基因沉默SIRT7抑制缺氧诱导的肺动脉平滑肌细胞增殖及迁移

Silencing of SIRT7 gene inhibits hypoxia-induced proliferation and migration of pulmonary artery smooth muscle cells

目的研究沉默信息调节因子2相关酶类7(silent information regulator 2 related enzyme 7,SIRT7)对低氧诱导的人肺动脉平滑肌细胞(human pulmonary artery smooth muscle cells,HPASMCs)增殖和迁移的调控作用,并探讨可能的分子作用机制。方法体外培养HPASMCs建立低氧模型。通过慢病毒介导的短发卡RNA(short hairpin RNA,shRNA)干扰技术沉默SIRT7在HPASMCs中的表达。实验分为4组:常氧对照组、低氧组、低氧+LV-sh-Ctrl组和低氧+LV-sh-SIRT7组。低氧+LV-sh-Ctrl组HPASMCs感染对照重组腺病毒LV-sh-Ctrl,然后进行低氧处理。低氧+LV-sh-SIRT7组HPASMCs感染表达SIRT7 shRNA的重组腺病毒LV-sh-SIRT7,然后进行低氧处理。采用实时定量PCR(real-time quantitative PCR,RT-qPCR)检测SIRT7的mRNA表达水平。采用Western blotting检测SIRT7、转化生长因子-β1(transforming growth factor-β1,TGF-β1)、转化生长因子β受体Ⅰ(transforming growth factor-βreceptorⅠ,TβRⅠ)、Smad2和Smad3的蛋白表达量。采用细胞计数试剂盒-8(cell counting kit-8,CCK-8)和5-乙炔基-2′-脱氧尿苷(5-ethynyl-2′-deoxyuridine,EdU)实验检测细胞增殖。采用流式细胞术检测细胞凋亡。采用Transwell实验检测细胞迁移。结果与常氧对照组比较,低氧组和低氧+LV-sh-Ctrl组SIRT7表达水平显著升高(P<0.01),细胞增殖和迁移水平显著提高(P<0.01),细胞凋亡率显著降低(P<0.05);与低氧组和低氧+LV-sh-Ctrl组比较,低氧+LV-sh-SIRT7组SIRT7表达水平显著降低(P<0.01),细胞增殖和迁移水平显著下降(P<0.01),细胞凋亡率显著增加(P<0.01)。与常氧对照组比较,低氧组和低氧+LV-sh-Ctrl组TGF-β1、TβRⅠ、p-Smad2和p-Smad3的蛋白表达量显著增加(P<0.01);与低氧组和低氧+LV-sh-Ctrl组比较,低氧+LV-sh-SIRT7组TGF-β1、TβRⅠ、p-Smad2和p-Smad3的蛋白表达量显著减少(P<0.01)。结论基因沉默SIRT7通过调控TGF-β1/Smad信号通路抑制缺氧诱导的HPASMCs增殖和迁移。

Objective To investigate the role of silent information regulator 2 related enzyme 7(SIRT7)in mediating the proliferation and migration of human pulmonary artery smooth muscle cells(HPASMCs)induced by hypoxia and explore the potential underlying mechanism.Methods HPASMCs were cultured in vitro to establish a hypoxia model.Lentivirus-mediated short hairpin RNA(shRNA)interfering technology was used to silence SIRT7 expression in HPASMCs.HPASMCs were divided into four groups:normoxic control group,hypoxia group,hypoxia+LV-sh-Ctrl group and hypoxia+LV-sh-SIRT7 group.HPASMCs in hypoxia+LV-sh-Ctrl group were infected with the control recombinant lentivirus(LV-sh-Ctrl)and then subjected to hypoxia treatment.HPASMCs in hypoxia+LV-sh-SIRT7 group were infected with the recombinant lentivirus expressing SIRT7 shRNA(LV-sh-SIRT7)and then subjected to hypoxia treatment.The mRNA expression of SIRT7 was measured by real-time quantitative PCR(RT-qPCR).The protein levels of SIRT7,transforming growth factor-β1(TGF-β1),transforming growth factor-βreceptorⅠ(TβRⅠ),Smad2 and Smad3 were measured by Western blotting.Cell proliferation was assessed by cell counting kit-8(CCK-8)and 5-ethynyl-2′-deoxyuridine(EdU)assays.Cell apoptosis was evaluated by flow cytometry.Cell migration was examined by Transwell assay.Results Compared with normoxic control group,the expression of SIRT7 was significantly increased in hypoxia group and hypoxia+LV-sh-Ctrl group(P<0.01),the cell proliferation and migration abilities were significantly upregulated(P<0.01),and the apoptotic rate was significantly decreased(P<0.05).Compared with hypoxia group and hypoxia+LV-sh-Ctrl group,the expression of SIRT7 was significantly reduced(P<0.01),the cell proliferation and migration abilities were significantly downregulated(P<0.01),and the apoptotic rate was significantly increased in hypoxia+LV-sh-SIRT7 group(P<0.01).Compared with normoxic control group,the protein levels of TGF-β1,TβRⅠ,p-Smad2 and p-Smad3 were significantly upregulated in hypoxia group and hypoxia+LV-sh-Ctrl group(P<0.01).Compared with hypoxia group and hypoxia+LV-sh-Ctrl group,the protein levels of TGF-β1,TβRⅠ,p-Smad2 and p-Smad3 were significantly decreased in hypoxia+LV-sh-SIRT7 group(P<0.01).Conclusion SIRT7 gene silencing can inhibit hypoxia-induced proliferation and migration of HPASMCs by regulating TGF-β1/Smad signaling pathway.