Research On The Role And Mechanism Of Long Non-Coding RNA H19 In Monocrotaline-Induced Pulmonary Arterial Hypertension

Pulmonary hypertension（PH）is a complex cardiopulmonary disease.Right heart floating catheter was used to measure the average pulmonary artery pressure（m PAP）of the patient at sea level and resting state.They were diagnosed when the m PAP≥25mm Hg.PH can be divided into five categories,of which pulmonary arterial hypertension（PAH）belongs to Group 1.At present,targeted drugs were used in the treatment of PAH in clinic.Targeted drugs act on the nitric oxide pathway,the prostacyclin pathway and the endothelin pathway.The use of targeted drugs has greatly improved the patient’s symptoms,increased exercise tolerance,and prolonged the patient’s survival time.Despite considerable progress in the treatment of PAH,the prognosis of this fatal disease is still worse than that of many cancers,with a 3-year survival rate of 68%-70%.Therefore,it was of great significance to explore new pathogenesis and therapeutic targets.The pathological features of PAH were pulmonary arteriolar remodeling,elevated pulmonary arterial pressure,and right ventricle hypertrophy.There were similar histopathological characteristics among the various subtypes of PAH,including intimal hyperplasia,excessive proliferation of medial smooth muscle cells,and thickening of adventitia,the most important of which is the excessive proliferation of pulmonary artery smooth muscle cells（PASMCs）.In recent years,researches on PAH has mainly focused on genetic transcription factors,non-coding RNA,inflammation,oxidative stress,DNA damage,metabolic disorders,mitochondrial function,stem cells and other fields.Long non-coding RNAs（LncRNAs）were a class of RNAs longer than 200 nt.They were involved in various biological processes such as gene modification,transcriptional regulation,and post-transcriptional regulation.They played roles in cell proliferation,migration,apoptosis,and phenotypic transformation.LncRNAs also played a critical role in the maintenance of vascular biology homeostasis,and participated in vascular remodeling and vasoconstriction.In recent years,studies have shown that lncRNAs were involved in the pathological process of PH.Inflammation,oxidative stress and other factors regulated the expression of lncRNAs,and differentially expressed lncRNAs further regulated the proliferation of PASMCs.H19 was one of the earliest discovered lncRNAs.H19 worked in three ways: sponge mi RNAs,recruitment proteins,and the H19/mi R-675 axis.H19 promoted the differentiation of aortic SMCs and repaired damaged arteries.H19 was reported to be upregulated by PDGF-BB in rheumatoid arthritis.PDGF-BB was a growth factor and promoted the proliferation of PASMCs,leading to pulmonary arteriolar remodeling and PAH.Therefore,we speculated that H19 may be involved in the development of PAH.Plasma from PAH patients and healthy subjects was used to explore the expression of H19.The expression of H19 was higher in PAH group than that in controls.Further,MCT-induced rats model of PAH was constructed.The expression of H19 in pulmonary arteries and plasma of PAH rats induced by MCT was higher than that in the control.Cytokines were used to stimulate PASMCs,and H19 was found to be upregulated by PDGF-BB.H19 was found to significantly promote the proliferation of PASMCs,but had no significant effect on migration and apoptosis.Through bioinformatics analysis,AGO2 RIP experiment,and dual luciferase report assay,we found that H19 can interact with Let-7b-5p and mi R-194-5p,and AT₁ R was a new target of Let-7b-5p and mi R-194-5p.The function recovery expriments showed that PDGF-BB-H19-Let-7b-5p/mi R-194-5p-AT₁ R axis promoted the proliferation of PASMCs