The Mechanisms Of M~6A Methyltransferase METTL3 In Hypoxic Pulmonary Vascular Remodeling By Targeting PTEN Via YTHDF2

Part Ⅰ Expression of METTL3 in lung tissue isolated from hypoxic pulmonary hypertension rats and hypoxia-induced pulmonary artery smooth muscle cells and the effect of METTL3 on pulmonary artery smooth muscle cells proliferation and migrationObjective:Hypoxic pulmonary hypertension(HPH)is a clinically common chronic cardiopulmonary disease characterized by irreversible pulmonary vascular remodeling(PVR).It is the leading cause of developing pulmonary heart disease and death among patients with chronic pulmonary disease.Hypoxia-induced hyperproliferation of pulmonary artery smooth muscle cells(PASMCs)plays a critical role in PVR.N~6-methyladenosine(m~6A),the most abundant endogenous RNA modification type in mammals,is widely found in a variety of messenger RNA(m RNA)and non-coding RNA.Recent studies have demonstrated that m~6A plays a substantial role in various cardiovascular diseases(CVDs),but the role m~6A modifications in HPH and the underlying mechanisms remain largely unexplored.This section aims to detect the expression of key m~6A regulators in pulmonary arteries of HPH rats and hypoxia-induced PASMCs,including methyltransferase-like 3(METTL3),methyltransferase-like 14(METTL14),Wilms tumour 1-associated protein(WTAP),Fat mass and obesity-associated protein(FTO)and Alkb homolog 5(ALKBH5),and further explore the effect of m~6A methyltransferase METTL3 on the proliferation and migration potential of PASMCs.Methods:Sprague-Dawley(SD)rats were placed in a hypoxic chamber to establish an HPH rat model.Right ventricular systolic pressure(RVSP)was measured by the right heart catheterization method.The right ventricle(RV),left ventricle(LV)and septum(S)were extracted and weighted.Then,right ventricular hypertrophy index(RVHI)was calculated through the formula RVHI=RV/(LV+S).The expression of the key m~6A core methyltransferase METTL3,METTL14,WTAP and demethylase FTO,ALKBH5m RNA and protein in the pulmonary artery tissues of HPH rats were detected by q-PCR and Western blot.Primary PASMCs were isolated,extracted and cultured by tissue adherent method and identified by immunofluorescence staining.PASMCs were cultured in a hypoxic incubator for 12 h and 24 h,respectively.m~6A content of PASMCs in the normoxic and hypoxic groups was measured using the Epi Quik m~6A RNA Methylation Assay Kit.The m RNA and protein expression of METTL3,METTL14,WTAP and demethylase FTO,ALKBH5 in PASMCs in hypoxic and normoxic groups were detected by q-PCR and Western blot.Small interfering RNA(si RNA)was used to inhibit METTL3 expression in PASMCs,and the proliferation and migration ability of PASMCs were assessed by CCK-8,Ed U cell proliferation,and transwell migration assay.Results:(1)HPH rat model was successfully established,chronic hypoxia led to a significant increase in right ventricular systolic pressure(RVSP)and right ventricular hypertrophy index(RVHI)in rats.(2)Compared with the normoxic group,pulmonary arteries of HPH rats showed significant pulmonary vascular remodeling characterized by vascular wall thickening and luminal narrowing pathological changes.(3)In Comparison with the control group,the key methyltransferase METTL3 m RNA and protein were significantly upregulated in the pulmonary arteries from rat models with HPH.(4)Hypoxia contributed to increased m~6A content in PASMCs compared with PASMCs cultured under normoxic condition.(5)Both METTL3 m RNA and protein levels were significantly upregulated in PASMCs exposed to hypoxia,the expression of demethylase FTO and ALKBH5 were mildly downregulated compared to the normoxic control group,and METTL14 and WTAP levels were statistically similar between the two groups.