MiR-126 Inhibits Vascular Endothelial Cell Apoptosis Through Targeting PI3K/Akt Signaling

[Objective]:Deep vein thrombosis (DVT) is the most common surgical complications, often lead to serious consequences, its mechanism is complex, the need to continue in-depth exploration. In recent years, the study found,Vascular endothelial cell (VEC) apoptosis (PI3K/AKT pathway) plays an important role in the pathogenesis of DVT, and this process may be related to the microRNAs. Studies show that microRNAs is involved in the regulation of cell differentiation, proliferation, apoptosis and other physiological and pathophysiological processes, and miR-126 is closely related to VEC and vascular function microRNA.In addition,the study showed that after the formation of PI3K/Akt, DVT signaling pathway related matrix metalloproteinases (MMP-1, MMP-13) and their inhibitors (TIMP-1) also showed differential expression.Therefore, further research on these molecules is more helpful to clarify the role of miR-126 in the PI3K/Akt signaling pathway. In this study, we constructed the miR-126 gene silencing/high expression in rat DVT model,using HE staining, TUNEL cell apoptosis test to observe the influence of miR-126 expression on apoptosis of endothelial cells and the formation of DVT.And the expression of MMP-1, MMP-13 and TIMP-1 Akt in different groups was analyzed by bolt Western and Real time-PCR To explore the relationship between miR-126 and DVT, TIMP-1, MMP13 and MMP1 in venous endothelial cells,provide theoretical basis and new target for the new DVT control method.[Materials and methods]:1. groups and modeling:the 72 SD rats (250±20 g), male and female, irrespective of age, adaptive feeding 4 Day, were randomly divided into four groups. A The normal control group (12):To give the proximal thigh injection of 20ml normal saline, no other treatment.B The DVT model group (20 rats):to be every day thigh proximal to the injection 20ml physiological saline, four days after the quantitative impact of bilateral thighs proximal and hip spica plaster external fixation way building a rat model of traumatic limb DVT, normal feeding.C the miRNA-126 silence group (20 rats):Proximal thigh local four consecutive days, daily injection 20ml containing antagomir-126 400 pmol of transfected 20ml, miR-126 gene silencing in a rat model of construction, four days after treatment with DVT model group.D The miRNA-126 high expression group (20 rats):Proximal thigh local four consecutive days, the daily injection 20ml containing agomir-126 400 pmol of transfected 20ml, miR-126 gene high expression in a rat model of construction, four days after treatment with DVT model group.2. Venous wall acquisition and pathological examination.According to preliminary experimental results, respectively in modeling after 2.5h, 25h randomly selected normal group, DVT model group, miRNA-126 silent group, miRNA-126 high expression of rats of each half, after disinfected after anesthesia incision and bilateral medial thigh skin, and blunt dissection of the subcutaneous tissue, exposure to shares of venous blood vessel wall, and naked eye observation whether thrombosis, each side take long approximately 3cm femoral vein and part of fresh tissue with 4% paraffin sections of formaldehyde fixed evacuation of PL A, mirror inspection thrombosis grading. The rest of the saline flush, remove the tube inside and outside of the thrombus after the rapid storage of frozen tube, placed in liquid nitrogen tank.3. The expression of MMP-1, MMP-13, TIMP-1 and miR-126 in venous wall tissues was detected with Real time-PCR.A、Using the tissue lysis solution for the extraction of total protein from the venous wall materials of different groups at different time points in liquid nitrogen, and the total protein concentration was determined by BCA method.B、Through the PubMed database and the primer 5.0 designed to detect gene primers and design reference gene; cDNA was synthesized by reverse transcription; fluorescence quantitative PCR and Real time-PCR related experimental procedures.C、Using SPSS 17.0 statistical software package for data processing, every value with mean+standard deviation (+s) and between the groups compared with single factor variance analysis (ANOVA); the results of each group P< 0.05 for statistical significance.4. TUNEL method to detect the apoptosis of vascular endothelial cells.From the liquid nitrogen tank removed at different time points in each group organization rapid paraffin sections were made, and in accordance with the TUNEL apoptosis detection kit operating instructions for detection of venous endothelial