| Objective: 1.To clarify the regulatory effect of Lnc RNA HOTAIR on the apoptosis of pulmonary vascular endothelial cells in chronic obstructive pulmonary disease(COPD).2.To further explore the mechanism of HOTAIR hypermethylation of Bcl-2 gene mediated by DNMT1 in pulmonary vascular endothelial cell apoptosis in COPD,so as to provide a new theoretical background for the treatment and pathogenesis of smoking-induced COPD.Methods:1.Lung tissues from patients undergoing lobar resection due to lung diseases in the Department of Thoracic Surgery in the Second Xiangya Hospital of Central South University were collected,and they were divided into nonsmoking,Non-COPD Smoker,and COPD Smoker.Baseline data of each group were analyzed.Hematoxylin and Eosin(HE)staining observed pathological changes of lung tissues in each group,and calculated Mean linear intercept(MLI)and alveolar destruction index(Destructive index).Hematoxylin and Eosin(HE)staining.DI)to assess the degree of emphysema in lung tissue;The protein expressions of DNMT1 and Bcl-2 in lung tissues were detected by Western blot.real-time fluorescence quantitative reverse transcription(q RT-PCR)was used to detect RNA expression of HOTAIR in lung tissue.RNA in situ hybridization technique(RNA-ISH)HOTAIR expression in lung tissue and positioning.2.Human pulmonary vascular endothelial cells(HPVEC)were cultured and divided into four groups: Control: cells were treated without any treatment.CSE+ transfection no-load group(CSE+Vector):5% Cigarette Smoke Extract(CSE)intervention + cell transfection no-load plasmid.CSE+HOTAIR Silencing(CSE+si-HOTAIR): 5%CSE intervention + Cell Transfection SI-Hot AIR-007.CSE+HOTAIR silenced+DNMT1 overexpression group(CSE+si-HOTAIR+OE-DNMT1):5%CSE intervention + cell transfection with Si-Hotair-007 and DNMT1 overexpression plasmid.RNA-ISH detection HOTAIR in HPVEC expression and localization,and restructuring marks HPVEC false von willebrand factor(VWF);The protein expressions of DNMT1,Bcl-2,Bax and Cleaved caspase 3 were detected by Western blot.The RNA expressions of HOTAIR,DNMT1,Bcl-2 and Bax were detected by q RT-PCR.TUNEL Apoptosis Detection Kit(FITC)was used to detect apoptosis in each group.Methylation-specific PCR(MSP)was used to detect the methylation of Bcl-2 promoters in each group.The interaction between HOTAIR and DNMT1 was detected by RNA pull down method.3.The mouse model of COPD was constructed and divided into five groups: Control group(Control),smoking COPD group(CSE),CSE+lentivirus silencing HOTAIR group(CSE+si-HOTAIR),control lentivirus intervention group(CSE+Vector),and CSE+HOTAIR silencing+DNMT1 overexpression intervention group(CSE+si-HOTAIR+OE)-DNMT1).HE staining was used to observe the pathological changes of lung tissues in each group,and MLI and DI were calculated to evaluate the degree of emphysema in lung tissues of mice.The protein expressions of DNMT1,Bcl-2,Bax and Cleaved caspase 3 were detected by Western blot.The RNA expressions of HOTAIR,DNMT1,Bcl-2 and Bax were detected by q RT-PCR.TUNEL+CD31 double fluorescence method was used to detect cell apoptosis in each group.The methylation status of Bcl-2 promoter was detected by MSP method.Results:1.The FEV1%pred and FEV1/FVC in COPD smoker group were significantly lower than those in Nonsmoker group and Non-COPD Smoker group(P<0.01).2.HE staining showed that the degree of emphysema in the lung tissues of patients with COPD smoker was more obvious than that of patients with Nonsmoker and Non-COPD Smoker.The specific pathological morphology shows that the alveolar wall of the lung tissue becomes thinner,and the alveolar cavity expands,ruptures or forms pulmonary bulla.Emphysema was more evident in patients with COPD smoker,with higher MLI(μm)and DI(%)than those in the Nonsmoker group(P<0.01).3.The protein expression level of Bcl-2 in lung tissues of patients in COPD smoker group was significantly decreased compared with that in non COPD Smoker group and Non-COPD smoker group(P<0.05).The protein expression level of DNMT1 in lung tissues of patients with COPD smoker group was significantly increased compared with those in non COPD Smoker group and Non-COPD smoker group(P<0.05).4.The content expression of HOTAIR in lung tissues of COPD smoker group was significantly higher than that of Nonsmoker group and Non-COPD Smoker group(P<0.05).5.RNA-ISH in the lung tissue of COPD smoker group detected the expression of HOTAIR more,with less relevant signal detected in Nonsmoker(P < 0.05).HOTAIR positive tissue section analysis showed that it was located in the nucleus.6.RNA-ISH detection found HOTAIR positioning in the nuclei of HPVEC.7.The protein expressions of DNMT1,Bax and Cleaved caspase 3were significantly up-regulated in HPVEC with 5%CSE.The expression of anti-apoptotic protein Bcl-2 was significantly