Hoxa1通过MEK/ERK信号通路促进低氧下大鼠肺动脉平滑肌细胞增殖、迁移和表型转换的研究

Hoxa1 promoting proliferation,migration and phenotypic transition of rat pulmonary artery smooth muscle cells via MEK/ERK signaling pathway

背景 低氧性肺动脉高压(hypoxic pulmonary arterial hypertension,HPH)是一种以血管阻力持续性升高和血管重塑为特征的进展性疾病。研究表明Hoxa1参与血管生成过程,但Hoxa1在调控肺动脉平滑肌细胞(pulmonary artery smooth muscle cells,PASMCs)中的作用尚不明确。目的 本研究旨在确定Hoxa1对PASMCs增殖、迁移和表型转化的调控作用。方法 使用Ⅱ型胶原酶消化分离培养原代大鼠PASMCs。采用1%低氧处理细胞构建低氧诱导的细胞模型,并检测24 h、48 h、72 h低氧处理的PASMCs中Hoxa1 mRNA和蛋白表达水平。采用干扰或过表达Hoxa1慢病毒下调或上调细胞中Hoxa1表达。慢病毒干扰实验分为常氧组(Normoxia)、低氧组(Hypoxia)、低氧空病毒组(Hypoxia+shRNA-NC)、低氧Hoxa1干扰病毒组(Hypoxia+shRNA-Hoxa1);慢病毒过表达实验分为对照组(Control)、过表达空病毒组(LV-NC)、过表达Hoxa1组(LV-Hoxa1)。采用EdU检测细胞增殖能力;RT-qPCR检测Hoxa1 mRNA表达;Western blot检测Hoxa1、表型转化标志蛋白、MEK/ERK信号通路蛋白表达;免疫荧光检测Hoxa1、α-actin表达;免疫共沉淀实验确定Hoxa1与MEK的相互作用。结果 成功分离培养PASMCs,并鉴定细胞表达α-SMA。低氧诱导下PASMCs中Hoxa1表达上调(P<0.05)。低氧下Hoxa1沉默抑制PASMCs增殖和迁移,而常氧下Hoxa1过表达促进PASMCs增殖和迁移(P均<0.05)。低氧下沉默Hoxa1导致Hypoxia+shRNA-Hoxa1组收缩型标志蛋白表达升高,合成型标蛋白表达降低(P<0.05)。而在常氧下过表达Hoxa1导致LV-Hoxa1组收缩型标志蛋白降低,合成型标志蛋白升高(P均<0.05)。此外,Hoxa1表达增加促进MEK/ERK信号通路激活。免疫共沉淀结果显示,Hoxa1与MEK存在相互作用。结论 Hoxa1可能通过激活MEK/ERK通路促进PASMCs增殖、迁移和表型转化。

Background Hypoxic pulmonary arterial hypertension(HPH)is a progressive disease characterized by persistent elevation of vascular resistance and vascular remodeling.Studies have shown that Hoxa1 is involved in the angiogenic process,but the role of Hoxa1 in regulating pulmonary artery smooth muscle cells(PASMCs)is unclear.Objective To determine the role of Hoxa1 in the regulation of proliferation,migration and phenotypic transformation on PASMCs.Methods Primary rat PASMCs were isolated and cultured using type II collagenase digestion.Hypoxia-induced cell model was constructed using 1%hypoxia-treated cells,and Hoxa1 mRNA and protein expression levels were examined in PASMCs cells treated with hypoxia for 24 h,48 h and 72 h.Lentiviral interference experiments were divided into Normoxia group,Hypoxia group,Hypoxia empty virus group(Hypoxia+shRNA-NC),and Hypoxia Hoxa1 interference virus group(Hypoxia+shRNA-Hoxa1).Lentiviral overexpression experiments were divided into control group,overexpression empty virus group(LV-NC),and overexpression Hoxa1 group(LV-Hoxa1).EdU was used to detect cell proliferation.Hoxa1 mRNA expression was detected by RT-qPCR.Western blot was used to detect Hoxa1,phenotypic transformation marker protein and MEK/ERK signaling pathway protein.The expression of Hoxa1 andα-actin was detected by immunofluorescence.The interaction between Hoxa1 and MEK was determined by co-immunoprecipitation assay.Results PASMCs were isolated and cultured successfully,andα-SMA expression was identified.Hoxa1 expression was upregulated in PASMCs under hypoxia induction(P<0.05).Hoxa1 silencing under hypoxia inhibited the proliferation and migration of PASMCs,whereas Hoxa1 overexpression under normoxia promoted the proliferation and migration of PASMCs(all P<0.05).Hoxa1 silencing under hypoxia led to increased expression of contractile marker proteins and decreased expression of synthetic marker proteins in the Hypoxia+shRNA-Hoxa1 group(P<0.05).In contrast,Hoxa1 overexpression under normoxia led to a decrease in contractile marker proteins and an increase in synthetic marker proteins in the LV-Hoxa1 group(all P<0.05).In addition,increased Hoxa1 expression promoted activation of the MEK/ERK signaling pathway.Immunoprecipitation results showed that Hoxa1 interacted with MEK.Conclusion Hoxa1 may promote proliferation,migration and phenotypic transformation of PASMCs through activation of the MEK/ERK pathway.