目的:构建中草药来源的目标微小RNA(microRNA,miR)在小鼠肺组织中的靶基因谱检测方法,利用该方法检测清肺排毒汤所含miR-320—靶基因谱及其生物学功能,为清肺排毒汤治疗病毒性肺炎的分子机制补充miR维度的数据支撑。方法:采用二代高通...目的:构建中草药来源的目标微小RNA(microRNA,miR)在小鼠肺组织中的靶基因谱检测方法,利用该方法检测清肺排毒汤所含miR-320—靶基因谱及其生物学功能,为清肺排毒汤治疗病毒性肺炎的分子机制补充miR维度的数据支撑。方法:采用二代高通量测序测定目标miR(miR-320)在清肺排毒汤中的表达情况,利用“结合态miR—靶基因抓取测序技术(capturing and sequencing of miRNA-target complex technology,CSCT)”初步检测其在小鼠肺组织中的靶基因谱,并采用Alphafold3验证检测结果,取其交集作为目标miR的靶基因谱;进而分析靶基因谱的功能,阐释清肺排毒汤通过miR-320在小鼠肺组织中的调控功能;此外,将检测结果与TargetScan的预测结果进行比对,验证本检测方法(CSCT+Alphafold3序贯检测法)的优势。结果:高通量测序结果表明清肺排毒汤中富含miR-320,其表达含量居前50位。miR-320在小鼠肺组织中可作用于26类靶基因,其中19类为已知基因(18类为mRNA以及1类为转录增强子),其主要通过miR经典作用模式识别靶基因,与26类靶基因具有良好的碱基互补性,最小自由能在-35.8~-21.8 kcal·mol^(-1)之间;Alphafold3预测的靶基因的iPTM和PTM之和最小值为1.1,位于高置信度区域,100%验证了CSCT的检测结果,据此确证26类靶基因为miR-320作用谱;与TargetScan相比,本研究构建的“CSCT+Alphafold3序贯检测法”对于靶基因谱的检测准确率更高。这些靶基因具有多种功能,主要富集于白介素介导的免疫信号通路,在抗原处理与提呈、免疫因子或细胞介导的细胞凋亡、淋巴细胞增殖与活化等生物学过程中发挥作用。结论:清肺排毒汤所含miR-320在小鼠肺组织内主要富集于免疫调节相关通路,这可能是清肺排毒汤治疗病毒性肺炎的miR分子机制之一;本研究构建的“CSCT+Alphafold3”法对于miR靶基因谱的检测具有较高的可靠性和准确性,可作为中药汤剂中miR—靶基因互作谱检测技术。展开更多
Objective Placental dysfunction induced by fetal cardiopulmonary bypass(CPB)imposes limitations on the clinical application of this procedure.The potential impact of microRNA-mediated autophagy in placental endothelia...Objective Placental dysfunction induced by fetal cardiopulmonary bypass(CPB)imposes limitations on the clinical application of this procedure.The potential impact of microRNA-mediated autophagy in placental endothelial cells on overall placental function remains elusive,necessitating a comprehensive exploration of the underlying mechanisms involved.Methods We established fetal sheep CPB models and employed immunohistochemistry to assess the placental expression of ATG7.Bioinformatic analysis,coupled with dual-luciferase reporter assays,was used to elucidate the intricate relationship between miR-320a and ATG7.Changes in ATG7 expression were further investigated through Western blotting and quantitative polymerase chain reaction(qPCR).Human umbilical vein endothelial cells(HUVECs)were cultured,and in vitro experiments were conducted to evaluate their regulatory effects on endothelial function.Immunoblotting was used to measure the expression levels of ATG7,endothelin-1(ET-1),SIRT1,and FOXO1,whereas enzyme-linked immunosorbent assay(ELISA)was used to quantify nitric oxide(NO)production.Results Sixty minutes after CPB,a substantial decrease in ATG7 expression in placental tissue was observed.The downregulation of ATG7 expression led to increased ET-1 production in HUVECs,concomitant with decreased NO production.miR-320a was identified as a specific regulator of ATG7 expression,with subsequent experiments demonstrating a significant reduction in placental ATG7 levels upon injection of the miR-320a agomir compared with the miR-320a antagomir during fetal sheep CPB.In HUVECs,miR-320a downregulated ATG7,resulting in increased ET-1 production and diminished NO production.Treatment with the miR-320a mimic/miR-320a inhibitor revealed that miR-320a inhibited the SIRT1/FOXO1 pathway in HUVECs by downregulating ATG7 expression,culminating in increased ET-1 production and reduced NO levels.Conclusion The observed downregulation of placental ATG7 expression subsequent to fetal CPB is intricately associated with endothelial dysfunction.Furthermore,our findings underscore the specific regulatory role of miR-320a in modulating ATG7 expression within the placenta.At the cellular level,increasing the level of miR-320a has emerged as a potential strategy for modulating endothelial function through the inhibition of ATG7 and the SIRT1/FOXO1 pathway.展开更多