cell apoptosis occurrence, DAPI after double staining under fluorescence microscope observation and photography.5. Western Bolt detection of MMP-I, MMP-I3, TIMP-1 expression in the vascular wall and the expression of PI3K, AKT,PI3K/AKT in the expression level of apoptosis pathway.A、The total protein was extracted from the venous wall materials of different groups at different time points in liquid nitrogen by using the tissue lysis solution and the total protein concentration was determined by BCA method.B、Preparation of SDS-PAGE electrophoresis after transfer membrane, blocking, antibody incubation and other operations; Series ChemiScop 3600 chemiluminescence imaging system to take pictures of the results, and use Analysis Chemi analysis to calculate the gray value.C、Using SPSS 17.0 statistical software package for data processing, every value with mean+standard deviation (+s) and between the groups compared with single factor variance analysis (ANOVA); the results of each group P< 0.05 for statistical significance.[Results]1. visual observation of thrombosis2.5h locus, rat DVT group, group of silence and high expression of group naked eye visible vein wall edema, deepen the color, no obvious thrombosis; in 25h sites, in addition to the normal group, the vein in the other three groups of tube wall edema, bilateral venous swelling and bruising, there are large thrombus formation, and the DVT model group and miRNA-126 silent group of most obvious.2. HE staining was used to observe the formation of thrombosis.2.5h locus, the rats in each group were stained with hematoxylin and eosin (he) staining showed no thrombosis, but DVT model group and miRNA-126 silent group showed more than the amount of red cell aggregation, in 25h site visible DVT group and miRNA-126 silent group complete thrombosis formation, miRNA-126 high expression group did not complete thrombosis formation.3. Tunel apoptosis experimentIn different sites and by observing the fluorescence density (i.e., the degree of apoptosis) determine apoptosis; in 2.5h, miR-126 silence group endothelial cells apoptosis of DVT in the model group, and normal group and miR-126 high expression group did not see obvious apoptosis; to 25h, except the normal group were visible apoptosis of endothelial cells. Compared with the DVT model group, the degree of apoptosis in miR-126 silencing group was heavy, and the degree of apoptosis was less than that in the miR-126 group.4. Real time-PCR, MMP-1, miR-126, MMP-13, TIMP-1 protein expression levels in the venous wall.Normal group of miR-126 expression level was significantly higher than that in the other groups at different time points; DVT model group, with the passage of time, miR-126 expression levels were significantly decreased; in the same time site of miR-126 expression group of miR-126 expression level was significantly higher than that of the model control group (P< 0.01), and inhibition of miR-126 groups, miR-126 expression level significantly lower than that of the model control group (P < 0.05).When miR-126 expression, and 2.5 and 25 hours sites with the model control group compared to the MMP-1 and MMP-13 gene expression level was decreased significantly (P< 0.05); when miR-126 is inhibited, in the specific time site with the model control group compared to the MMP-1 and MMP-13 expression was significantly higher. Compared with miR-126 group, the expression of MMP-1 and high expression of MMP-13 protein in miR-126 group decreased significantly (P<0.01), but the expression of TIMP-1 was significantly increased.5. Bolt Western detection of MMP-1, MMP-13, TIMP-1 protein, as well as the level of Akt expression.The expression levels of MMP-1, MMP-13 and TIMP-1 were consistent with the results of Real time-PCR detection.When miR-126 inhibited and Akt phosphorylation in the pS473, pT308 sites by degree of inhibition compared with model control group at the same time site significantly decreased; when miR-126 over expression and Akt in pS473, pT308 sites of phosphorylation level enhanced, at the same time site the enhancement degree was significantly higher in the model control group (P< 0.01).[In conclusion]1. MiR-126 and DVT were negatively correlated with apoptosis of vein endothelial cells2. MiR-126 and MMP-1, MMP-13 expression was positively correlated with the level of TIMP-1 expression was negatively correlated.3. MiR-126 was positively correlated with the level of AKT phosphorylation and MiR-126 inhibits the apoptosis of vascular endothelial cells and the formation of deep venous thrombosis by regulating the phosphorylation level of PI3K/Akt transduction pathway.