by activating the ERK1/2 signaling pathway.H19 knockout mice were used to investigate the effects of H19 on PAH in vivo.Our results showed that H19 knockout protected mice from pulmonary artery remodeling and PAH following MCT treatment.In summary,H19 may become a potential circulating diagnostic marker and therapeutic target of PAH.This study provides new ideas for the pathogenesis,diagnosis and treatment of PAH.Part Ⅰ Expression of lncRNA H19 in PAH patients, MCT-induced PAH rats and cytokine-stimulated PASMCs Objective⑴ To explore the expression of lncRNA H19 in plasma of PAH patients and healthy controls.⑵ To explore the expression of lncRNA H19 in pulmonary arteries and plasma of MCT-induced PAH rats.⑶ To explore the expression of lncRNA H19 in cytokine-stimulated PASMCs.MethodsThe expression of H19 in plasma of PAH patients and healthy subjects was detected by RT-q PCR.The PAH rats model induced by MCT was constructed,and was confirmed by HE staining of lung tissue,RVSP measurement,and RVHI.Pulmonary arteries and plasma of MCT-induced PAH rats were used to detect the expression of H19 and cytokines by RT-q PCR.The primary rat PASMCs were isolated by using enzymatic hydrolysis method,identified by α-SMA staining,and purified by the differential adherence method.IL-1α,IL-1β,IL-6,PDGF-AA,PDGF-BB,TNF-α and VEGF were used to stimulate PASMCs,and the expression of H19 was detected by RT-q PCR.Results⑴ The expression of H19 in the plasma of PAH patients was significantly higher than that in healthy subjects.⑵ The expression of H19 in pulmonary arteries and plasma of MCT-induced PAH rats was significantly higher than that in the control group.⑶ The expression of H19 in the IL-1β and PDGF-BB groups was higher compared with the control group.With the increase of IL-1β and PDGF-BB concentrations,the expression of H19 was gradually increased.Conclusions⑴ The expression of H19 in PAH patients’ plasma,pulmonary arteries and plasma of MCT-induced PAH rats was significantly increased.⑵ The expression of H19 was significantly upregulated by IL-1β and PDGF-BB.Part Ⅱ Effect of lncRNA H19 on the proliferation,migration and apoptosis of PASMCs ObjectiveTo explore the effects of lncRNA H19 on the proliferation,migration and apoptosis of PASMCs.MethodsThe plasmid pc DNA3.1（-）-H19（pH19）and small interfering RNA si H19 were constructed.The negative controls were pc DNA3.1（-）（Vector）and si RNA control（si Con）,respectively.pH19 and si H19 were transfected into PASMCs,and the expression of H19 was detected by RT-q PCR.The effect of H19 on the proliferation of PASMCs was detected by flow cytometry using the CCK-8 method;the effect of H19 on the migration of PASMCs was detected by scratch and transwell experiments;the effect of H19 on the apoptosis of PASMCs was detected by flow cytometry.Results⑴ Flow cytometry results showed that the proportion of S+G2/M phase cells in the pH19 group was significantly higher than that in the Vector group.CCK-8 results showed that cells exhibited higher proliferative activity in 100 ng,500ng and 1000 ng pH19 groups than that in the Vector group.At 12 h,24h,48 h,and 72 h,the cells proliferation activity of the pH19 group was higher than that in the Vector group.Compared with the si Con group,si H19 significantly reduced the proportion of S+G2/M cells and proliferative activity,and PDGF-BB stimulation significantly increased the proportion of S+G2/M cells and proliferative activity,while si H19 partially offset the effect of PDGF-BB on the proliferation of PASMCs.⑵ The width of wound healing and the number of migrating cells in the pH19 or si H19 group were not significantly different from those in the Vector group or si Con group,respectively.