| [Background]Pulmonary hypertension(PH)is a group of severe progressive pulmonary artery remodeling diseases.The diagnostic criterion is that the average pulmonary artery pressure is?25 mmHg,which will eventually lead to increased pulmonary vascular resistance and right ventricular failure.According to its etiology,it can be divided into arterial pulmonary hypertension,pulmonary hypertension caused by left heart disease,pulmonary hypertension caused by hypoxia and/or pulmonary disease,chronic thromboembolic pulmonary hypertension,pulmonary arteries caused by multiple mechanisms and/or unknown mechanisms Hypertension,of which arterial pulmonary hypertension(Pulmonary arterial hypertension,PAH)is the first of the five major categories of pulmonary hypertension.Clinically,calciumion antagonists,endothelin receptor antagonists and other drug treatments are the main treatments,but most patients have poor efficacy,poor prognosis,and a median survival time of only 2-8 years.Therefore,in-depth exploration of the pathogenesis of PAH and active exploration of new targets for the treatment of PAH will be of great significance for the prevention and treatment of PAH and improving the quality of life of patientsThe main histopathological feature of PAH is the remodeling of the distal tiny pulmonary artery.Abnormal cell metabolism,abnormal endoplasmic reticulum stress,calcium homeostasis,mitochondrial dysfunction and structural disorders,and abnormal modification of epigenetics can all lead to the transformation of the vascular smooth muscle of the pulmonary vascular media from contractile to synthetic,and synthetic smooth muscle has more Strong proliferation and migration ability,and eventually form the typical vascular plexus lesions of PAH,showing small blood vessels as plexiform,reticulate or spherical,or fissure-like disordered hyperplasia and reconstruction.Therefore,further study of the function and regulation mechanism of pulmonary vascular smooth muscle cells will provide more theoretical and experimental basis for the treatment of PAH.Recent studies have confirmed that PAH is related to estrogen(E2)levels in the body.Epidemiological data show that PAH occurs more frequently in women,and the prevalence of women is 2-4 times that of men.It is suggested that high level of E2 may promote the occurrence and development of PAH.Further research on the role of E2 in the occurrence and development of PAH and its molecular mechanism are of great significance for the selection of effective interventions(estrogen replacement therapy or antagonist therapy)and the exploration of new targets for PAH prevention and treatment.Studies have shown that PFKFB3 plays a key role in promoting PAH.PFKFB3(Phosphofructose kinase-2/Fructose-2,6-bisphosphatase 3,Phofructo kinase-2/fructose-2,6-bisphosphatase 3)is a key promoter of glycolysis(conversion of glucose to lactic acid and energy)Increased expression of enzymes promotes increased lactate,which in turn stimulates angiogenesis.In addition,PFKFB3 can activate cyclin-dependent kinases and promote cell proliferation.Pulmonary vascular smooth muscle cells PFKFB3 can increase glycolysis levels,activate ERK1/2-dependent calcium-activated neutral protease 2 phosphorylation,promote abnormal proliferation of smooth muscle cells,accelerate and aggravate pulmonary vascular remodeling.Studies have shown that E2 can induce the expression of PFKFB3 in MCF-7 cells and promote the proliferation and migration of cancer cells.It is suggested that E2 may also promote the proliferation and migration of pulmonary vascular smooth muscle cells by affecting the expression of PFKFB3 protein in pulmonary vascular smooth muscle cells,thereby affecting the occurrence and development of pulmonary hypertension.Therefore,this topic will use PFKFB3 as an entry point to explore the key role of E2 in promoting the proliferation and migration of pulmonary vascular smooth muscle cells.E2 can exert the classic gene effect through the estrogen receptor ?,?