down-regulated(P<0.05).After si RNA interference silenced HOTAIR,DNMT1 protein expression was significantly down-regulated,followed by pro-apoptotic proteins Bax and Cleaved caspase 3,and Bcl-2 protein expression was significantly up-regulated(P<0.05).DNMT1,Bax and Cleaved caspase 3 were upregulated after plasmid transfection with DNMT1 overexpression.The expression of Bcl-2 was significantly down-regulated(P<0.05).8.Under 5%CSE,the RNA expressions of HOTAIR,DNMT1 and Bax in HPVEC were significantly up-regulated.The expression of anti-apoptotic gene Bcl-2 was significantly down-regulated(P<0.05).When silencing HOTAIR with si RNA interference,the RNA expression of HOTAIR was significantly down-regulated,along with DNMT1 and Bax,while the Bcl-2 RNA expression was up-regulated(P<0.05).The RNA expressions of DNMT1 and Bax were significantly up-regulated after plasmid transfection with overexpressed DNMT1.The expression of Bcl-2 was significantly down-regulated(P<0.05).9.Compared with the control group,CSE treatment can significantly increase the apoptosis of HPVEC cells.After si RNA interference silencing HOTAIR,apoptosis was significantly reduced(P<0.05).After plasmid transfection with DNMT1 overexpression,cell apoptosis increased significantly again(P<0.05).10.MSP results showed that the methylation level of Bcl-2 gene promoter region in HPVEC was significantly up-regulated under the effect of 5%CSE(P<0.05).After si RNA interference silencing HOTAIR,the methylation level of Bcl-2 gene promoter region was significantly down-regulated(P<0.05).After plasmid transfection with DNMT1 overexpression,the methylation level of Bcl-2 showed a more obvious upward trend(P<0.05).11.RNA pull down results showed that endogenous Lnc RNA HOTAIR was specifically enriched in DNMT1 detection compared with the control group.12.HE staining showed that the degree of emphysema in the lung tissues of CSE,CSE+Vector and CSE+si-HOTAIR+OE-DNMT1 groups was more obvious than that of Control group and CSE+si-HOTAIR group.The specific pathological morphology of mice in CSE group showed that the alveolar wall of lung tissue was thinner,and the alveolar cavity was enlarged,ruptured or bulla formed.MLI(μm)and DI(%)in the first three groups were significantly higher than those in Control group and CSE+si-HOTAIR group(P<0.05).13.The expression of DNMT1,Bax and Cleaved caspase 3 in mouse lung tissue was significantly upregulated under the action of CS.The expression of anti-apoptotic protein Bcl-2 was significantly down-regulated(P<0.05).After lentivirus transfection silenced HOTAIR,DNMT1 protein expression was significantly down-regulated,followed by pro-apoptotic proteins Bax and Cleaved caspase 3,and Bcl-2 protein expression was significantly up-regulated(P<0.05).When DNMT1 was overexpressed by HOTAIR+,DNMT1,Bax and Cleaved caspase 3 were significantly up-regulated.The expression of Bcl-2 was significantly down-regulated(P<0.05).14.The RNA expressions of HOTAIR,DNMT1 and Bax in mouse lung tissue were significantly up-regulated under the action of CS;The expression of anti-apoptotic gene Bcl-2 was significantly down-regulated(P<0.05).After lentivirus transfection was performed to silence HOTAIR,the RNA expression of HOTAIR was significantly down-regulated,along with DNMT1 and Bax,while the RNA expression of Bcl-2 was up-regulated(P<0.05).When HOTAIR+ overexpression DNMT1 was silenced,the RNA expressions of DNMT1 and Bax were significantly up-regulated.The expression of Bcl-2 was significantly down-regulated(P<0.05).15.The TUNEL molecule was closely associated with CD31 in the fluorescence image,thus demonstrating the presence of apoptosis in mouse pulmonary vascular endothelial cells.Compared with the control group,CS intervention can significantly increase the apoptosis of vascular endothelial cells.When HOTAIR was silenced,apoptosis was significantly reduced(P<0.05).After overexpression of DNMT1,apoptosis was increased again(P<0.05).16.The results of MSP showed that the methylation level of Bcl-2gene promoter region in mouse lung tissue was significantly up-regulated under the action of CS(P<0.05).When HOTAIR was silenced,the methylation level of Bcl-2 gene promoter region was significantly down-regulated(P<0.05).After overexpression of DNMT1,the methylation level of Bcl-2 showed a more obvious upward trend(P<0.05).Conclusion:1.The expression of Lnc RNA HOTAIR was up-regulated in COPD lung tissues,and HOTAIR could directly interact with DNMT1 to mediate hypermethylation of Bcl-2 gene and participate in apoptosis of pulmonary vascular endothelial cells in COPD.2.HOTAIR knockdown can down-regulate the hypermethylation of Bcl-2 gene mediated by DNMT1,thereby reducing the apoptosis of pulmonary vascular endothelial cells in COPD.Graphs 18,Table 7,Reference 98. |
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