⑶ There was no significant difference in the apoptotic ratio of cells in the pH19 or si H19 group compared with the Vector group or si Con group,respectively.Conclusions⑴ H19 increased the proliferation activity of PASMCs and promoted the transition of PASMCs to proliferation phase.⑵ siH19 reduced the pro-proliferation effect of PDGF-BB.⑶ H19 had no significant effect on the migration and apoptosis of PASMCs.Part Ⅲ Research on the mechanism of lncRNA H19 regulating the proliferation of PASMCs ObjectiveTo explore the mechanism of lncRNA H19 in promoting the proliferation of PASMCs.MethodsNuclear and cytoplasmic separation experiment was used to extract RNA from nucleus and cytoplasmic,and the expression of H19 was detected by RT-q PCR.Bioinformatics analysis was performed to analyze H19-associated mi RNAs（H19-mi RNAs）and MCT-induced mi RNAs（MCT-PAH-mi RNAs）in PAH rat lung mi RNAs chips.AGO2 RIP and dual luciferase reporter assay were used to detect the binding of H19 with Let-7b-5p and mi R-194-5p.GO and KEGG pathways were used to analyze the target genes of Let-7b-5p and mi R-194-5p.Bioinformatics analysis were used to analyze the binding structural basis of H19 and AT₁ R with Let-7b-5p and mi R-194-5p.Dual luciferase reporter assay,Western Blot,and RT-q PCR were used to detect the regulation of AT₁ R by Let-7b-5p and mi R-194-5p.H19 overexpression plasmid was transfected,then Western Blot and RT-q PCR were used to analyze the effects of H19 on AT₁ R and ERK1/2 pathway.Western Blot was used to detect the effect of inhibition of ERK1/2 phosphorylation（U0126）on the expression of Cyclin A2,CDK1,and PCNA.Co-transfection of si H19 and Let-7b-5p/mi R-194-5p inhibitor was used for rescue experiments,the expression of AT₁ R,ERK1/2,cell cycle and proliferation activity of PASMCs was detected.Co-transfection of p AT₁ R with Let-7b-5p/mi R-194-5p mimic was performed for functional rescue experiments to detect changes in cell cycle and proliferation activity of PASMCs.Co-transfection of pH19 and si AT₁R/U0126 was used for functional rescue experiments to detect changes in cell cycle and proliferation activity of PASMCs.Results⑴ The results showed that 79% of H19 RNA was enriched in the cytoplasm of PASMCs.Dual luciferase reporter assay showed that the luciferase activity of Let-7b/mi R-301a/mi R-185/mi R-194 mimic group was significantly reduced compared with mi Con group.AGO2 RIP experiment showed that Let-7b-5p mimic and mi R-194-5p mimic group had the highest enrichment of H19.Dual luciferase reporter assay showed that with the increase of Let-7b-5p/mi R-194-5p mimic concentration,the luciferase activity was gradually decreased,and there was no significant change in luciferase activity after the binding sequence was knocked out or mutated.⑵ KEGG pathway analysis found that the target genes were mainly enriched in the renin pathway.Dual luciferase reporter assay showed that with the increase of Let-7b-5p/mi R-194-5p mimic concentration,the luciferase activity was gradually decreased.Moreover,there was no significant change in luciferase activity after the binding sites was mutated.Western Blot and RT-q PCR showed that AT₁ R m RNA and protein level were significantly reduced after transfected with Let-7b-5p/mi R-194-5p mimic compared with the control group;after transfected with Let-7b-5p/mi R-194-5p inhibitor,AT₁ R m RNA and protein level were significantly increased compared with the control group.⑶ Western Blot and RT-q PCR showed that the expression of AT₁ R,Cyclin A2,CDK1,PCNA were significantly increased,the degree of phosphorylation of ERK1/2 was significantly increased,and the degree of phosphorylation of AKT was not significantly changed in the pH19 group.Western Blot showed that the degree of phosphorylation of ERK1/2 in the U0126 group was significantly reduced,and the expression of Cyclin A2,CDK1 and PCNA protein was also significantly reduced.