Research On Effects And Mechanisms Of MicroRNA-21 Deficiency In Mouse Model Of Thoracic Aortic Aneurysm

Part I Effects and mechanisms of microRNA-21 regulate TGF-β-Smad3 pathway to exacerbate angiotensin II induced thoracic aortic aneurysmObjective:To determine the dynamic change of microRNA-21 in thoracic aortic aneurysm (TAA), and to explore the effect of intervention in microRNA-21 expression of thoracic aortic aneurysm.Methods:We infused angiotensin Ⅱ on Smad3+/- mice and used RT-PCR and hybridization in situ to detect the change of microRNA-21 in pathological tissues. Meanwhile, RT-PCR analyzed microRNA-21 in aneurysm patients tissues Next to, we also used miR-21-/-:Smad3+/- double knock out (DKO) mice to duplicate animal model. Echocardiography and histology detected the lesion characteristic and downstream signal path. And cultured VSMC in vitro to test the response of TGF-β stimulation.Results:Angiotensin II would induced thoracic aortic aneurysm Smad3 mutant mice. RT-PCR and ISH detect that the microRNA-21 was up-expression in intermediate VSMC, which was consistent with specimen of thoracic aortic aneurysm patients. Moreover, the miR-21-/-:Smad3+/- DKO mice developed TAA rapidly. The phenotype seemed expansion and dissection faster than Smad3+/- mice, and have higher mortality. In HE and EVG stains, the aorta diameter was increase and intermediate was thickness with degradative elastin. IHC showed the lesion location with down-regulation pSMAD2 expression. The TGF-P signal descended in the cultured VSMC in vitro.Conclusion:In the lesion tissue of TAA, microRNA-21 was highly expression in VSMC. Artificially reduce microRNA-21 would lead TAA worsen in short time. We hypothesis that the mechanism was TGF-β signal down-regulation.Part II The mechanisms of SMAD7 play as the key factor of TGF-P signal in thoracic aortic aneurysmObjective:To clarify the mechanism of miR-21-/-:Smad3+/- DKO mice TAA and using si Smad7 to rescue TAAMethods:Using IHC and RT-PCR to study the VSMC phenotypes switching related molecular expression in vivo and in vitro. Lentiviruses transfected si smad7 into VSMC try to recover TGF-β signal and to treat TAA in DKO mice.Results:IHC and RT-PCR analysis showed that the contraction protein αSMA, SM22, SM-MHC were down-regulated and the inflammation relation factor CCL2, CXCL12 and IL-6 were up-regulated through VSMC phenotypes switching. VSMC secretion chemokine leaded inflammation cells infiltration into adventitia of aneurysm. After silence smad7 using lentiviruses, TGF-β signal restored and the VSMC phenotypes switching reduced. TAA of DKO mice was improved.Conclusion:The primary cause of TAA in miR-21-/-:Smad3+/- DKO mice was unusual low activation of TGF-β signal in VSMC. The VSMC tend to phenotypes switching in this state. Using lentiviruses decrease smad7 gene would rescue the TAA in DKO mice.