(ER?,ER?)to promote the expression of target protein.Therefore,whether E2 can promote the expression of PFKFB3 protein through genetic effects is the first question we explore.In addition,E2 can also regulate gene expression through post-transcriptional modification.Post-transcriptional regulation includes splicing and processing of mRNA precursors,nucleation and localization of mRNA,stability and degradation of mRNA.m6A,as the most common modification method of mRNA,plays an important role in post-transcriptional regulation.m6A refers to the methylation modification of adenine(A)on mRNA.This modification process is reversible,and it is jointly participated by methyltransferases(Writers),demethylases(Erasers)and methylated readers(Readers),and is widely involved in mammalian reproductive development,immunity,metabolism,tumor The process of generation and transfer.Recent studies have shown that methyltransferase 3(METTL3)can promote the expression of several key oncoproteins,and its high expression enhances the proliferation,migration and invasion of several human cancer cells.There is no report that E2 affects m6A modification to regulate target protein.Therefore,we speculate whether E2 affects the proliferation and migration of pulmonary vascular smooth muscle cells by regulating the expression of m6A mRNA methylase METTL3,and thereby regulating the expression of PFKFB3 protein,which is the content of this researchIn order to further verify the effect of E2's promotion of PFKFB3 expression through METTL3 on the occurrence and development of PAH,this topic confirmed the important role of METTL3/PFKFB3 in the promotion and development of PAH by E2 at the overall animal level,namely in MCT-induced pulmonary hypertension In model+ovarian castration(OVX)model,the expression level of METTL3/PFKFB3 in each group of rat models was detected by immunohistochemical method,Western blot,m6A detection kit and real-time RT-PCR method.The key role in the occurrence and development of PAH[Objectives]The study is to explore the role of E2 in upregulating the expression of PFKFB3 protein by regulating METTL3,thereby promoting pulmonary vascular smooth muscle function and the development of pulmonary hypertension,and to explore its molecular regulatory mechanism,confirming that METTL3/PFKFB3 plays a role in the development and development of pulmonary hypertension on important role of E2[Methods and Results]1.E2 promotes the development of MCT-induced PAH rat models1.1 E2 promoted the progression of disease in MCT-induced PAH rat modelsAdult female sprague-dawley(SD)rats weighing 200-250g were given normal diet at the age of 6-8 weeks and randomly divided into 6 groups(6 rats in each group).B)the OVX group;C)OVX+E2 group:OVX group was treated with 17 everl-estradiol(E2)sustained-release tablets(0.25mgE2/60days)implanted subcutaneous in the neck Days The above processing complete recovery after 1 week,the trade is often oxygen PAH building:the female SD rats disposable hypodermic injection of the side before ham monocrotaline(MCT,50mg/kg)and control group in SD rats(control group,the OVX group,the OVX+E2 group)injection volume dose of normal saline,same in SPF animal breeding,given a normal diet.These groups of rats receiving treatment after 4 weeks,testing related indicators1.1.1 Detection of serum E2 levels in rat models of PAH groups induced by MCTA total of 36 adult female Sprague-Dawley rats in each group,with an average age of 6-8 weeks,were collected and serum levels of 17?-estradiol(E2)in the serum of rats in each group were detectedThe results showed that compared with the Sham group,the serum E2 level of rats in the OVX group was significantly lower,and the serum E2 level of rats in the OVX+E2 group was significantly higher than that in the OVX group,with an increase rate of(165.3±23.7)%(n=6,P<0.01).Compared with the Sham group,the serum E2 level of rats in the Sham+MCT group was significantly increased,with an increase rate of(155.3±26.7)%(n=6,P<0.01).The serum E2 level of rats in the OVX+MCT group was lower than that in the Sham+MCT group,but still higher than that in the Sham group.The serum E2 level of rats in the OVX+MCT+E2 group was significantly higher than that in the OVX+MCT group,indicating that the serum E2 level in the MCT-induced pulmonary arterial hypertension rat model was higher than that in normal rats1.1.2 Effect of E2 on right ventricular systolic pressure induced by MCT in PAH rat modelThe results showed that the RVSP of rats in the OVX+MCT group was significantly reduced(168.6±20.7)%(n=6,P<0.01)when compared with Sham+MCT group(n=6)Compared with the OVX+MCT group,after supplementation with E2 sustainedrelease