Study On The Mechanisms Of Type ? Alveolar Epithelial Cell Senescence Regulated By LncRNA-mediated SIRT1 Signaling Networks In Chronic Obstructive Pulmonary Disease

Objective Lung tissue samples from COPD and control groups were collected to explore the level of senescence biomarker of type?alveolar epithelial cell.We isolation,culture and identification of AEC II in vitro,and study the mechanisms of type?alveolar epithelial cell senescence regulated by lncRNA-mediated SIRT1signaling networks in chronic obstructive pulmonary disease.Method 26 cases of COPD patients combined with non-small cell lung cancer and 31 cases of non-small cell lung cancer patients without COPD were enrolled in the present study.The basic clinical characteristics of the COPD group?26 patients?and control group?31 patients?such as age,gender,body mass index,smoking index and pulmonary function index?FVC,FEV₁/FVC,FEV₁,%predicted?were analyzed.The lung tissue specimens of two groups were stained with hematoxylin and eosin?H&E?in order to assess the morphological changes in the lungs.Senescence-associated?-galactosidase activity in lung tissue specimens were assessed using an in situ?-galactosidase staining kit.Real time quantitative PCR was used to detect the expression levels of lncRNA SAL-RNA1,lncRNA SAL-RNA2,lncRNA SAL-RNA3,SIRT1,FOXO3?,P53 and P21.Immunohistochemical analysis and western blot were employed to detect the protein expression of SIRT1,FOXO3?,P53and P21 in lung tissue specimens.The AECII was isolated,cultured and identified by electron microscopy in vitro.We successfully established a model of AECII senescence induced by cigarette smoke extract.We successfully established the lentiviral vector,which mediated overexpression of lncRNA SAL-RNA1.The SA-?-gal activity was assessed in the four groups:AEC?group,AEC?+CSE group,SAL-RNA1+CSE group,SAL-RNA1+CSE group.Results The forced vital capacity?FVC?,ratio of forced expiratory ventilation in1 sec to FVC?FEV₁/FVC?and FEV₁?%predicted?in patients with COPD was significantly reduced compared with the control group?P<0.05?.Quantitative analyses of lung histomorphology revealed that the mean linear interval?MLI?and mean alveoli area?MAA?of the COPD group was significantly higher compared with the control group?P<0.05?and the mean alveoli number?MAN?was significantly decreased in the COPD group compared with the control group?P<0.05?.Compared with the control group,SA-?-gal activity was visibly increased in the COPD group.Most SA-?-gal-positive cells were located at the edges of alveoli.RT-PCR results showed that SAL-RNA2 and SAL-RNA3 mRNA expression levels were significantly increased in the lung tissues of the COPD group compared with the control group?P<0.05?.However,the SAL-RNA1 level in the COPD group was significantly decreased compared with the control group?P<0.05?.p53 and p21 mRNA expression levels were significantly increased in lung tissues in the patients with COPD compared with the control group?P<0.05?.By contrast,SIRT1 and Fox O3?mRNA expression levels in the COPD group were significantly decreased compared with the control group?P<0.05?.Immunohistochemical analysis and western blot showed that SIRT1 and FoxO3a protein levels were visibly reduced in the COPD group compared with the control group,while p53 and p21 protein levels were visibly upregulated in the COPD group compared with the control group.Accompanying the downregulation in SIRT1 protein levels,there was a significant reduction of SIRT1activity in the COPD group compared with the control group?P<0.05?.The primarily isolated and cultured AEC?were able to grow adhering to the wall in DMEM medium containing 10%FBS.The structure of the characteristic lamellar body in AECII cells was clearly observed by transmission electron microscopy,and it was concentric round.CSE could induce down regulation of SIRT1 mRNA in AEC II cells dose-dependently and increased activity of SA-?-gal dose-dependently and time-dependently.The lentiviral vector-mediated overexpression of lncRNA SAL-RNA1 could delay the up-regulated activity of SA-?-gal induced by CSE.Conclusion The level of senescence biomarkers in COPD group were up-regulated and the lentiviral vector-mediated overexpression of lncRNA SAL-RNA1 can delayed the up-regulated activity of SA-?-gal induced by CSE.