CircRNA-Tbpl1海绵吸附miR-214-3p调控自噬在低氧性肺动脉高压血管重塑中的作用及其机制

The role and mechanism of CircRNA-Tbpl1 sponge adsorbing miR-214-3p to regulate autophagy in vascular remodeling in hypoxic pulmonary hypertension

目的 探讨CircRNA-Tbpl1通过海绵吸附miR-214-3p调控自噬在低氧性肺动脉高压(HPH)血管重塑中的作用,并分析其机制。方法 12只8周龄C57BL/6雄性小鼠根据随机数字表法分为两组:对照组和HPH组,每组6只,通过低氧构建HPH模型小鼠,观察两组小鼠血流动力学差异,HE染色检测肺组织病理学改变,蛋白免疫印迹(Western blot)检测选择性自噬接头蛋白(p62)、微管相关蛋白1轻链3(LC3)和重组人自噬效应蛋白(Beclin-1)水平,实时荧光定量PCR(qRT-PCR)检测CircRNA-Tbpl1和miR-214-3p表达水平。分离小鼠原代肺动脉平滑肌细胞(PASMCs)并进行高、低氧分组培养,低氧处理PASMCs,细胞计数试剂盒-8(CCK-8)检测细胞活性,Western blot检测自噬相关蛋白表达,qRT-PCR检测CircRNA-Tbpl1的相对表达水平。通过转染和自噬诱导剂雷帕霉素干预,分析CircRNA-Tbpl1与PASMCs自噬的相关性。通过共转染shCircRNA-Tbpl1和miR-214-3p抑制物,分析CircRNA-Tbpl1/miR-214-3p对低氧诱导的PASMCs自噬和细胞增殖活性的影响。结果 与对照组小鼠比较,HPH组小鼠的右心室收缩压(RVSP)[(39.50±3.69)mmHg比(21.00±3.42)mmHg,P<0.01]、右心室肥厚指数(RVHI)[(0.43±0.05)比(0.24±0.03),P<0.01]显著增加,且HPH组小鼠肺血管出现明显的中膜肥大和外膜增生,肺血管重塑。同时,HPH组小鼠的肺组织中LC3Ⅱ、Beclin-1表达水平显著上调,p62表达下调,CircRNA-Tbpl1水平[(2.99±0.28)比(1.00±0.15),P<0.01]升高,miR-214-3p水平[(0.49±0.28)比(1.00±0.11),P<0.01]降低。与高氧组比较,低氧组PASMCs细胞增殖活力[(152.02±7.81)%比(100.00±2.08)%,P<0.05]增加,LC3Ⅱ和Beclin-1表达水平上调,p62表达下调,CircRNA-Tbpl1水平[(2.84±0.34)比(1.00±0.12),P<0.01]升高,而miR-214-3P水平[(0.52±0.07)比(1.00±0.15),P<0.01]降低。此外还发现,下调CircRNA-Tbpl1可抑制低氧诱导的PASMCs自噬,减弱细胞增殖活性[(70.23±7.30)%比(100.00±4.56)%,P<0.05]。双荧光素酶报告基因检测结果显示,CircRNA-Tbpl1海绵吸附miR-214-3p。与sh-NC组比较,sh-CircRNA-Tbpl1组的自噬水平减弱,细胞增殖活力降低[(71.13±8.17)%比(100.00±5.32)%,P<0.05]。与sh-CircRNA-Tbpl1+miRNA-NC组比较,sh-CircRNA-Tbpl1+miR-214-3p抑制物组的自噬水平升高,细胞增殖活力增强[(85.78±5.37)%比(69.80±6.34)%,P<0.05]。结论CircRNA-Tbpl1可能通过海绵吸附miR-214-3p抑制低氧诱导的自噬,从而缓解HPH血管重塑。