tablets(n=6)in the OVX+MCT+E2 group,RVSP was significantly increased,with an increase rate of(150.6±18.9)%(n=6,P<0.01)1.1.3 Effect of E2 on right cardiac hypertrophy index in rat model of PAH induced by MCTAfter collecting the hemodynamic indexes of rats in each group,the chest was opened and the cardiopulmonary tissue was removed,and the right cardiac hypertrophy index of rats in each group was measuredRight cardiac hypertrophy index(RI)was significantly reduced in the OVX+MCT group(n=6)compared with Sham+MCT group(n=6)(n=6,P<0.01).Compared with OVX+MCT group,after adding E2 zyban(OVX+MCT+E2)group(n=6),RI increased significantly,and increase rate(223.3±21.8)%,respectively(n=6,P<0.01)1.1.4 Effect of E2 on pathological changes of PAH in rat model induced by MCTAfter the determination of right cardiac hypertrophy index,paraffin sections of lung tissue were performed and eosin(HE)staining was performed to observe the pathological changes of lung tissues in each group of rats and the thickness of medium membrane of distal pulmonary small blood vesselsResults showed that rats by subcutaneous injection of physiological saline group(n=6)group of distal lung small rat vascular intima complete,smooth muscle cells arranged rules,no inflammatory cell infiltration,full-thickness without thickening.Relatively than Sham+saline(n=6),the OVX+saline(n=6),the OVX+E2+saline group(n=6),immunohistochemical study lung tissue biopsy showed Sham+MCT group(n=6)rat smooth muscle cells and the arrangement of the nuclei is disorder,smooth muscle layer thickening,blood vessel lumen stenosis,pulmonary vascular and more neutrophils in the lung tissue and lymph Infiltration of cells;Compared with Sham+MCT group(n=6),the vascular media thickness of rats in the OVX+MCT group(n=6)was significantly reduced,with a reduction rate of(218.6±20.7)%(n=6,P<0.01).Compared with the OVX+MCT group(n=6),the vascular media thickness of the OVX+MCT+E2 group was significantly increased with an increase rate of(226.6±19.8)%(n=6,P<0.01)after the addition of E2 sustainedrelease tablets,and the pathological changes in the pulmonary small vascular tissue above were significantly worsened1.2 E2 promoted the expression of PFKFB3 protein in the smooth muscle layer of the pulmonary artery in rat models of PAH induced by MCTThe above each rat distal lung small artery was isolated from lung tissue,using microscopic stripped clean tweezers to the outer and inner membrane,frozen exist-80?refrigerator,then separated out good pulmonary arteriolar smooth muscle layer and weighed respectively according to certain proportion to join cracking lung tissue fluid,using mechanical homogenate device for homogenate after placed in the ice,using ultrasonic broken instrument for crushing,centrifugal,return the supernatant and repackaging in 1.5ml centrifuge tube,placed in-80? freezer to save.Through the BCA method after the determination of protein concentration,use of Western The protein expression of PFKFB3 in the distal pulmonary arteriole of rats in each group was detected by blot,Trizol method was used to extract the smooth muscle layer RNA of pulmonary arteriole,and RT-PCR method was used to detect the mRNA expression of PFKFB3 in the distal pulmonary arteriole of rats in each groupThe results showed that the protein expression of PFKFB3 in the distal pulmonary arteriole of Sham+MCT group was significantly increased by Western blot and rt-pcr compared with that of the rats injected with normal saline through the neck,with an increase rate of(151.6±18.6)%(n=6,P<0.01),and the mRNA level of PFKFB3 showed no significant change.Compared with Sham+MCT group,the protein expression of PFKFB3 in the distal pulmonary arteriole of rats in the OVX+MCT group was significantly reduced(152.8±19.6)%(n=6,P<0.01),and there was no significant change in the mRNA level of PFKFB3.Compared with the OVX+MCT group,the protein expression of PFKFB3 in the distal pulmonary arteriole of rats increased significantly after estrogen supplementation(OVX+MCT+E2)(P<0.01),with an increase rate of(268.6±25.7)%(n=6,P<0.01),and the mRNA level of PFKFB3 showed no significant change2.E2 increased the expression of PFKFB3 protein in pulmonary vascular smooth muscle cells2.1 E2 up-regulated PFKFB3 protein expression in pulmonary vascular smooth muscle cells2.1.1 The expression of PFKFB3 protein in