⑷ Rescue experiment showed that the expression of AT₁ R,Cyclin A2,CDK1,and PCNA was downregulated when H19 was knocked-down.Let-7b-5p/mi R-194-5p inhibitor partially offseted the down-regulated effect,and partially offseted the proliferation of PASMCs by si H19.Let-7b-5p/mi R-194-5p mimic inhibited the proliferation of PASMCs,but p AT₁ R partially offseted this inhibited effect.pH19 promoted the proliferation of PASMCs,si AT₁ R and U0126 partially offseted this inhibition.Conclusions⑴ H19 can interact with Let-7b-5p and mi R-194-5p.⑵ AT₁ R is a novel target of Let-7b-5p and mi R-194-5p.⑶ PDGF-BB-H19-Let-7b-5p/mi R-194-5p-AT₁ R axis promotes the proliferation of PASMCs by activating the ERK1/2 signaling pathway.Part Ⅳ The role of H19 in the pathogenesis of MCT-induced PAH mice model ObjectiveTo investigate the role of H19 in the pathogenesis of MCT-induced PAH mice model.MethodsC57/BL6 mice and H19 knockout mice were used to construct the MCT-induced PAH mice model.Mice were divided into 4 groups: the C57/BL6 saline control group（WT Control）,the C57/BL6 monocrotaline group（WT MCT）,H19 knockout mice in saline control group(H19^-/-Control),H19 knockout mice in the monocrotaline group(H19^-/-MCT),6 mice in each group.RVSP was measured by the right heart catheter.Pulmonary arterial remodeling was measured by HE staining and α-SMA immunohistochemistry and calculated by percentage of muscular arteries and WT%.Western Blot and RT-q PCR were used to detect the effect of H19 knockout on Let-7b-5p,mi R-194-5p,AT₁ R,and ERK1/2 signaling pathways.Results⑴ After eight weeks of MCT injection in wild-type mice,RVSP was significantly higher than it in the control group（33.41 mm Hg±2.19 mm Hg vs 19.32 mm Hg±0.79 mm Hg,P<0.001）.No significant difference was detected in RVSP between H19^-/-Control group and H19^-/-MCT group,but the RVSP of the H19^-/-MCT group was significantly lower than that in the WT MCT group（17.72 mm Hg±1.54 mm Hg vs 33.41 mm Hg±2.19 mm Hg,P<0.001）.⑵ Compared with the WT Control group,the WT MCT group showed a significant increase in WT%（40.60%±3.79% vs 19.11%±2.56%,P<0.001）and a significant increase in the proportion of myogenic pulmonary arterioles（PM: 28.55%±2.65% vs 18.89%±2.66%,P<0.001;FM: 41.91%±4.34% vs 17.13%±2.25%,P<0.001）.WT% and myogenic pulmonary arterioles showed no significant difference between H19^-/-Control group and H19^-/-MCT group.But WT%（40.60%±3.79% vs 23.92%±3.17%,P<0.001）and the proportion of arterioles was significantly decreased（PM: 28.55%±2.65% vs 21.74%±0.72%,P<0.01;FM: 41.91%±4.34% vs 20.22%±1.80%,P<0.001）in the H19^-/-MCT group compared with the WT MCT group.⑶ RVHI in the WT MCT group was significantly higher than that in the WT Control group（0.23±0.03 vs 0.15±0.01,P<0.001）.Compared with H19^-/-Control,RVHI in the H19^-/-MCT group did not show significant changes.RVHI in H19^-/-MCT group was significantly lower than that in WT MCT group（0.17±0.02 vs 0.23±0.03,P<0.01）.⑷ IL-1β,PDGF-AA,PDGF-BB,IL-6,TNF-α and VEGF were significantly increased in the pulmonary arteries of WT MCT and H19^-/-MCT mice;AT₁R and ERK1/2 significantly increased in WT MCT mice,while it showed no significant difference in H19^-/-MCT mice compared with H19^-/-Control group.Let-7b-5p and mi R-194-5p did not show significant changes in the pulmonary arteries of WT MCT and H19^-/-MCT mice.Conclusions⑴ H19 knockout protected mice from pulmonary artery remodeling and PAH following MCT treatment.⑵ After H19 knocked out,the expression of PDGF-BB was increased with MCT induction,but the expression of AT₁ R,Let-7b-5p,mi R-194-5p,and ERK1/2 did not change significantly.