| Background : Pulmonary hypertension(PAH)is a clinical and pathophysiological syndrome caused by changes in pulmonary vascular structure or function caused by a variety of heterologous diseases and different pathogenesis,resulting in increased pulmonary vascular resistance and pulmonary artery pressure.Its main characteristics are progressive increase of pulmonary vascular resistance and progressive failure of right heart function.With the increase of pulmonary artery pressure,the heart load of patients will continue to increase,eventually leading to right ventricular hypertrophy,failure and even death.Pulmonary vascular remodeling in patients with pulmonary hypertension often occurs vascular endothelial injury,vascular middle membrane thickening,peripheral vascular muscle fibrosis and extracellular matrix increase,which leads to conformational changes such as pulmonary vascular lumen stenosis,vascular wall thickening,and even occlusive lesions.It is reported that LncRNA PAXIP1-AS1 is involved in cell proliferation and is highly expressed in lung tissue and PASMC of IPAH.Therefore,this topic will study whether PAXIP1-AS1 can regulate PAH pulmonary vascular remodeling and its molecular mechanism,in order to provide a new target and theoretical basis for the study of PAH.Part I Expression of LncRNA PAXIP1-AS1 and RhoA in MCTInduced PAH Rat ModelObjective: to determine the differential expression of paxip1-as1 and RhoA in MCT induced PAH rat model.Methods:(1)PAH rat model was established by subcutaneous injection of MCT and the model was evaluated on the 28 th day after injection,including the measurement of right ventricular systolic pressure by right heart floating catheter method and the observation of pulmonary artery thickening and fibrosis in PAH rat model by HE staining and Masson staining.(2)Immunohistochemical staining was used to observe the in situ expression of RhoA in the lung tissue of PAH rat model.(3)The expression level of RhoA protein in rat lung was detected by Western blot.(4)RT-q PCR was used to detect the gene expression level of PAXIP1-AS1 in lung tissue and plasma of PAH rats.(5)Pearson correlation analysis of PAXIP1-AS1 and RhoAResults:(1)the pulmonary artery pressure of rats was observed by right ventricular manometry.It was found that the PAH model induced by MCT could significantly increase the pulmonary artery pressure compared with the normal group,suggesting that the model was established successfully.(2)He staining of lung tissue sections showed that in PAH group,the pulmonary artery wall was significantly thickened,vascular smooth muscle cells proliferated and hypertrophy,the lumen became significantly smaller,and even the pulmonary arterioles formed lumen atresia.(3)Masson staining of lung tissue sections showed that in PAH group,there were obvious blue fibrous tissue,intimal thickening and fibrosis,adventitia thickening,and fibrous tissue growing to alveolar septum.(4)The expression of RhoA protein in pulmonary artery was detected by immunohistochemistry and Western blot.It was found that the expression of RhoA protein was significantly increased in PAH model.(5)QRT PCR showed that the gene expression level of PAXIP1-AS1 in lung tissue and plasma of PAH model group was significantly up-regulated,which may be closely related to the pathological mechanism of PAH.(6)Pearson correlation analysis showed that the expression level of PAXIP1-AS1 was positively correlated with the content of RhoA.Conclusion: the PAH rat model induced by MCT is accompanied by the increase of pulmonary artery pressure and pulmonary artery thickening;In addition,PAXIP1-AS1 and RhoA were significantly overexpressed in PAH rat model.Part Ⅱ The possible mechanism of paxip1-as1 in proliferation and migration of human pulmonary vascular smooth muscle cellsObjective: To clarify the regulatory role of PAXIP1-AS1 in hypoxia induced h PASMC.Methods:(1)h PASMC hypoxia model was established and transfected with sh-PAXIP1-AS1 and sh NC.(2)The protein expression PCNA was detected by Western blot(3)The expression o PAXIP1-AS1 was analyzed by reverse transcription PCR.(4)The effect of PAXIP1-AS1 on the viability of h PASMC cells was detected by CCK-8method.(5)Brd U cell proliferation experiment to measure the effect of PAXIP1-AS1 on the proliferation of h PASMC cells was detected.