pulmonary vascular smooth muscle cells was increased after the treatment of E2 at different concentrations for the same time,and the expression of PFKFB3 mRNA in pulmonary vascular smooth muscle cells was not affected.PASMCs were cultured in vitro and E2 treatment was given to rats with different concentration gradients(1nM-1?M)for 24h.The expression of PFKFB3 protein was observed by Western blot and RT-PCRResults show that:compared with control group,were given different concentrations of E2(1nM-1?M)24 h after processing,can increase PFKFB3 protein expression,increase rate(205.3±28.5)%,respectively(n=3,P<0.01),(225.6±25.7)%(n=3,P<0.01),(180.3±23.5)%(n=3,P<0.01),(165.6±22.7)%(n=3,P<0.01),while the PFKFB3 mRNA expression had no obvious effect2.1.2 The expression of PFKFB3 protein in pulmonary vascular smooth muscle cells was increased after the treatment of E2 at the same concentration at different time,and the expression of PFKFB3 mRNA in pulmonary vascular smooth muscle cells was not affected.PASMCs were cultured in vitro and treated with E2(10nM)for 2h,4h,6h,8h,12h,24h,respectively.The protein expression levels of PFKFB3 were observed by Western blot and mRNA expression levels of PFKFB3 were observed by RT-PCRResults show that:compared with control group,respectively give E2(10nM)after treatment,all can increase PFKFB3 protein expression,increase rate(165.3±28.5)%,respectively(n=3,P<0.01),(180.6±25.7)%(n=3,P<0.01),(220.3±23.5)%(n=3,P<0.01),(265.6±25.7)%(n=3,P<0.01),(320.3±21.5)%(n=3,P<0.01),(150.6±23.7)%(n=3,P<0.01),while the PFKFB3 mRNA expression had no obvious effect(n=3,P<0.01)2.2 E2 upregulated protein expression of PFKFB3 in pulmonary vascular smooth muscle cells via ERaPASMCs were cultured in vitro and treated with E2(10nM),PPT(ER radiation agonist,1nM),DPN(ER radiation agonist,1nM),ICI182780(estrogen receptor inhibitor),MPP(ER radiation inhibitor,1nM),PHTPP(ER radiation inhibitor,1nM),and G15(GPR30 inhibitor,1nM)for 24 h respectively.The expression of PFKFB3 protein was detected by Western blotThe results showed that the expression of PFKFB3 protein could be upregulated by E2 and PPT,respectively(150.3±18.5)%(n=3,P<0.01)and(280.6±21.7)%(n=3,P<0.01),but the upregulated effect of DPN was not obvious.E2 upregulation of PFKFB3 can be inhibited by MPP,but not by PHTPP or G15.The effect of E2 and PPT could be inhibited by ICI 182,780,an estrogen receptor inhibitor(figure 2-2)(n=3,P<0.01).This indicated that ER,rather than ER,was involved in E2 upregulation of PFKFB3 expression2.3 E2 promoted the proliferation and migration of pulmonary vascular smooth muscle cells by upregulating the expression of PFKFB3 proteinPASMCs were cultured in vitro and transfected with PFKFB3 overexpressed plasmid or the corresponding no-load plasmid or treated with PFKFB3 small molecule inhibitor 3PO for 24h,then treated with E2(10nM)for 24h.The proliferation and migration of PASMCs were observed by CCK8 detection kit and cell staining experimentsThe results showed that E2 could promote the proliferation and migration of pulmonary vascular smooth muscle cells.After inhibiting PFKFB3,3PO significantly inhibited the above effects of E2(n=3,P<0.01)In addition,the cells were treated with E2(10nM)for 24h,or the PFKFB3 overexpressed plasmid was transfected at the same time.Compared with the control group,E2 treatment promoted the proliferation and migration of pulmonary artery smooth muscle cells.Overexpression of PFKFB3 can further promote the proliferation and migration of pulmonary vascular smooth muscle cells(n=3,P<0.01)3.The mechanism by which METTL3-mediated E2 up-regulates PFKFB3 protein expression in pulmonary vascular smooth muscle cells3.1 The effect of E2 on the m6A level of PFKFB3 mRNA in pulmonary vascular smooth muscle cellsTotal RNAs were extracted from rat pulmonary artery smooth muscle cells(PASMCs)in vitro after the cells had grown to 90%fusion,and the operation was performed according to the instructions of Magna MeRIPTM m6A Kit:a)cell mRNA was extracted and purified;b)mRNA fragmentation:mRNA was heated at 94? for 6min by a thermal cycling apparatus to break the mRNA into fragments less than 200nt;c)immunoprecipitation:1×IP buffer was used to resuscitate Protein AAG Magnetic d)RNA elution and purification:RNA elution solution was added to the precipitation obtained above,and the supernatant