Objective To investigate the role of CircRNA-Tbpl1 in regulating autophagy in vascular remodeling in hypoxic pulmonary hypertension(HPH)through sponge-like adsorption of miR-214-3p,and to analyze it mechanism.Methods Twelve 8-week-old C57BL/6 male mice were divided into two groups according to the random number table method:the control group and the HPH group,with 6 mice in each group.An HPH mouse model was induced by hypoxia.The differences in hemodynamics between the two groups were observed.Pathological changes in lung tissue were assessed using HE staining.Selective autophagy adapter protein(p62),microtubule-associated protein 1 light chain 3(LC3),and recombinant human autophagy effect Protein(Beclin-1)levels were determined using Western blot.The expression levels of CircRNA-Tbpl1 and miR-214-3p were measured using real-time fluorescence quantitative PCR(qRT-PCR).Mouse primary pulmonary artery smooth muscle cells(PASMCs)were isolated from mice and cultured under hypoxia and hyperoxia conditions.PASMCs were subjected to hypoxia treatment,and cell viability was assessed using a cell counting kit-8(CCK-8).Western blot was used to detect the expression of autophagy-related proteins and qRT-PCR was used to measure the relative expression levels of CircRNA-Tbpl1.The correlation between CircRNA-Tbpl1 and autophagy in PASMCs was analyzed through transfection and intervention with the autophagy inducer rapamycin.By co-transfecting sh-CircRNA-Tbpl1 and miR-214-3p inhibitors,the effects of CircRNA-Tbpl1/miR-214-3p on hypoxia-induced autophagy and cell proliferation activity of PASMCs were analyzed.Results Compared with the control group,mice in the HPH group exhibited significantly increased right ventricular systolic pressure[(39.50±3.69)mmHg vs.(21.00±3.42)mmHg,P<0.01]and right ventricular hypertrophy index[(0.43±0.05)vs.(0.24±0.03),P<0.01].Additionally,the pulmonary blood vessels of mice in the HPH group showed obvious medial hypertrophy and adventitial hyperplasia,indicating pulmonary blood vessel remodeling.At the same time,mice in the HPH group exhibited significantly up-regulated expression levels of LC3 Ⅱ and Beclin-1,and down-regulated expression levels of p62.Furthermore,the level of CircRNA-Tbpl1 was significantly increased[(2.99±0.28)vs.(1.00±0.15),P<0.01],while the level of miR-214-3p was significantly decreased[(0.49±0.28)vs.(1.00±0.11),P<0.01].Compared with the hyperoxia group,PASMCs in the hypoxia group showed significantly increased cell proliferation activity[(152.02±7.81)vs.(100.00±2.08)%,P<0.05],accompanied by up-regulated the expression levels of LC3 Ⅱ and Beclin-1,and down-regulated expression of p62.Moreover,the level of CircRNA-Tbpl1 was significantly increase[(2.84±0.34)vs.(1.00±0.12),P<0.01],while the level of miR-214-3P was significantly decreased[(0.52±0.07)vs.(1.00±0.15),P<0.01].In addition,it was also found that down-regulation of CircRNA-Tbpl1 could inhibit hypoxia-induced autophagy in PASMCs and weaken cell proliferation activity[(70.23±7.30)vs.(100.00±4.56)%,P<0.05].Dual-luciferase reporter gene detection results showed that CircRNA-Tbpl1 acted as a sponge to adsorb miR-214-3p.Compared with the sh-NC group,the autophagy level of the sh-CircRNA-Tbpl1 group was weakened and the cell proliferation activity was reduced[(71.13±8.17)vs.(100.00±5.32)%,P<0.05].Furthermore,compared with the sh-CircRNA-Tbpl1+miRNA-NC group,the autophagy level of the sh-CircRNA-Tbpl1+miR-214-3p inhibitor group was increased,and the cell proliferation activity was enhanced[(85.78±5.37)vs.(69.80±6.34)%,P<0.05].Conclusion CircRNA-Tbpl1 may inhibit hypoxia-induced autophagy by sponging miR-214-3p,thereby alleviating vascular remodeling in HPH.