| Objectives We used Microarray expression profile analysis to find that the expression of miR-22 in the aortic tissue samples in patients with aortic dissection was significantly lower than that in the normal aortic tissue samples.In animal experiments,the effects of miR-22 on the occurrence of aortic dissection,the biomechanical properties of the wall of the tube and the expression of mechanical properties related proteins were studied by the model of aortic dissection.In cell experiments,the effects of miR-22 in smooth muscle cells and endothelial cells on the function of proliferation and apoptosis of smooth muscle cells and endothelial cells and the expression of mechanical related proteins were detected.In the non contact co-culture model of endothelial cells and smooth muscle cells,the expression of miR-22 in the endothelial cells and the secretory function of endothelial cells was interfered.The content of mechanical related proteins in smooth muscle cells were detected respectively,in order to reveal the effect of miR-22 on the biomechanical properties of the aortic wall and the mechanism of aortic dissection.Methods According to the previous Microarray expression profile analysis,we screened the differentially expressed miRNAs in human tissue samples and confirmed that miR-22 was significantly lower in the aortic dissection samples than in the normal aortic tissue samples by realtime PCR.In animal experiments,we used 3 weeks male FVB mice to make a model of aortic dissection,and the mouse were fed with beta aminopropane in drinking water.After continuous feeding for 1 months,the miR-22 Agomir/antagomir was given via tail vein.After continuing to feed for one week,we implanted the angiotensin II micropump in the mice subcutaneously to establish the aortic dissection model by 72 h.After modeling,the aortic tissue samples of mice were taken to observe the pathological changes of aorta and record the incidence of aortic dissection.The thickness of the middle aorta,the diameter of the lumen of the aorta,the ratio of the thickness of the middle membrane were measured by HE staining in the aortic specimens,and the wall elasticity was measured by EVG staining.The content of collagen fiber was measured by Masson staining.The effect of miR-22 on vascular remodeling of aortic wall was detected.The mechanical test system was used to perform the mechanical testing of mouse aorta.The values of aortic thickness,circumference,stiffness,ultimate load,limit displacement,ultimate stress,ultimate strain and tensile ratio were recorded,and stress strain curves were drawn.The elastic modulus of the aorta was calculated by linear fitting analysis with Origin software.To clarify the effect of miR-22 on the mechanical properties of aortic wall.The effect of miR-22 on apoptosis of cells in aortic wall was detected by TUNEL staining of aorta tissue,and the effect of miR-22 on the expression of Rho/ROCK1 related protein in aortic wall was detected by immunohistochemistry.In the cell experiment,the up-regulated and down regulated adenovirus of miR-22 were constructed.The smooth muscle cells and endothelial cells were infected respectively.The proliferation function was detected by CCK-8,and the apoptosis function was detected by Annexin V-APC and 7-AAD double staining.After interfering the expression of miR-22 in smooth muscle cells,the total protein was extracted and the effect of miR-22 on the expression of mechanical related protein Rho/ROCK1 of smooth muscle cells was detected by Western-blot.The bioinformatics method was used to predict the secretory proteins that could be targeted by miR-22,and the expression of protein in the endothelial cell culture medium weith miR-22-interfered was detected by ELISA to determine the target protein FBXW7 which is one of the targets of miR-22.In the non-contact co-culture model of endothelial cells and smooth muscle cells,the total protein of smooth muscle cells was extracted with the miR-22 expression of endothelial cells interfering,and the effect of miR-22 on the expression of mechanical related protein Rho/ROCK1 of smooth muscle cells was detected by Western-blot.SiRNA,which is used to knock down the secretory protein FBXW7 of endothelial cells in endothelial cells,is used to interfere with endothelial cells in co-culture model,to detect the expression of mechanical related proteins in smooth muscle cells,and to determine whether miR-22 can change the secretory function of endothelial cells to change the expression of mechanical related protein ROCK1 in smooth muscle cells.Results We found that the expression of miR-22 in aortic dissection tissue was significantly lower than that in normal aortic tissue samples by realtime PCR of human aortic tissue samples.The results of animal experiment showed aortic dissection rate:the up regulation group was 4/12(33.3%),the control group was 8/12(66.7%),the down regulation group was 7/12(58.3%),and