGF-β could up-regulate the activity of miR-494 promoter. Conclusions: The activity of TGF-β signal pathway was up-regulated in AP. TGF-β/Smad3 pathway could regulate the expression level of miR-494 by up-regulating the activity of miR-494 promoter.