The Effect Of MicroRNA-223 On Diabetic Myocardial Fibrosis About EndMT

Background:Diabetic myocardial fibrosis as one of the important complications of diabetes is closely related to high morbidity and mortality of cardiovascular disease. Fibrosis is characterized by the accumulation of fibroblasts and extracellular matrix, leading to the disorder of cardiac structure and function and finally resulting in the occurrence of cardiovascular disease development. Several evidences report that EndMT plays an important role in the pathologic conditions of cardiac fibrosis. EndMT is a process of losing features of endothelial cells such as cell junction, the ability of invasion and migration and transforming into fibroblast-like cells. Data from literature reports microRNA-223 plays a certain role in a variety of cardiovascular disease such as coronary artery disease, acute coronary syndrome, pulmonary hypertension and the like. In the meantime, reports show that microRNA-223 is concerned with EMT in pancreatic cancer. However, there is no exact research whether microRNA-223 is involved in cardiac fibrosis. The main aim of the experiment is to probe the relationship and mechanism of microRNA-223 and EndMT in diabetic myocardial fibrosis.Objectives:(1) Explore the function of microRNA-223 in cardiac fibrosis;(2) Explore the relationship and the mechanism between microRNA-223 and EndMT.Methods:1. The establishment of cells:Studies show 30mmol/L high glucose(HG) can induce endothelial injury by EndMT as well as EndMT can lead to cardiac fibrosis. In this experiment, human aortic endothelial cells are selected and cultured by special culture medium of endothelial cells called ECM. The overexpression of LV-hsa-mir-223-3psponge (miR-223) and negative control virus are transfected into six well plates or culture flasks. HAECs are stimulated at the density of eighty percent and divided into four groups(NG, HG, HG+miR-223, HG+LV-CN). HAECs are disposed at 72-hour sustained stimulation.2. Western BlotThe expression of PARP-l(the target of miR-223), the markers of EndMT(a-SMA, VE-cadherin, eNOS, CD31), the markers of inflammation(TNF-a, VCAM1, ICAM1) are observed after protein extraction, electrophoresis, trarsmembrane, immunoreaction and luminescence.3. RT-PCRThe expression of miR-223 and PARP-1 are observed after RNA extraction, reverse transcription and amplification. In addition, the level of expression is contrasted with LV-CN group.4. ImmunocytochemistryThe slides of HAECs in good condition are transferred into 24-well plates. After a series of reaction, the markers of EndMT and cellular morphology are observed by Inversed Fluorescent Microscope.5. Cell viability assayThe viability of HAECs are measured by Multiscan Spectrum using CCK-8 Kits. The viability and the quantity of each group are observed whether there is a corresponding change.6. Cell wound scratch assayThe ability of migration is observed by scratching three parallel lines in 6-well plates covered with HAECs.7. Transwell migration assayThe number of HAECs is counted as HAECs in the upper chamber with serum-free medium penetrate into low chamber by polycarbonate membrane. The quantities may reflect the ability of migration in HAECs.8. Statistical analysisAll the experimental data are presented as mean ± SD. Differences between each group are analyzed for statistical significance using the Student’s t test (two-tailed). The differences are regarded as statistically significant at the value of P< 0.05. Statistical analysis is conducted via SPSS 19.0.Results:1. The expression of miR-223 in HG-induced HAECs The expression level of miR-223 is observed by PCR. Compared with NG group, miR-223 decreases at the stimulation of high glucose. MiR-223 rises after the transfection of LV-hsa-mir-223-3psponge. The LV-CN group have no significant changes compared with HG group.2. MiR-223 improves cell viability and affect the ability of cell migration The cell viability is examined by CCK8 assay. In contrast with the group of NG, the cell viability of HG group decline significantly. The overexpressed miR-223 group enhance cell viability compared to HG group. However, the LV-CN group decreases in contrasted with the overexpressed miR-223 group. In the transwell experiment, the number of HAECs in HG group penetrating into low chamber increases clearly compared to NG group after 10-hour incubation. To further probe the migratory effect HAECs, the cell wound scratch assay is applied. The results are consistent with transwell’s, simultaneously with the extension of time the wound get closer.3. PARP1 is latently one of miR-223 targeted genes PARP1 is highly expressed in HG group and become lower in overexpressed miR-223 group by PCR. However, the expression level of miR-223 is contrary to PARP-1. This may implies that miR-223 may have a significant negative correlation with PARP-1.4. MiR-223 may suspend the development of EndMT Endothelial markers such as VE-cadherin, eNOS, CD31are downregulated and mesochymal markers like a-SMA are upregulated in HG group compared with NG group by Western Blot and Immunofluorescence. However, after transfection of LV-hsa-mir-223-3psponge, a-SMA decreases and the process of transition from endotheliocyte to mesenchyma mitigates in miR-223 group.5. MiR-223 may reduce inflammation stimulation The inflammatory factors such as TNF-a, VCAM, ICAM rise at the stimulation of high glucose. Nevertheless, the level of inflammatory factors decreases after the transfection of LV-hsa-mir-223-3psponge. This suggests that miRNA-223 lightens the progress of inflammation.Conclusion:1. The overexpression of miR-223 can delay the process of EndMT by targeting PARP-1.2. The up-regulation of miR-223 can alleviate the extent of diabetic myocardial fibrosis.