the control group was 5/12(41.7%).Statistical analysis showed that compared with the control group,the up regulation group significantly decreased the incidence of aortic dissection.The HE staining of the mouse aortic tissue samples was measured and the results were statistically analyzed.It was found that the ratio of the thickness of the middle membrane in the agomir-22 group was significantly higher than that in the agomir-NC group.The statistical analysis of the EVG staining showed that the agomir-22 group were significantly higher than the agomir-NC group.The results of the Masson staining were measured.Statistics and analysis showed that the collagen fiber content in group agomir-22 was significantly higher than that in group agomir-NC.Immunohistochemical results showed that the expression of Rho and ROCK1 in the aortic tissue of mice,the up regulation group of miR-22 was significantly lower than down regulation group of miR-22 group.The results of the mechanical test of the aorta wall showed that the average thickness of the aortic wall in the agomir-22 group was significantly higher than that in the agomir-NC group,and there was no significant difference in the circumference of the aorta wall of the mice.The limit load,the limit displacement,the ultimate stress,the ultimate strain,the initial modulus of elasticity and the tensile ratio of the agomir-22 group were all significant higer than the agomir-NC group.There was no significant difference between the limit elastic modulus and the area under the curve.The limit load,ultimate stress,ultimate modulus of elasticity and stiffness of group antagomir-22 decreased while the limit displacement,ultimate strain,tensile ratio,area under curve and initial modulus of elasticity had no significant difference.The results showed that the up regulation of miR-22 played a protective role in the occurrence of aortic dissection.Cell experiment results: after interfering miR-22 in smooth muscle cells and endothelial cells,the results showed that down regulation of miR-22 could significantly inhibit the proliferation of HASMC and promote its apoptosis function,and the intervention of miR-22 had no effect on the content of Rho/ROCK1 in smooth muscle cells.Intervention miR-22 had no effect on endothelial cell proliferation and apoptosis function.By bioinformatics,we predicted the possible secreted proteins FBXW7 and TMED4 that miR-22 might target.The culture solution of miR-22 with endothelial cells was collected,and the content of FBXW7 and TMED4 was detected by ELISA.It was found that the up regulation of miR-22 could significantly reduce the content of FBXW7,and the downregulation of miR-22 could significantly increase the content of FBXW7,but the intervention miR-22 had no effect on the content of TMED4.The expression of mechanical related protein Rho/ROCK1 in smooth muscle cells was measured in the endothelial cells and smooth muscle cells non-contact co-culture model.The results showed that down regulated the miR-22 of endothelial cells increased the content of ROCK1 in the smooth muscle cells.Then we synthesized siRNA,which was used to knock down the expression of FBXW7,and interfered miR-22 down regulate group of endothelial cells in co-culture model,and then detected the expression of ROCK1 in smooth muscle cells.The results showed that FBXW7 could weaken the increase of ROCK1 protein expression in smooth muscle cells induced by miR-22 down regulation.Conclusions The purpose of this study is to investigate the effect of miR-22 on the mechanical properties of aortic wall and its role in the pathogenesis of aortic dissection.1.miR-22 decreased significantly in aortic dissection than normal aorta.2.Upregulation of miR-22 decreased the incidence of aortic dissection in mice.3.with mechanical test,upregulation of miR-22 could significantly increase the limit load,limit displacement,ultimate stress,ultimate strain,initial modulus of elasticity and tension of the aorta.4.the results of immunohistochemistry showed that down regulation of miR-22 could increase the Rho/ROCK1 content in the aorta of mice;5.downregulation of mir-22 can inhibit the proliferation of smooth muscle cells and promote the ability of apoptosis,but the intervention of miR-22 on the mechanical protein content of smooth muscle cells has no effect.6.miR-22 has no effect on the proliferation and apoptosis function of endothelial cells;7.Upregulation of miR-22 can inhibit the secretion of FBXW7 in endothelial cells but have no effect on the secretion of TMED4;8.Downregulation of miR-22 of endothelial cells in endothelial cells and smooth muscle cells non-contact co-culture model can increase the content of mechanical related protein ROCK1 in smooth muscle cells;9.miR-22 indirectly affects the expression of ROCK1 in smooth muscle cells by affecting the secretion of FBXW7 in endothelial cells. |
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