文摘目的:构建中草药来源的目标微小RNA(microRNA,miR)在小鼠肺组织中的靶基因谱检测方法,利用该方法检测清肺排毒汤所含miR-320—靶基因谱及其生物学功能,为清肺排毒汤治疗病毒性肺炎的分子机制补充miR维度的数据支撑。方法:采用二代高通量测序测定目标miR(miR-320)在清肺排毒汤中的表达情况,利用“结合态miR—靶基因抓取测序技术(capturing and sequencing of miRNA-target complex technology,CSCT)”初步检测其在小鼠肺组织中的靶基因谱,并采用Alphafold3验证检测结果,取其交集作为目标miR的靶基因谱;进而分析靶基因谱的功能,阐释清肺排毒汤通过miR-320在小鼠肺组织中的调控功能;此外,将检测结果与TargetScan的预测结果进行比对,验证本检测方法(CSCT+Alphafold3序贯检测法)的优势。结果:高通量测序结果表明清肺排毒汤中富含miR-320,其表达含量居前50位。miR-320在小鼠肺组织中可作用于26类靶基因,其中19类为已知基因(18类为mRNA以及1类为转录增强子),其主要通过miR经典作用模式识别靶基因,与26类靶基因具有良好的碱基互补性,最小自由能在-35.8~-21.8 kcal·mol^(-1)之间;Alphafold3预测的靶基因的iPTM和PTM之和最小值为1.1,位于高置信度区域,100%验证了CSCT的检测结果,据此确证26类靶基因为miR-320作用谱;与TargetScan相比,本研究构建的“CSCT+Alphafold3序贯检测法”对于靶基因谱的检测准确率更高。这些靶基因具有多种功能,主要富集于白介素介导的免疫信号通路,在抗原处理与提呈、免疫因子或细胞介导的细胞凋亡、淋巴细胞增殖与活化等生物学过程中发挥作用。结论:清肺排毒汤所含miR-320在小鼠肺组织内主要富集于免疫调节相关通路,这可能是清肺排毒汤治疗病毒性肺炎的miR分子机制之一;本研究构建的“CSCT+Alphafold3”法对于miR靶基因谱的检测具有较高的可靠性和准确性,可作为中药汤剂中miR—靶基因互作谱检测技术。
基金supported by the National Key Research and Development Program of China(No.2022YFC2407406)Guangzhou Science and Technology Planning Project(No.2023B03J0596)2023 Stability Support for Innovative Capacity Building of Guangdong Provincial Scientific Research Institutions(No.KD022023019).
文摘Objective Placental dysfunction induced by fetal cardiopulmonary bypass(CPB)imposes limitations on the clinical application of this procedure.The potential impact of microRNA-mediated autophagy in placental endothelial cells on overall placental function remains elusive,necessitating a comprehensive exploration of the underlying mechanisms involved.Methods We established fetal sheep CPB models and employed immunohistochemistry to assess the placental expression of ATG7.Bioinformatic analysis,coupled with dual-luciferase reporter assays,was used to elucidate the intricate relationship between miR-320a and ATG7.Changes in ATG7 expression were further investigated through Western blotting and quantitative polymerase chain reaction(qPCR).Human umbilical vein endothelial cells(HUVECs)were cultured,and in vitro experiments were conducted to evaluate their regulatory effects on endothelial function.Immunoblotting was used to measure the expression levels of ATG7,endothelin-1(ET-1),SIRT1,and FOXO1,whereas enzyme-linked immunosorbent assay(ELISA)was used to quantify nitric oxide(NO)production.Results Sixty minutes after CPB,a substantial decrease in ATG7 expression in placental tissue was observed.The downregulation of ATG7 expression led to increased ET-1 production in HUVECs,concomitant with decreased NO production.miR-320a was identified as a specific regulator of ATG7 expression,with subsequent experiments demonstrating a significant reduction in placental ATG7 levels upon injection of the miR-320a agomir compared with the miR-320a antagomir during fetal sheep CPB.In HUVECs,miR-320a downregulated ATG7,resulting in increased ET-1 production and diminished NO production.Treatment with the miR-320a mimic/miR-320a inhibitor revealed that miR-320a inhibited the SIRT1/FOXO1 pathway in HUVECs by downregulating ATG7 expression,culminating in increased ET-1 production and reduced NO levels.Conclusion The observed downregulation of placental ATG7 expression subsequent to fetal CPB is intricately associated with endothelial dysfunction.Furthermore,our findings underscore the specific regulatory role of miR-320a in modulating ATG7 expression within the placenta.At the cellular level,increasing the level of miR-320a has emerged as a potential strategy for modulating endothelial function through the inhibition of ATG7 and the SIRT1/FOXO1 pathway.