(6)Ki-67 immunofluorescence staining was used to detect the effect of PAXIP1-AS1 on the proliferation of h PASMC cells.(7)Transwell chamber test was used to detect the effect of PAXIP1-AS1 on the invasive ability of h PASMC cells.(8)The effect of PAXIP1-AS1 on the migration ability of h PASMC cells was detected by scratch test.Results:(1)the gene expression of PAXIP1-AS1 in h PASMC cells was detected by RT-q PCR.It was found that the gene expression level of PAXIP1-AS1 was significantly up-regulated 12 hours after hypoxia.(2)Chromatin immunoprecipitation analysis showed that sh-PAXIP1-AS1 could significantly inhibit the expression of PAXIP1-AS1.(3)The results of CCK-8 showed that the survival rate of h PASMC cells decreased significantly 72 hours after transfection of shpaxip1-as1.Inhibiting the expression of PAXIP1-AS1 can be stored in the survival of h PASMC cells.(4)Brd U detection of cell proliferation showed that silencing PAXIP1-AS1 could significantly reduce the cell proliferation rate of h PASMC.(5)The number of proliferating cells was calculated by Ki-67 immunofluorescence staining.The transfection of sh-PAXIP1-AS1 could significantly reduce the Ki-67 positive rate of h PASMC cells and the proliferation ability of h PASMC cells.(6)Western blot showed that sh-PAXIP1-AS1 could significantly reduce the protein expression of PCNA in h PASMC cells.(7)The results of cell scratch test showed that sh-PAXIP1-AS1 could inhibit the migration of h PASMC cells,that is,inhibiting the expression of PAXIP1-AS1 could inhibit the migration of h PASMC cells.(8)Transwell chamber invasion experiment showed that sh-PAXIP1-AS1 could inhibit the invasion of h PASMC cells,that is,silencing PAXIP1-AS1 could inhibit the invasion of h PASMC cells.Conclusion: Silencing PAXIP1-AS1 can significantly reduce the cell survival rate of normoxic and hypoxic h PASMC,effectively inhibit the proliferation,migration and invasion of h PASMC cell.Part III The possible mechanism of paxip1-as1 regulating ETS1 /wipf1 to promote the proliferation and migration of pulmonary vascular smooth muscle cellsObjective: To clarify the role and molecular mechanism of PAXIP1-AS1 regulating ETS-1/WIPF1 in promoting the proliferation and migration of human vascular smooth muscle cells.Methods:(1)the expression levels of WIPF1 and ETS1 protein in lung tissue of PAH rats induced by MCT and h PASMC induced by hypoxia were detected by Western blot.(2)Rip(RNA immunoprecipitation)was used to analyze the interaction between PAXIP1-AS1 and ETS-1.(3)The interaction between ETS1 and WIPF1 was detected by double Luciferase Report experiment.(4)Chip detected the effect of knockdown of PAXIP1-AS1 on ETS-1 and WIPF1 promoters.(5)CCK-8 was used to detect the effects of overexpression of PAXIP1-AS1 and silencing ETS-1 or WIPF1 on cell viability.(6)Brd U and Ki67 were used to detect the proliferative activity of h PASMC cells.(7)The effects of overexpression of PAXIP1-AS1 and silencing ETS-1 or WIPF1 on the migration of h PASMC cells were detected by cell scratch test.(8)Transwell chamber experiment was used to investigate the effects of PAXIP1-AS1,ETS-1 and WIPF1 on the invasive ability of h PASMC cells.(9)Pearson correlation analysis of WIPF1 and PAXIP1-AS1 and WIPF1 and RhoA.Results:(1)WIPF1 protein was significantly overexpressed in PAH and hypoxia induced h PASMC cells.(2)Pearson correlation analysis showed that the protein expression level of WIPF1 was significantly positively correlated with PAXIP1-AS1.(3)Western blot showed that overexpression of PAXIP1-AS1 could significantly up regulate the protein expression of WIPF1;When PAXIP1-AS1 was silenced,the expression level of WIPF1 decreased significantly.(4)Rip analysis confirmed that ETS1 could directly bind to PAXIP1-AS1.(5)Luciferase Report showed that WIPF1 was the target gene of ETS1.(6)Chip analysis showed that ETS-1 could bind to wipf1 promoter.(7)Western blot results showed that over-expression of PAXIP1-AS1 could significantly increase the protein expression level of ETS-1,and silencing ETS-1 could significantly reduce the protein level of ETS-1 in h PASMC cells.