| Background and Objective:PASMCs proliferation and perivascular inflammation are key factors in the development of PAH disease.the pathophysiological characteristics of PAH are pulmonary vascular remodeling caused by excessive proliferation of pulmonary artery smooth muscle cells.hypoxia is the main cause of PAH.Current PAH underlying mechanisms remain unclear.lnc RNA are key regulators of many pathophysiological processes,including but not limited to cellular functional regulation,immune and inflammatory responses,and angiogenesis.There is still little research on the mechanism of the relationship between abnormal expression level and PAH in lnc RNA.UCA1 plays an important role in cell proliferation,invasion,migration,apoptosis,metastasis and other cellular processes.Studies have shown the role of UCA1 in PAH development,and studies have demonstrated that AKT/m TOR pathway activation promotes PASMCs proliferation.downregulation of AKT/m TOR signaling pathway plays an important role in inhibiting cell proliferation and reducing PVR.Studies have confirmed the role of UCA1 in various diseases and the role of the AKT / m TOR signaling pathway in PVR.However,the effect of UCA1 on PAH is unclear,and in PAH studies,UCA1 interacts with AKT / m TOR signaling pathways very little.Through the research in this thesis,we will deeply understand the role of Lnc RNA UCA1 in the phenotypic transformation of PASMCs induced by hypoxia and study its mechanism,and then provide new targets for PAH treatment.MethodsIn this study,the expression of UCA1 was judged by q RT-PCR in rat healthy tissues and PAH tissues.Western bolt was used to judge the effect of UCA1 on AKT/m TOR signaling pathway and the expression of p-AKT,AKT,p-ERK,ERK,pm TOR,m TOR.Flow cytometry analysis,MTT and Brd U assays were used to investigate the cell cycle,viability and cell proliferation,respectively.Moreover,cell cycle related proteins,such as,CDK4,CDK6,cyclin A2,cyclin D1 and cyclin E were analyzed by western bolt.Wound-healing analysis and transwell experiment were used to measure cell migration.Result:?The SD rat model of pulmonary hypertension takes 3 weeks.The mean pulmonary artery pressure(m PAP)>25mm Hg meets the diagnostic criteria of PAH,and it is statistically different from the control group,the 0 week group and the 1week group(P <0.05),The results show that the modeling is successful;? Under laser confocal fluorescence microscopy,the cultured PASMCs showed positive response to smooth muscle ?-actin immunofluorescence.Brown-red fluorescence-colored actin was visible in the cytoplasm of PASMCs,and the filaments were aligned parallel to the long axis of PASMCs.? Under PAH condition,UCA1 was up-regulated in lung tissue and PAs tissue compared with normal tissue(P <0.01).Under hypoxic conditions,the expression of UCA1 was up-regulated as treatment time increased(P <0.01).Relative to UCA1,the expressions of si-UCA1-1(P <0.01)and si-UCA1-2(P <0.05)were significantly down-regulated;? Cell viability was higher in hypoxia than in normoxic conditions(P <0.01).Compared with the blank group and the si-NC group,the cell viability was upregulated under hypoxia,while the down-regulation of UCA1 expression decreased the relative viability of the cells(P <0.01).Compared with the blank group and the siNC group,the number of positive cells in si-UCA1-1 decreased under hypoxia;?Compared with the si-NC group and the blank group,the proportion of cells in the G0/G1 phase increased under hypoxia,while the proportion of the S-phase cells in the si-UCA1-1 group decreased,but the cell cycle in the blank group and si-NC No significant difference.Compared with normoxic conditions,the expression of cyclin D1 in the blank group and si-NC group was significantly up-regulated under hypoxia(P <0.01).Compared with the blank group,the relative expression of cyclin D1 in the si-UCA1-1 group was down-regulated under hypoxia.In addition,compared with normoxic conditions,cyclin E1 was significantly up-regulated under hypoxic conditions,and the relative expression of cyclin A2 was not changed(P> 0.05);?Under hypoxic conditions,the cell migration ability of the blank and si-NC groups was significantly higher than that of normoxic conditions.At the same time,si-UCA1-1 can significantly inhibit the migration ability under normoxic and hypoxic conditions;?The expression levels of p-AKT,p-ERK,p-TOR,CDK4 and CDK6 in the cells of the blank group and si-NC group were higher than those of normoxic conditions under hypoxia.At the same time,under normoxic and hypoxia conditions,si-UCA1-1could significantly inhibit the expression of the above proteins(P <0.01),but under normoxic and hypoxia conditions,there was no significant difference between the si-NC group and the blank group.Difference(P> 0.05);? Compared with the control group and the blank group,p-UCA1 enhanced the relative expression of UCA1(P < 0.01),but periposine had no effect on the relative expression of UCA1.And periposine did not inhibit p-uca1.p-UCA1 can increase the relative value of p-AKT/ Akt(P < 0.01),while periposine can inhibit the relative value of p-Akt / Akt(P < 0.01).Compared with p-UCA1 group,Perifosine combined with p-uca1 could inhibit the relative value of p-Akt / Akt(P < 0.01)and regulate the cell viability to normal level;?In the p-UCA1 group,the number of G0 / G1 cells was lower than that in the blank group and the control group(P<0.05).G0 / G1(%)in periposine group was higher than that in blank group and control group(P<0.05).At the same time,pUCA1 combined with periposine can regulate the number of G0 / G1(%)cells to normal levels.p-UCA1 can significantly increase the cell migration distance and cell number(P<0.01),while periposine can inhibit cell migration(P<0.01).p-UCA1 combined with periposine can regulate cell migration distance and number to normal levels.conclusion(1)Lnc RNA UCA1 is involved in the phenotypic transformation of PASMCs inducedby hypoxia;(2)Lnc RNA UCA1 promotes hypoxia-induced viability,proliferation and migration of PASMCs;(3)Lnc RNA UCA1 activates the AKT/m TOR signaling pathway to enhance hypoxia-induced phenotypic transformation of PASMCs.In general,the Lnc RNA UCA1-AKT/m TOR regulatory pathway is an important regulatory mechanism for phenotypic transformation of PASMCs induced by hypoxia,and UCA1 is expected to become a new target for PAH therapy. |
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