摘要
目的探讨长链非编码RNA-Ang362(lncRNA-Ang362)在妊娠期高血压(PIH)中的表达及其对血管平滑肌细胞(VSMC)增殖、迁移的影响。方法收集2017年7月-2020年12月荆州市中心医院妇产科收治的PIH患者180例为实验组,同期180名正常孕妇为对照组。体外培养人主动脉VSMC,使用Lipofectamine 3000将用于lncRNA-Ang362和第10号染色体缺失性磷酸酶-张力蛋白同源物基因(PTEN)沉默的小干扰RNA(si-Ang362、si-PTEN)及其相应的阴性对照载体分别转染至细胞,并分为对照组、血小板衍生生长因子(PDGF)-BB组、si-NC组、Ang362沉默组、Ang362沉默+si-PTEN-NC组、Ang362沉默+si-PTEN组;除对照组外,其余组均给予20 ng/mL的PDGF-BB处理。RT-qPCR检测细胞中lncRNA-Ang362和PTEN、增殖细胞核抗原(PCNA)、基质金属蛋白酶2(MMP-2)、基质金属蛋白酶9(MMP-9)的mRNA表达;Western blot检测细胞中PTEN/蛋白激酶B(AKT)/哺乳动物雷帕霉素靶蛋白(mTOR)通路相关蛋白[PTEN、磷脂酰肌醇3-激酶(PI3K)、磷酸化PI3K(p-PI3K)、AKT、磷酸化AKT(p-AKT)、mTOR、磷酸化mTOR(p-mTOR)]的表达。结果实验组血浆中lncRNA-Ang362的表达水平[(1.62±0.21)vs.(1.00±0.00)]显著高于对照组,差异有统计学意义(t=39.610,P<0.001)。与对照组比较,PDGF-BB组细胞中lncRNA-Ang362表达水平、VSMC增殖活力、划痕愈合率和PCNA、MMP-2和MMP-9 mRNA水平以及p-PI3K/PI3K、p-AKT/AKT和p-mTOR/mTOR比值显著升高,PTEN mRNA和PTEN蛋白水平显著降低,差异均有统计学意义(P均<0.05);与si-NC组比较,Ang362沉默组细胞中lncRNA-Ang362表达水平、VSMC增殖活力、划痕愈合率和PCNA、MMP-2和MMP-9 mRNA水平以及p-PI3K/PI3K、p-AKT/AKT和p-mTOR/mTOR比值显著降低,PTEN mRNA和PTEN蛋白水平显著升高,差异均有统计学意义(P均<0.05);与Ang362沉默+si-PTEN-NC组比较,Ang362沉默+si-PTEN组VSMC增殖活力、划痕愈合率和PCNA、MMP-2、MMP-9 mRNA水平以及p-PI3K/PI3K、p-AKT/AKT和p-mTOR/mTOR比值显著升高,PTEN mRNA和PTEN蛋白水平显著降低,差异均有统计学意义(P均<0.05)。结论lncRNA-Ang362在PIH中高表达,并可能通过下调PTEN,激活PI3K/AKT/mTOR信号通路,促进VSMC增殖、迁移,进而参与PIH的发生发展。
Objective To investigate the expression of long non-coding RNA-Ang362(lncRNA-Ang362)in pregnancy-induced hypertension(PIH)and its effects on the proliferation and migration of vascular smooth muscle cells(VSMC).Methods During July 2017 to December 2020,180 cases of PIH patients admitted to the Department of Obstetrics and Gynecology in our hospital were collected as the experimental group and 180 normal pregnant women as the control group.Human aortic VSMC were cultured in vitro,and using lipofectamine 3000,they were transfected with small interfering RNA(si-Ang362,si-PTEN)and the corresponding negative control vectors which were used for lncRNA-Ang362 and phosphatase and tensin homolog deleted on chromosome 10(PTEN)silencing,and then they were divided into control group,platelet-derived growth factor(PDGF)-BB group,si-NC group,Ang362 silence group,Ang362 silence+si-PTEN-NC group,and Ang362 silence+si-PTEN group;except for the control group,all other groups were treated with PDGF-BB at 20 ng/mL.RT-qPCR was used to detect the mRNA expression of lncRNA-Ang362 and PTEN,proliferating cell nuclear antigen(PCNA),matrix metalloproteinase 2(MMP-2)and matrix metalloproteinase 9(MMP-9)in cells;western blot was used to detect the expression of PTEN/protein kinase B(AKT)/mammalian target of rapamycin(mTOR)pathway related proteins[PTEN,phosphatidylinositol 3-kinase(PI3K),phosphorylated-PI3K(p-PI3K),AKT,phosphorylated-AKT(p-AKT),mTOR,phosphorylated-mTOR(p-mTOR)]in cells.Results The expression level of lncRNA-Ang362 in the plasma of PIH patients[(1.62±0.21)vs.(1.00±0.00)]was significantly higher than that of normal pregnant women,and the difference was statistically significant(t=39.610,P<0.05).Compared with the control group,the expression level of lncRNA-Ang362,the proliferation activity of VSMC,the scratch healing rate,the mRNA levels of PCNA,MMP-2 and MMP-9,and the ratios of p-PI3K/PI3K,p-AKT/AKT and p-mTOR/mTOR in the PDGF-BB group were significantly increased,and the levels of PTEN mRNA and PTEN protein were significantly decreased,with statistical significance(all P<0.05);compared with the si-NC group,the expression level of lncRNA-Ang362,proliferation activity of VSMC,scratch healing rate,mRNA levels of PCNA,MMP-2 and MMP-9,p-PI3K/PI3K,p-AKT/AKT and p-mTOR/mTOR ratio in the Ang362 silting group were significantly decreased,PTEN mRNA and PTEN protein levels were significantly increased,and the differences were statistically significant(all P<0.05);compared with Ang362 silencing+si-PTEN-NC group,the proliferative activity,scratch healing rate,mRNA levels of PCNA,MMP-2 and MMP-9,p-PI3K/PI3K,p-AKT/AKT and p-mTOR/mTOR ratio of VSMC were significantly increased in Ang362+si-PTEN group,while the levels of PTEN mRNA and PTEN protein were significantly decreased,the differences were statistically significant(all P<0.05).Conclusion LncRNA-Ang362 was highly expressed in PIH,it might down-regulate PTEN and activate PI3K/AKT/mTOR signaling pathway to promote proliferation and migration of VSMC,and also participate in the occurrence and development of PIH.
作者
梅小琴
陈耀光
MEI Xiaoqin;CHEN Yaoguang(Department of Obstetrics and Gynecology,Jingzhou Central Hospital,Jingzhou,Hubei 434022,China;Department of Laboratory Medicine,Jingzhou Central Hospital,Jingzhou,Hubei 434022,China)
出处
《热带医学杂志》
CAS
2023年第7期925-931,共7页
Journal of Tropical Medicine
基金
湖北省自然科学基金(2020CFA613)
作者简介
梅小琴(1984-),女,硕士,主治医师,研究方向:产科重症,E-mail:mxq198406@126.com