was taken.RNA was purified by RNA purification kit,and was dissolved in DEPC water.The results showed that the m6A methylation level of PFKFB3 mRNA increased after E2(10nM)treatment,with an increase rate of(203.6±20.9)%(n=3,P<0.01)3.2 E2 up-regulated the expressive level of m6A-related protein METTL3 in pulmonary vascular smooth muscle cellsPASMCs were cultured in vitro.PASMCs were E2 treated with different concentration gradient(1nM-1?M)for 24h or E2(10nM)for different time(lh,3h,6h,12h,24h).Expression of m6A related proteins(METTL3,METTL14,FTO,ALKBH5)in PASMCs was observed by Western blotResults showed that in different concentration gradient of E2(1nM-1?M)processing PASMCs after 24h,METTL3 protein expression level increased,increasing the rate(165.3±22.5)%,respectively(n=3,P<0.01),(155.6±15.7)%(n=3,P<0.01),(205.3±21.5)%(n=3,P<0.01),(210.6±23.7)%(n=3,P<0.01);With the same concentration of E2(10 nM)dealing with different time(1h,3h,6h,12h,24h)after METTL3 protein expression level increased,increasing the rate(145.3±28.5)%,respectively(n=3,P<0.01),(155.6±26.7)%(n=3,P<0.01),(148.3±21.6)%(n=3,P<0.01),(205.6±23.8)%(n=3,P<0.01),(195.3±22.5)%However,the expression levels of METTL14,FTO and ALKBH5 were basically unchanged or decreased(n=3,P<0.01)3.3 E2 promoted the expression of METTL3 in the smooth muscle layer of the pulmonary artery in rat models of PAH induced by MCTThe results showed that compared with the rats injected with normal saline through the neck,Western blot and R T-PCR showed that the expression of METTL3 protein and mRNA in the distal pulmonary arterioles of rats in the sham operation group+MCT group were significantly increased(205.623.6)%(n=3,P<0.01)and(165.6±21.9)%(n=3,P<0.01).Compared with the sham operation group+MCT group,the expression of METTL3 protein and mRNA in the distal pulmonary arteriole of rats in the OVX+MCT group were significantly reduced(175.6±20.6)%(n=3,P<0.01)and(195.6±20.8)%(n=3,P<001).Compared with the OVX+MCT group,the protein and mRNA expressions of METTL3 in the distal pulmonary arterioles of rats were significantly increased after estrogen supplementation(OVX+MCT+E2 group),with the increase rates of(285.6±21.6)%(n=3,P<0.01)and(280.6±25.9)%(n=3,P<0.01),respectively3.4 METTL3-mediated E2 up-regulated the level of PFKFB3 mRNA m6A in pulmonary vascular smooth muscle cellsTotal RNAs were extracted from PASMCs of cultured rats and operated according to the instructions of Magna MeRIPTM m6A Kit.After E2 treatment was detected by the m6A RNA methylation immunoprecipitation Kit,m6A methylation of PFKFB3 mRNA by METTL3 was analyzedThe results showed that the PFKFB3 m6A levels in PASMCs of rats with METTL3 knockdown were evaluated by MeRIP and RT-PCR and the decrease rate of PFKFB3 m6A methylation was confirmed,with a decrease rate of(805.6±22.9)%(n=3,P<0.01),and the m6A level of PFKFB3 mRNA was significantly increased after E2 treatment,with an increase rate of(208.6±20.7)%(n=3,P<0.01)3.5 E2 upregulated protein expression of METTL3 in pulmonary vascular smooth muscle cells via ERaPASMCs were cultured in vitro and treated with E2(10nM),PPT(ER radiation agonist,1nM),DPN(ER radiation agonist,1nM),ICI182780(estrogen receptor inhibitor),MPP(ER radiation inhibitor,1nM),PHTPP(ER radiation inhibitor,1nM)and G15(GPR30 inhibitor,1nM)for 24h,respectively.The expression of METTL3 protein was detected by Western blot.The results showed that the expression of METTL3 protein could be upregulated by E2 and PPT,respectively(180.3±18.9)%(n=3,P<0.01)and(240.6±20.7)%(n=3,P<0.01),but the upregulated effect of DPN was not obvious.E2 upregulation of METTL3 can be inhibited by MPP,but not by PHTPP and G15(GPR30 inhibitor,1nM).The effect of E2 and PPT could be inhibited by ICI182,780,an estrogen receptor inhibitor(figure 2-4)(n=3,P<0.01).Therefore,ER?,rather than ER?,played a role in E2 upregulation of METTL3 protein in pulmonary vascular smooth muscle cells.3.6 Effect of METTL3-mediated on promoting protein expression of PFKFB3 by E2 in pulmonary vascular smooth muscle cellsPASMCs were cultured in vitro,transfected with rat negative control virus,METTL3 overexpressed lentivirus for 48h,and METTL3 siRNA for 24h,then treated with E2(10 nM)for 24h.The expression of PFKFB3 protein was detected by Western blot,and the expression of PFKFB3 mRNA was detected by RT-PCR.The results