Study On Action Mechanism Of BRG1and Relative Long Noncoding RNA In Thoracic Aortic Dissection

Part1Expression of BRG1and related protein in thoracic aorticdissection【Objective】 The chromatin remodeling complex containing BRG1involved in thecardiovascular system has been confirmed by many studies. Researches proved that thechromatin remodeling enzyme BRG1was involved in the regulation of matrixmetalloproteinase-9/-2expression, and played an important role in the phenotypetransformation, cell proliferation and apoptosis of vascular smooth muscle cells. As themain cellular components of aortic media, vascular smooth muscle cells are themain source of extracellular matrix components, and are involved in thoracic aorticdissection because of the phenotype conversion and imbalance of cell proliferation andapoptosis. In this part, the expression of BRG1, specific mark proteins of contractilevascular smooth muscle cells, matrix metalloproteinase-9/-2expression in aortic mediaacquired from thoracic aortic dissection patients have been detected. The apoptosis ofvascular smooth muscle cells in the aortic dissection was also evaluated. The correlationbetween BRG1and matrix metalloproteinase-9/-2expression, phenotype conversion ofvascular smooth muscle cells as well as cell apoptosis were analysed.【Methods】 The experiment was divided into two groups. The aortic mediaspecimens acquired from20thoracic aortic dissection patients were defined as thoracicaortic dissection group, and the aortic media specimens acquired from10normal donorswere defined as normal control group. The expression level of BRG1mRNA and proteinwere evaluated by qRT-PCR and western blot analysis. The expression level of BRG1,matrix metalloproteinase-9/-2and α-SMA protein were analysed by immunohistochemicalmethod. The terminal deoxynucleotidyl transferase (TdT) mediated dUTP Nick-EndLabelling (TUNEL) technique was used to evaluate the apoptosis rate of aortic smoothmuscle cells. The correlation between BRG1and matrix metalloproteinase-9/-2expression,percentage of α-SMAhighvascular smooth muscle cells as well as cell apoptosis wereanalysed.【Results】 The expression level of BRG1mRNA(3.18±0.74vs1.25±0.23，P<0.01)and protein (0.66±0.05vs0.34±0.05，P<0.01) in the aortic media was significantlyincreased in the thoracic aortic dissection group by qRT-PCR and Western blot analysis. Inimmunohistochemical analysis, it was found that BRG1was mainly localized in the nucleus of vascular smooth muscle cells, and the average optical densities weresignifcantly higher in thoracic aortic dissection tissues (577.88±41.27vs264.00±25.50，P<0.01). Immunohistochemistry assay also showed that the expressions of matrixmetalloproteinase-9/-2in thoracic aortic dissection tissues were significantly increasedand that the expression of BRG1was positively correlated with the expression of matrixmetalloproteinase-9/-2in thoracic aortic dissection tissues (r=0.479, P=0.007�'�r=0.418,P=0.022). The percentage of α-SMA-positive vascular smooth muscle cells was decreasedin thoracic aortic dissection tissues, and the expression of BRG1was significantlynegatively related to the percentage of α-SMA-positive vascular smooth muscle cells (r=﹣0.403，P=0.027). TUNEL assay showed that the apoptotic rate of vascular smooth musclecells were significantly increased in the thoracic aortic dissection group (25.52±4.87vs2.54±1.48，P<0.01), and that the expression of BRG1was significantly positivelycorrelated with the rate of TUNEL-positive vascular smooth muscle cells in thoracic aorticdissection (r=0.408，P=0.025).