(6)The proliferation and migration levels of PASMCs were significantly increased after hypoxia exposure.Silencing METTL3 effectively inhibited the proliferation and migration potential of PASMCs both under normoxic and hypoxic conditions.Conclusion:METTL3 expression was significantly increased in the pulmonary arteries from HPH rats,and no statistically differences were found in the expression of other methyltransferases and demethylases in the pulmonary arteries of control and HPH rats.Hypoxia exposure significantly upregulated m~6A content and METTL3 expression in PASMCs.METTL3 inhibition attenuated hypoxia-induced PASMCs proliferation and migration.It is suggested that METTL3-mediated m~6A modifications might be involved in the development of HPH.Part Ⅱ Effect of METTL3/YTHDF2/PTEN axis on hypoxia-induced proliferation and migration of pulmonary artery smooth muscle cells and the underlying mechanismsObjective: The oncogene phosphatase and tensin homolog(PTEN),an upstream inhibitor of the PI3K/Akt pathway,plays a vital role in the regulation of cell proliferation,apoptosis and other biological behaviors.Decreased PTEN expression in PASMCs is a key driving factor underlying hypoxia-induced PASMCs overproliferation.PTEN inhibition contributes to increased PASMCs proliferation and irreversible PVR.The first part of the study showed that METTL3 and m6 A levels were significantly upregulated in HPH both in vitro and in vivo,and silencing METTL3 inhibited hypoxia-induced proliferation and migration of PASMCs.However,it remains unknown on the underling mechanisms of METTL3 mediated m6 A RNA modification in regulation of PASMCs biological behaviors.This section aims to investigate whether METTL3 could regulate PTEN m RNA,and then affect the proliferation and migration of PASMCs and explore the underlying molecular mechanisms.Methods: Primary PASMCs were isolated and cultured for this part of the study.A sequence-based N6-methyladenosine modification site predictor(SRAMP)online website was used to predict the m6 A modification sites of PTEN m RNA.The HPH rat model was constructed by incubating SD rats in a hypoxic incubator for 4 weeks,and the PASMCs were incubated in a hypoxic incubator(O2=1%,CO2=5%,N2=94%)to construct the hypoxic cell model.PASMCs were transfected with METTL3 overexpression plasmid and si METTL3,then Western blot was used to detect the expression of PTEN m RNA and protein.RNA immunoprecipitation-PCR(RIP-PCR)was performed to verify the combination between METTL3 and PTEN m RNA.The expression of YT521-B homology domain family 2(YTHDF2)in pulmonary arteries of HPH rats and hypoxic PASMCs was detected by q-PCR and Western blot.After transfection with METTL3 overexpression plasmid and/or si YTHDF2 in PASMCs,qPCR,Western blot,and actinomycin D half-life assay were performed to detect PTEN expression and PTEN m RNA half-life.Western blot was used to detect the expression of PI3K/Akt pathway associated proteins and PCNA.CCK-8,Ed U cell proliferation assay and transwell migration assay were used to assess the proliferation and migration ability of PASMCs,respectively after transfection with si METTL3,si PTEN,and the inhibitor of PI3K/Akt signaling pathway LY294002.Results:(1)A sequence-based N6-methyladenosine modification site predictor(SRAMP)online site prediction showed that there were multiple possible m6 A modification sites for PTEN m RNA,and multiple predicted sites were with high confidence.(2)Overexpression of METTL3 led to downregulated expression of PTEN m RNA and protein,while METTL3 inhibition caused upregulated PETN m RNA and protein levels.(3)METTL3 directly interacts with PTEN m RNA.(4)YTHDF2 expression was significantly upregulated in pulmonary artery tissues of HPH rats and hypoxia-induced PASMCs compared to controls.(5)Overexpression of METTL3 promoted PTEN m RNA degradation and reduced its stability,resulting in a decrease in PTEN expression.However,silencing YTHDF2 could reverse the decreased PTEN expression caused by METTL3 overexpression.(6)Silencing METTL3 could inhibit hypoxia-induced proliferation and migration of PASMCs,and PTEN inhibition could reverse the decreased cell proliferation ability caused by METTL3 deletion.