(8)CCK-8 results showed that overexpression of PAXIP1-AS1 significantly increased cell viability,while silencing ETS1 or WIPF1 reversed the effect of hypoxia on h PASMC cell viability under hypoxia and normal conditions.(9)Brd U and Ki67 detected the proliferation activity of h PASMC cells.It was found that silencing the expression of ETS-1 and WIPF1 could significantly reduce the proliferation activity of h PASMC cells under normoxia and hypoxia.(10)Scratch test showed that overexpression of PAXIP1-AS1 could significantly improve the cell migration ability of h PASMC under normoxia and hypoxia;Silencing the expression of ETS-1 and WIPF1 could significantly reduce the cell migration ability of h PASMC under normoxia and hypoxia.(11)Transwell chamber experiment showed that overexpression of PAXIP1-AS1 could significantly improve the cell invasion ability of h PASMC under normoxia and hypoxia;Silencing the expression of ETS-1 and WIPF1 could significantly reduce the cell invasion ability of h PASMC under normoxia and hypoxia.(12)Pearson correlation analysis showed that the protein expression level of WIPF1 was significantly positively correlated with RhoA.Conclusion: PAXIP1-AS1 can promote its expression in h PASMC by binding ETS-1.ETS-1 further binds to the promoter region of WIPF1 and regulates the expression of WIPF1.Overexpression of PAXIP1-AS1 can improve the cell activity,promote cell proliferation,migration and invasion of h PASMC,and its mechanism may be regulated by ETS-1/WIPF1 axis.Part IV PAXIP-AS1 regulates ETS-1/WIPF1/RhoA axis to promote pulmonary vascular reconstructionObjective: to verify that PAXIP-AS1 regulates ETS-1/WIPF1/RhoA axis and promotes pulmonary vascular remodeling at the level of experimental animalsMethods:(1)Establishment,administration and transfection of PAH rat model.(2)Right ventricular systolic pressure was measured by right ventricular floating catheter(3)The pathological changes of lung tissue in PAH rats were observed by HE staining.(4)Masson staining was used to observe the pulmonary fibrosis in PAH rats.(5)Immunohistochemical observed the expression of α-SMA and RhoA proteins in lung tissue.(6)The protein expression levels of ETS-1,WIPF1 and RhoA in rats were detected by Western blot.Results:(1)the measurement results of right heart floating catheter method showed that silencing PAXIP-AS1 could significantly reduce the pulmonary artery pressure of rats;Inhibition of RhoA can also effectively reduce pulmonary artery pressure in rats and relieve pulmonary artery pressure in PAH rats.(2)He staining showed that silencing paxip-as1 and inhibiting RhoA expression could significantly reduce the thickening of pulmonary artery wall in rats.(3)Masson staining showed that silencing PAXIP-AS1 and inhibiting RhoA expression could significantly reduce pulmonary fibrosis.(4)Pulmonary artery RhoA and α-SMA were detected by immunohistochemistry,it was found that silencing PAXIP-AS1 could significantly inhibit the protein expression level of RhoA;Silencing PAXIP-AS1 and inhibiting the expression of RhoA would significantly decrease the expression of α-SMA protein in lung tissue of PAH rats.(5)RT q PCR detected the gene expression level of PAXIP-AS1.It was found that sh-PAXIP-AS1 and inhibition of RhoA expression could significantly reduce the gene expression of PAXIP-AS1 in the lung tissue of PAH rats.(6)Western blot showed that silencing the expression of PAXIP-AS1 could significantly reduce the protein expression levels of WIPF1,ETS-1 and RhoA in the lung tissue of PAH rats;Inhibition of RhoA expression can significantly reduce the protein expression levels of WIPF1,ETS-1 and RhoA in the lung tissue of PAH rats.Conclusion: Silencing PAXIP-AS1 can reduce pulmonary artery pressure and pulmonary vascular remodeling in PAH rats by regulating ETS-1/WIPF1/ RhoA.Conclusion:(1)PAXIP1-AS1 was significantly and highly expressed in hypoxia-induced h PASMC cells models and MCT-induced PAH rat models;(2)Silencing of PAXIP1-AS1 can significantly reduce the cell survival rate of normoxic and hypoxic h PASMC,and effectively inhibit the proliferation,migration and invasion of h PASMCs cells;(3)PAXIP1-AS1 is able to regulate the proliferation,apoptosis,and migration of pulmonary artery vascular smooth muscle cells by regulating the ETS1 / WIPF1 / RhoA axis. |
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