【Conclusions】 BRG1mRNA and protein are increased in the thoracic aorticdissection. Significantly，the expression of BRG1was positively correlated with the rate ofTUNEL-positive vascular smooth muscle cells and with the expressions of matrixmetalloproteinase-9/-2in thoracic aortic dissection. Meanwhile, the expression of BRG1was significantly negatively related to the percentage of α-SMA-positive vascular smoothmuscle cells in thoracic aortic dissection tissues. BRG1may be involved in thedevelopment of thoracic aortic dissection by regulating matrix metalloproteinase-9/-2expressions, apoptosis and phenotype transition of vascular smooth muscle cells. Part2Effects of BRG1over-expression on biological properties ofhuman aortic smooth muscle cells【Objective】 Upregulation of BRG1mRNA and protein in aortic media of thoracicaortic dissection patients indicated that BRG1may be involved in the pathophysiologicalprocess of thoracic aortic dissection. Vascular smooth muscle cells, which are the mainsource of extracellular matrix, are involved in the pathophysiological process of thoracicaortic dissection. In order to further explore the regulatory mechanism of BRG1in aortic smooth muscle cells and its role in thoracic aortic dissection, we transfected the humanaortic smooth muscle cells with recombinant adenovirus overexpressing BRG1andinvestigated the effects on biological properties including the secretion of matrixmetalloproteinase-9/-2, phenotype conversion as well as the apoptosis of human aorticsmooth muscle cells.【Methods】 BRG1over-expression in human aortic smooth muscle cells wasachieved via adenovirus mediated gene transfer. The upregulation effects of BRG1mRNAand protein expressions were detected by qRT-PCR and western blot．The expression ofmatrix metalloproteinase-9/-2and α-SMA mRNA and protein were also detected byqRT-PCR and western blot after transfection in human aortic smooth muscle cells. Thepercentage of apoptotic and contractile cells were determined through flow cytometryanalysis of cells stained by annexin V-FITC and α-SMA, respectively.【Results】 After transfected with recombinant adenovirus vector containing BRG1,the BRG1mRNA(0.61±0.12) and protein(0.42±0.05) expression were increasedsignificantly when compared with the empty vector transfected control group (Ad.Nullgroup)(0.13±0.02and0.18±0.04, both P<0.01). In human aortic smooth muscle cellsline, the percentage of apoptosis in Ad.BRG1group was significantly higher than that inAd.Null group(17.25±2.23vs8.26±2.14, P<0.01). It was found that the expression ofmatrix metalloproteinase-2mRNA and protein in Ad.BRG1infected cells weresignificantly increased than those in Ad.Null-infected cells (0.22±0.02vs0.12±0.01and0.19±0.03vs0.13±0.02, both P<0.01). The expression of matrix metalloproteinase-9mRNA and protein were also increased in Ad.BRG1group(0.11±0.01vs0.02±0.01and0.10±0.01vs0.04±0.01, both P<0.01compared with Ad.Null group). Compared withAd.Null group, the expression of α-SMA mRNA (0.36±0.05vs0.57±0.12，P<0.01) andprotein (0.32±0.08vs0.43±0.06，P<0.05) were significantly decreased in Ad.BRG1group. In addition, the percentage of α-SMA-positively cell in Ad.BRG1treated group wassignificantly decreased than that in Ad.Null treated cells (33.52±3.56vs43.68±0.3.28,P<0.01).【Conclusions】 Adenovirus vector containing BRG1could be transfected intohuman aortic smooth muscle cell line effectively, and BRG1mRNA and protein wereover-expressed significantly. BRG1overexpression in human aortic smooth muscle cellscan promote matrix metalloproteinase-9/-2secretions, apoptosis and phenotypic transitionfrom contractile to synthetic type. These results further prove that BRG1may be involved in the pathological process of human aortic smooth muscle cells and play a key role in thedevelopment of thoracic aortic dissection. Part3Construction of shRNA lentiviral vector targeting humanBRG1gene and identification of RNAi eficiency【Objective】 BRG1may be involved in the pathological process of human aorticsmooth muscle cells. To make further study of BRG1，we constructed shRNA lentiviralvector targeting human BRG1gene and detected its efect on gene silencing in humanaortic smooth muscle cells.【Methods】 The specific siRNA sequences targeting human BRG1gene werecloned into pLKO.1lentiviral vector. The recombined plasmid pLKO.1-BRG1-shRNAwere transfected into293T cell line with psPAX2and pMD2.G. Lentiviral Particlesproduced in293T cells were then transfected into human aortic smooth muscle cells.Silence eficiency of BRG1mRNA and protein was determined by qRT-PCR and Westernblot after transfection.