showed that compared with the control group,when lentivirus overexpressed METTL3 with METTL3,E2 up-regulated PFKFB3 protein level increased,with an increase rate of(250.8±22.7)%(n=3,P<0.01).The effect of E2 up-regulated PFKFB3 mRNA expression was basically not affected,while when METTL3 siRNA was used to silence METTL3 expression,the effect of E2 up-regulated PFKFB3 expression was inhibited,but for PFKFB3 MRNA expression also had no significant effect(n=3,P<0.01).Thus,E2 up-regulated PFKFB3 protein expression in pulmonary vascular smooth muscle cells through METTL3(figure 2-6).3.7 E2 can promote the transcriptional activity of METTL3 in pulmonary vascular smooth muscle cells3.3.7.1 the expression of METTL3 mRNA was increased in PASMCs treated with E2 at different concentrations and PASMCs treated with E2 at the same concentration(10nM)at different times.Rat pulmonary artery smooth muscle cells(PASMCs)were cultured in vitro and treated with E2 at different concentration gradients for 24h,or with E2 at 10nM concentration for 1h,3h,6h,12h,and 24h,respectively.The expression of METTL3 mRNA was observed by RT-PCRResults show that:compared with control group,were given different concentrations of E2(1nM-1?M)processing PASMCs 24h increased significantly in rats PASMCs METTL3 mRNA expression,increase rate is respectively:(160.3±23.5)%(n=3,P<0.01),(235.6±24.7)%(n=3,P<0.01),(228.3±21.6)%(n=3,P<0.01),(201.6±23.8)%(n=3,P<0.01),(195.3±22.5)%(n=3,P<0.01);And giving E2(10nM)dealing with different time(1h,3h,6h,12h,24h)after METTL3 mRNA expression of rat PASMCs increased significantly,increase rate(185.3±26.5)%,respectively(n=3,P<0.01),(195.6±24.6)%(n=3,P<0.01),(220.3±21.8)%(n=3,P<0.01),(235.6±22.9)%(n=3,P<0.01),(245.3±21.7)%(n=3,P<0.01).3.3.7.2 PASMCs were inoculated into a 24-well plate and grown to 80%.METTL3 ERE luciferase plasmid(METTL3-luc,750ng),ERE expressing plasmid(pLV-ERE-Luc,750ng)and empty plasmid PGL3(250ng)were transfected into PASMCs,respectively.Then the cells were treated with E2(10nM),PPT(1nM)and ICI(10nM)for 24h,and the fluorescence values of the corresponding luminous units were detected.The results showed that E2 and PPT could significantly increase the transcriptional activity of METTL3,with the increase rates of(285.6±21.7)%(n=3,P<0.01)and(268.6±23.8)%(n=3,P<0.01),respectively,which could be reversed by ICI(figure 2-6).3.8 Inhibition of METTL3 expression on promoting the proliferation and migration of pulmonary vascular smooth muscle cells by E2.Rats in vitro cultivation of PASMCs,transfection METTL3 siRNA or the corresponding Scrambled after siRNA,giving E2(10nM)cells for 24h,through CCK8 detection kits and scratch experiment observation PASMCs proliferation and migration.Results show that:compared with control group,the transfection Scrambled siRNA exert E2 treatment can promote the proliferation and migration of pulmonary artery smooth muscle cells,and transfection METTL3 siRNA,significantly inhibited the E2 treatment after the effect of increasing the pulmonary artery smooth muscle cell proliferation and migration(n=3,P<0.01).3.9 Inhibition of the expression of METTL3 and the effect of overexpression of PFKFB3 on promoting the proliferation and migration of pulmonary vascular smooth muscle cells by E2.PASMCs were cultured in vitro,transfected with rat METTL3 siRNA or transfected with rat METTL3 siRNA at the same time.PFKFB3 overexpressed plasmid was transfected and treated with E2(10nM)for 24h.The proliferation and migration of PASMCs were observed by CCK8 detection kit and cell scratching experiment.Results showed that:compared with control group,the transfection Scrambled siRNA exert E2 treatment can promote the proliferation and migration of pulmonary artery smooth muscle cells,and transfection METTL3 siRNA,inhibit that the effect of E2 promoted pulmonary artery smooth muscle cell proliferation and migration.Covering PFKFB3 protein,can promote the ability of cell proliferationand migration.(n=3,P<0.01)[Conclusion]On the one hand,E2 can promote the development of MCT-induced pulmonary hypertension.On the other hand,E2 promotes the PFKFB3 mRNA m6A methylation by promoting the progressive level of METTL3,promotes protein expression of PFKFB3,and ultimately promotes the proliferation and migration of smooth muscle cells. |
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