| Purpose: To investigate whether miR-22 plays a role in aortic dissection and the regulatory mechanisms in vascular smoothe muscle invoved vascular remodeling.Methods: 1.Based on the ethical requirements,collect 8 aortic tissues from normal donor people and 10 aortas by aortic dissection patients,total RNA was extracted and detected the expression of miR-22 by qRT-PCR;miR-22 distribution in aortic tissue was examined by miRNA fluorescence in situ hybridization and immunofluorescence co-localized in aortic tissue frozen section;2.Tg(Flik1?egfp)zebrafish embryos were injected under microscope at single-cell stage with 16 ng of MO-22,embryos were collected at 20 phf,24phf,30 phf to extract total RNA,miR-22 was detected by qRT-PCR;zebrafish embryos were observed the growth of blood vessels and dorsal artery using a fluorescence microscop at 24 phf and 30 phf respectively;3.Construct Ad-miR-22 and Anti-miR-22 to overexpression and down expression miR-22,green fluorescent protein(GFP)adenovirus vector was used as control virus,respectively;HASMC was transfected by Ad-miR-22 and Anti-miR-22,HASMC proliferation was measured by CCK-8 at 0h?12h?24h?36h?48h after 48 h transfection,in parallel experiments,cell counts at 36 h under microscope;Annexin V-APC and 7-AAD stained apoptotic cell to examine HASMC apoptosis;miR-22 target was predicted by several bioinformatics algorithms,Western-blot was used to confirm miR-22 target protein expression in the aortic dissection and HASMC,transfected by Ad-miR-22 and Anti-miR-22;Over-expression of miR-22 not only reduced the luciferase activity of the 3'untranslated region of MAPK14 as reported,but also suppreased the endogenous protein expression of MAPK14,indicating that MAPK14 is the direct target of miR-22.MAPK14 was repressed in HASMC by siRNA,and miR-22 was restrained by Anti-miR-22,Annexin V-APC and 7-AAD stained apoptotic cell to examine HASMC apoptosis after both MAPK14 siRNA and Anti-miR-22 transfection;4.Three weeks old FVB male mice were fed on a normal diet and BAPN was dissolved in drinking water for four weeks by 1g/kg/day.At five weeks age,miR-22 Agomir/antagomir and both control Agomir/antagomir were injected through caudal vein at 15 OD per mouse,and 1?g/kg/min Ang?were implanted subcutaneously for 24 h two weeks later,the mice were euthanized by overdose anesthetics.Collect mice aortas from ascending aorta to common iliac artery and record the incidence of aortic dissection,media thickness(MT)and lumen diameter(LD)were evaluated by mice aorta HE staining,the elastic fibers and collagen were observed by EVG and Masson staining respectively.Results: 1.With qRT-PCR and miRNA fluorescence in situ hybridization,we revealed that miR-22 was knockdown in the aortic dissection aortas and was predominantly decreased in the media VSMCs;2.MO-22 significantly inhibit zebrafish embryos miR-22 expression in 24-30 hpf,reduced expression of miR-22 in zebrafish embryos can significantly inhibit the development of zebrafish blood vessels in the 24 phf and dorsal artery growth in the 30-hpf.3.MiR-22 expression was increased by Ad-miR-22 and reduced by Anti-miR-22 compared with their control adenovirus after transfection 48 h to 72 h,and the best transfection MOI value was 100.At the time of 36 h after transfection,Ad-miR-22 promote HASMC proliferation,whereas Anti-miR-22 inhibit HASMC proliferation by cell counting;Anti-miR-22 was significantly accelerate HASMC apoptosis(P<0.05);MAPK14 was predicted as a potential target of miR-22,the expression of MAPK14 not only increased in aortic dissection aortas,but also supressed by Anti-miR-22 in the HASMC,indicating that MAPK14 is the direct target of miR-22.More importantly,inhibition the expression of MAPK14 and miR-22,HASMC apoptosis was reduced demonstrating miR-22 regulate HASMC apoptosis by MAPK14.4.Ago-miR-22 was identified increased mice aortic miR-22 expression and reduced MAPK14 simultaneously,whereas antago-miR-22 knockdown miR-22 and promote MAPK14 expression.The incidence rate of aortic dissection model group,agomir-22 group,agomir –NC group,antagomir-22 group,antagomir –NC group were 6/18(33.3%),12/18(66.7%),11/17(64.7%),7/18(38.9%)separately,aortic dissetion was reduced in the agomir-22 group comparaed with control group(P<0.05),there was no significantly difference between the antagomir-22 group and antagomir –NC group(P=0.1267).The average media thickness in aortic dissection model group,agomir-22 group,agomir –NC group,antagomir-22 group,antagomir –NC group were 74.76±4.59?m,118.94±5.76?m,81.23±3.05?m,65.59±3.74?m,94.76±4.02?m and average lumen diameter were 2.54±0.21 mm,1.55±0.11 mm,3.12±0.2mm,3.74±0.2mm,1.55±0.11 mm,2.94±0.14 mm,respectively.Over-expression of miR-22 increased aorta MT and MT/LD,wheras down-expression of miR-22 reduced MT and MT/LD;The elastic fibers and collagen were augmented in the agomir-22 group and reduced in the antagomir-22 group.Conclusion: Our results demonstrate that the expression of miR-22 is down-regulated in aortic dissection,and indicate that miR-22 is down expression in the aortic dissection media VSMC.MO-22 significantly inhibit the development of zebrafish blood vessels in the 24 phf and dorsal artery growth in the 30-hpf.The mitogen-activated protein kinase 14 protien is identified as a novel target of miR-22 in HASMC.We not only report for the firsrt time that inhibit miR-22 could suppressed HASMC proliferation and aggravated HASMC apoptosis,but also demonstrate that reduced miR-22 was promote HASMC apoptosis by regulated its target MAPK14.Furthermore,over-expression of miR-22 was reduced aortic dissection through increase aorta MT and MT/LD,augmente elastic fibers and collagen on the mice.Our work contributes to futher clarify the pathogenesis by which miR-22 regulates aortic diseetion,and broadens our knowledge about the mechanism of vascular remodeling diseases,provides a new potential therapeutic target. |
PDF Full Text Request