(7)Silencing METTL3 could attenuate the hypoxia-induced enhanced proliferation and migration potential,and p-PI3 K,p-Akt,and PCNA expression in PASMCs.Compared with hypoxia+si METTL3 group,the proliferation and migration potential and PCNA expression in PASMCs were increased in the hypoxia+si METTL3+si PTEN group.PI3K/Akt pathway inhibitor LY294002 reversed the increased PASMCs proliferation,and migration ability,and PCNA expression induced by silencing PTEN.Conclusion: METTL3 directly interacts with PTEN m RNA and affects its stability and protein expression.Hypoxia upregulated the expression of the m6A-binding protein YTHDF2.In hypoxia induced PASMCs,YTHDF2 recognized and interacts with PTEN m RNA,promoting the degradation of PTEN m RNA and decreased its m RNA stability,leading to reduced PTEN expression.Reduced PTEN contributed to activation of PI3K/Akt pathway,promoting the proliferation and migration potential of PASMCs.METTL3/YTHDF2/PTEN signaling axis plays an important role in hypoxia-induced proliferation and migration of PASMCs through activating its downstream PI3K/Akt signaling pathway.Part Ⅲ Exploring the role of METTL3 in hypoxic pulmonary vascular remodeling in vivoObjective: In this section,METTL3 knockdown mice model was constructed through intranasal pathway of administration of adeno-associated virus(AAV)-packaged sh METTL3(AAV-GP-1-METTL3-Mus-221),and then we assess whether METTL3 inhibition in mouse pulmonary arteries could effectively improve chronic hypoxiainduced HPH and PVR.Methods: Healthy male C57BL/6J mice were randomly divided into the following three groups: normoxia+AAV-NC group,hypoxia+AAV-NC group,and hypoxia+AAVGP-1-METTL3 group.The mice in the hypoxic group were kept in a fully automated normoxic chamber for 4 weeks to construct the HPH model,while the mice in the normoxic group were kept in a normoxic and normobaric environment.AAV-GP-1-METTL3-Mus-221 was packaged in an AAV vector with sh RNA expression driven by the U6 promoter.Mice in the hypoxia+AAV-GP-1-METTL3 group were given 50 μl of AAV-GP-1-METTL3-Mus-221 via intranasal pathway,while NC group mice received the same dose of AAV-NC intranasally,followed by kept in a fully automated normobaric hypoxic chamber for 4 weeks 2 weeks days later.The expression of METTL3 m RNA and protein in the pulmonary arteries of each group were detected by q-PCR,Western blot,and immunofluorescence.RVSP was measured by right heart catheterization,and RVHI was calculated.HE staining was used to assess the PVR in each group.PCNA and PI3K/Akt signaling associated proteins were detected through Western blot.Results:(1)Chronic hypoxia significantly upregulated METTL3 level in the pulmonary arteries of HPH mice,and administration of AAV-GP-1-METTL3-Mus-221 effectively downregulated METTL3 expression in the pulmonary artery tissues of mice.(2)Compared with the normoxia+AAV-NC group,RVSP and RVHI were significantly increased in the hypoxia+AAV-NC group,and RVSP and RVHI were remarkably decreased in the hypoxia+AAV-GP-1-METTL3 group compared with the Hypoxia+AAV-NC group.(3)Compared with the Normoxia+AAV-NC group,the Hypoxia+AAV-NC group displayed significant thickening of the pulmonary arterial wall and lumen narrowing.In contrast,compared with the Hypoxia+AAV-NC group,the Hypoxia+AAV-GP-1-METTL3 group mice manifested a thinner pulmonary artery wall and a wider lumen.(4)Compared with the Normoxia+AAV-NC group,PTEN expression was significantly reduced in the pulmonary artery tissues of mice in the Hypoxia+AAV-NC group,while the expression of PCNA,p-PI3 K and p-Akt were significantly increased.Compared with the Hypoxia+AAV-NC group,PTEN expression was significantly increased,and p-PI3 K and p-Akt expression were significantly decreased in the mice pulmonary arteries in the hypoxia+AAV-GP-1-METTL3 group.Conclusion: METTL3 expression was significantly upregulated in the pulmonary arteries of HPH mice.Inhibition of METTL3 expression in the pulmonary arteries of HPH mice effectively decreased the elevated RVSP,right ventricular hypertrophy,and alleviated PVR caused by chronic hypoxia.Downregulation of METTL3 expression in pulmonary arteries could reverse decreased PTEN expression and increased PCNA,pPI3 K and p-Akt expression in mice pulmonary arteries induced by hypoxia.Collectively,chronic hypoxia significantly upregulated METTL3 expression in the pulmonary arteries of HPH mice and promoted a decrease of PTEN expression,contributing to HPH and PVR through activation of the PI3K/Akt pathway.