Study Of The Role Of Integrin-β1on Airway Remodeling In Asthma And The Targeting Intervention Of ShRNA Expression Vector

Airway remodeling is a major character of asthma, while the mechanisms responsible forsuch structural alterations appear to be ambiguous. Integrins are dimeric glycoproteins composedof distinct α and β chains, which can mediate signal transduction and regulate severalphysiological processes including cell growth, differentiation, proliferation and apoptosis. It hasbeen proved presently that Integrin-β1generally exists on the surface of airway smooth musclecells(ASMC), and regulates a serial of physiological processes. It is indicated that Integrin-β1may play a key role in driving airway remodeling. This research attempts to conduct the shRNAexpression vector silencing Integrin-β1gene, and to reveal the role of Integrin-β1in airwayremodeling of asthma in vivo and in vitro and the regulating mechanisms of signal transduction.So that we can identify the novel therapeutic targets of asthma.PART IThe role of Integrin-β1in airway remodeling of asthmaObjective To investigate the role of Integrin-β1in airway remodeling of asthma.Methods The shRNA expression vector silencing Integrin-β1gene is designed, conducted,and transfected into ASMCs of asthma model in vivo and in vitro. The transfection efficienciesof the experiments are detected whih an immunofluorescence method. The mRNA and proteinexpressions of Integrin-β1were determined by RT-PCR, Real-time PCR, and Western-blotrespectively. Cell proliferation was measured by CCK-8and BrdU ELISA, and cell cycle byfluorescence-activated cell sorter. Cell apoptosis was detected by Annexin V-PE/7-AAD staining.Cell migration assays were evaluated by transwell assay and expression of IL-6and IL-8byELISA. The pathological changes of the lungs dyed with HE stain were observed through light microscope.Results The shRNA expression vector stably transfected ASMC of asthma, decreased themRNA and protein expressions of Integrin-β1(P<0.05), enhanced the proportion of cells inG0/G1phase(P<0.05), decreased the proportion in S-phase(P<0.05), promoted cell apoptosis,inhibited cell proliferation(P<0.05), migration(P<0.05), IL-6and IL-8secretion(P<0.05) in vitro.The airway remodeling of asthma model was improved after transfection in vivo.Conclusion The overexpression of Integrin-β1in ASMC is essential for airway remodelingdevelopment because it promotes proliferation, declines apoptosis, accelerates migration, andincreases the secretion of IL-6and IL-8in ASMC. PART IIThe role of Integrin-β1in signal network of ASMC proliferation and thetargeting intervention of shRNA expression vectorObjective To investigate the mechanisms about Integrin-β1effecting the airway remodelingof asthma and the targeting intervention of shRNA expression vector.Methods The shRNA expression vector silencing Integrin-β1gene was conducted, andtransfected into ASMCs of asthma model in vitro. The transfection efficiencies of theexperiments were detected whih an immunofluorescence method. The mRNA expressions ofCyclinD1and c-Fos were determined by RT-PCR and Real-time PCR. The protein expressionsof ERK1/2, STAT6, p-ERK1/2, p-STAT6, CyclinD1, and c-Fos were detected by Western-blot.Cell proliferation was measured by CCK-8and BrdU ELISA, and cell cycle was evaluated byfluorescence-activated cell sorter.Results The shRNA expression vector stably transfected ASMC of asthma in vitro, decreasedthe mRNA and protein expressions of CyclinD1and c-Fos(P<0.05), went no change on theprotein expressions of ERK1/2and STAT6(P>0.05), reduced the protein expressions of p-ERK1/2(P<0.05), while enhanced the protein expressions of p-STAT6(P<0.05). The cellproliferation was inhibited (P<0.05) and the cell cycle was arrested at the G0/G1phase (P<0.05).Conclusion There is a crosstalk between Integrin-β1/ERK1/2pathway andIL-4/IL-13/STAT6pathway in ASMC of asthma. Integrin-β1may be a molecular switch in thesignal network of ASMC proliferation, because it promotes the expressions of CyclinD1andc-Fos in the cells, and which are exactly the mechanisms in Integrin-β1effecting the airwayremodeling of asthma. The shRNA expression vector targeting Integrin-β1can be applied as anovel gene target for intervention to treat the airway remodeling of asthma.