摘要
目的探索环状RNA 0006104(circ_0006104)对血管紧张素Ⅱ(AngⅡ)诱导的心脏成纤维细胞(CFs)的增殖,活化和细胞外基质(ECM)合成的作用和机制。方法分离培养乳小鼠原代CFs,分为对照组、circ_0006104敲低组、AngⅡ组、AngⅡ+circ_0006104敲低组和AngⅡ+circ_0006104敲低+miR-370-3p抑制组。EdU免疫荧光染色检测CFs的增殖水平;Western blot检测活化相关基因(α-SMA)的表达;RT-qPCR检测collagenⅠ和Ⅲ(ColⅠ和ColⅢ)的表达。结果利用RNA-seq高通量测序结果,筛选得到circ_0006104。沉默circ_0006104对生理条件下CFs的增殖、活化和ECM合成没有显著影响;而沉默circ_0006104可显著抑制AngⅡ诱导的CFs增殖,活化以及ECM合成水平(P<0.05)。此外,circ_0006104可靶向结合并负向调控miR-370-3p;抑制miR-370-3p明显减轻circ_0006104沉默对AngⅡ诱导的CFs增殖,活化和ECM合成的的阻碍作用(P<0.05)。结论沉默circ_0006104通过结合并负向调控miR-370-3p抑制AngⅡ诱导的CFs纤维化作用。
Objective To explore the function and mechanism of circular RNA 0006104(circ_0006104)in regulating proliferation,activation and extracellular matrix(ECM)synthesis of cardiac fibroblasts(CFs)induced by angiotensinⅡ(AngⅡ).Methods The primary CFs of neonatal mice were isolated and divided into control group,circ_0006104 knockdown group,AngⅡgroup,AngⅡ+circ_0006104 knockdown group and AngⅡ+circ_0006104 knockdown+miR-370-3 p inhibitor.EDU immunofluorescence staining was used to detect the proliferation level of CFs;Western blot was used to detect activation related gene(α-SMA);RT-qPCR was used to detect the expression of collagenⅠandⅢ(Col and ColⅢ).Results Circ_0006104 was screened by RNA-seq high-throughput sequencing results.In the primary CFs of neonatal mice,silencing circ_0006104 had no effect on the proliferation,activation and ECM synthesis of CFs under physiological conditions,while silencing circ_0006104 could significantly inhibit the proliferation,activation and ECM synthesis of CFs induced by AngⅡ(P<0.05).The molecules of circ_0006104 could bind to and negatively regulate miR-370-3 p.The combined experimental results showed that inhibition of miR-370-3 p significantly reversed the inhibitory effects of circ_0006104 silencing on the proliferation,activation and ECM synthesis of CFs induced by AngⅡ(P<0.05).Conclusions Knockdown of circ_0006104 inhibits AngⅡ-induced CFs fibrosis by binding and negatively regulating miR-370-3 p.
作者
李亚飞
杜颖强
王泽穆
张俊
LI Ya-fei;DU Ying-qiang;WANG Ze-mu;ZHANG Jun(Department of Cardiology,Suzhou Hospital Affiliated to Nanjing Medical University,Suzhou 215000;Department of Cardiology,the First Affiliated Hospital of Nanjing Medical University,Nanjing 210000,China)
出处
《基础医学与临床》
2022年第11期1667-1674,共8页
Basic and Clinical Medicine
基金
国家自然科学基金青年科学基金(81703213)
江苏省自然科学基金(BK20210101)
南京医科大学姑苏学院科研项目(GSKY20210214)。
