miR-150-3p靶向CDH2基因对缺氧性肺动脉高压新生大鼠肺组织损伤的影响

Effects of miR-150-3p targeting CDH2 gene on lung tissue damage in neonatal rats with hypoxia-induced pulmonary hypertension

目的:探究miR-150-3p靶向CDH2基因对缺氧性肺动脉高压(HPH)新生大鼠肺组织损伤的影响。方法:将新生Wistar大鼠随机分为对照组、模型组、阴性对照组和miR-150-3p过表达组,每组10只。除对照组外,其余各组构建新生大鼠HPH模型。分别检测各组大鼠缺氧3、6、12 d时的平均肺动脉压(mPAP),酶联免疫吸附测定法(ELISA)检测大鼠血清中缺氧诱导因子-1α(HIF-1α)和内皮素-1(ET-1)水平;在缺氧12 d时,Western blotting检测大鼠肺组织HIF-1α、ET-1、诱导型一氧化氮合酶(iNOS)和钙黏蛋白2(CDH2)表达情况,实时荧光定量PCR(RT-PCR)检测大鼠肺组织miR-150-3p、CDH2 mRNA表达。双荧光素酶报告基因实验验证miR-150-3p与CDH2之间的靶向关系。结果:与对照组相比,模型组和阴性对照组大鼠出现反应迟钝、萎靡不振、食量减少、肺组织炎症细胞浸润、肺泡大小改变等现象;大鼠mPAP,血清HIF-1α、ET-1水平,肺组织HIF-1α、ET-1、iNOS和CDH2蛋白表达均明显升高(P<0.05);肺组织miR-150-3p表达明显降低,CDH2 mRNA表达明显升高(P<0.05)。与模型组、相比,阴性对照组上述指标均无显著差异(P>0.05)。与模型组、阴性对照组相比,miR-150-3p过表达组大鼠反应迟钝、炎症细胞浸润等不良现象均获得良好改善,且大鼠mPAP,血清HIF-1α、ET-1水平,肺组织HIF-1α、ET-1、iNOS和CDH2蛋白表达均降低(P<0.05);肺组织miR-150-3p表达明显升高,CDH2 mRNA表达明显降低(P<0.05)。双荧光素酶报告基因实验结果证明CDH2为miR-150-3p的靶基因。结论:miR-150-3p能够通过靶向CDH2基因减轻HPH新生大鼠肺组织损伤。

Objective:To explore the effect of miR-150-3 p targeting CDH2 gene on lung tissue damage in neonatal rats with hypoxia-induced pulmonary hypertension(HPH).Methods:Newborn Wistar rats were randomly divided into control group,model group,negative control group,and miR-150-3 p overexpression group,with 10 rats in each group.Except for the control group,the rest of the groups constructed a newborn rat HPH model.The mean pulmonary arterial pressure(mPAP)of rats in each group was measured when hypoxia for 3,6,and 12 d.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of hypoxia inducible factor-1α(HIF-1α)and endothelin-1(ET-1)in rat serum;on the 12 th day of hypoxia,Western blotting was used to detect the expression of HIF-1α,ET-1,iNOS and CDH2 in the lung tissue of rats,RT-PCR was used to detect the expression of miR-150-3 p and CDH2 mRNA in the lung tissue of rats.The dual luciferase reporter gene experiment was used to verify the targeting relationship between miR-150-3 p and CDH2.Results:Compared with the control group,rats in the model group and the negative control group showed slow response,malaise,reduced appetite,lung tissue inflammatory cell infiltration,and alveolar size changes;mPAP,the levels of serum HIF-1αand ET-1,the expression of HIF-1α,ET-1,iNOS and CDH2 proteins in lung tissue were all significantly increased(P<0.05),the expression of miR-150-3 p in lung tissue was significantly reduced,and the expression of CDH2 mRNA was significantly increased(P<0.05).Compared with the model group,there was no significant difference in the above indicators of the negative control group(P>0.05).Compared with the model group and the negative control group,the rats in the miR-150-3 p overexpression group showed a good improvement in slow response and inflammatory cell infiltration;mPAP,the levels of serum HIF-1αand ET-1,the expression of HIF-1α,ET-1,iNOS and CDH2 proteins in lung tissue were all significantly decreased(P<0.05),the expression of miR-150-3 p in lung tissue was significantly reduced,and the expression of CDH2 mRNA was significantly decreased(P<0.05).The results of the dual luciferase reporter gene experiment proved that CDH2 was the target gene of miR-150-3 p.Conclusion:MiR-150-3 p can reduce lung tissue damage in newborn rats with HPH by targeting CDH2 gene.