【Results】 It was confirmed by DNA sequencing that the BRG1siRNA sequenceswere corectly inserted into the pLKO.1lentiviral vector，and that human BRG1gene RNAilentivirus vectors were successfully constructed． Three BRG1gene RNAi lentivirusvectors can effectively downregulate the expression of BRG1mRNA in HASMCs (P＜0.01, respectively), and the BRG1shRNA-3had the highest silencing efficiency (88.8%).After transfection with BRG1shRNA-3，the expression of BRG1protein was significantlydecreased in human aortic smooth muscle cells when compared with the negtive controlgroup (0.11±0.01vs0.56±0.01，P<0.01)．【Conclusions】 Human BRG1gene RNAi lentivirus vectors has been successfullyconstructed. The expression of BRG1mRNA and protein levels can be significantlydownregulated in human aortic smooth muscle cells when transfected with BRG1shRNA-3, and it lays the foundation for the future study of pathophysiological process ofhuman aortic smooth muscle cells. Part4BRG1regulates proliferation and apoptosis of aortic smoothmuscle cells through the long noncoding RNA HIF1A-AS1【Objectives】 Increased apoptosis of vascular smooth muscle cell observed in theaortic walls of patients is considered to be an important cause for thoracic aortic dissection.Our study indicated that BRG1overexpression can promote apoptosis of aortic smoothmuscle cells in vitro. However, the underlying mechanism is still far from clear.Accumulating evidences indicate that long noncoding RNAs play critical roles in theregulation of cellular processes such as cell growth and apoptosis. Relevant studies alsoreported that long noncoding RNAs were regulated by epigenetic processes in some celllines. However, there have been few articles discussing the manner of the interactionbetween long noncoding RNAs and ATP-dependent chromatin remodelling complexes.Therefore, in this section, we used lncRNA RT-PCR Array method to identify the longnoncoding RNA regulated by BRG1in human aortic smooth muscle cells. We alsoexplored the effects of this long noncoding RNA on the proliferation and apoptosis ofaortic smooth muscle cells.【Methods】Human aortic smooth muscle cells were transfected with Lenti-shBRG1and Ad.BRG1, and subsequently lncRNA RT-PCR array method was used to identify thelong noncoding RNAs regulated by BRG1. We reduced the expression of identifiedlncRNA HIF1A-AS1by shHIF1A-AS1and the silence eficiency was determined byqRT-PCR. Finally, the effects on proliferation and apoptosis of human aortic smoothmuscle cells induced by long noncoding RNA interference were detected by caspase3activity assay, Western blot and CCK-8cell proliferation assay.【Results】 LncRNART-PCR array indicated that the expression of long noncodingRNA-HIF1A-AS1showed the most obvious change. We further transfected human aorticsmooth muscle cells with Lenti-shBRG1and Ad.BRG1and testified this change. Theexpression levels of HIF1A-AS1were upregulated by3.13-fold compared to control byBRG1overexpression, and downregulated to0.27-fold by BRG1silencing. Aftertransfection with shHIF1A-AS1, the expression level of HIF1A-AS1was significantlydecreased when compared with the negtive control group (0.29±0.02vs1.0±0.10，P＜0.01) and the silencing efficiency was about71.0%. The expression of caspase3proteinwas downregulated to0.7-fold and Bcl2protein was upregulated by1.3-fold compared tocontrol by HIF1A-AS1silencing (both P＜0.01). The caspase3activity was decreased (836.0±98.5vs1371.3±102.6，P＜0.05) and cell proliferation was increased significantlyby HIF1A-AS1interference at2days (0.31±0.01vs0.22±0.01),3days (0.51±0.07vs0.30±0.02) and4days (0.60±0.04vs0.45±0.02)(P＜0.05, respectively).【Conclusions】 BRG1can positively regulate the expression of long noncodingRNA-HIF1A-AS1. HIF1A-AS1interference in human aortic smooth muscle cells canpromotes proliferation and suppress apoptosis. Although more studies are clearly needed toidentify a causal relationship between BRG1and HIF1A-AS1, the delicate balance ofproliferation and apoptosis in aortic smooth muscle cells is clearly essential for a normalaorta, and the HIF1A-AS1associated with BRG1may play a vital role in this dynamicequilibrium.