目的构建稳定表达成熟miRNA-30a和miRNA-30e腺病毒表达载体。方法小鼠基因组中分别扩增出带有酶切位点的mmu-miR-30a、mmu-miR-30e目的基因,目的基因连接到穿梭质粒pSES-HUS,带有目的基因的穿梭质粒重组到骨架质粒AdEasy1上,重组质粒转...目的构建稳定表达成熟miRNA-30a和miRNA-30e腺病毒表达载体。方法小鼠基因组中分别扩增出带有酶切位点的mmu-miR-30a、mmu-miR-30e目的基因,目的基因连接到穿梭质粒pSES-HUS,带有目的基因的穿梭质粒重组到骨架质粒AdEasy1上,重组质粒转染到Hek-293细胞中包装成腺病毒载体,反复扩增之后获得高滴度的pAd-mmu-miR-30a、pAd-mmu-miR-30e腺病毒。用目的腺病毒分3组(RFP对照组,miR-30a组,miR-30e组)感染小鼠Mefs细胞,用荧光定量PCR方法检测mmu-miR-30a、mmu-miR-30e表达情况。结果分别扩增出带有酶切位点的357 bp mmu-miR-30a,324 bp mmu-miR-30e,并成功连接到pSES-HUS上,进一步与AdEasy1重组,包装得到腺病毒表达载体,通过荧光定量PCR检测,Mefs细胞中mmu-miR-30a升高26.46±7.46倍,mmu-miR-30e升高2.76±0.25倍。结论成功构建了成熟miRNA的腺病毒表达载体。展开更多
BACKGROUND Exosomal miRNAs play crucial roles in many central nervous system diseases.Cerebral small vessel disease(CVSD)is a small vessel disease that is affected by various factors.This study aimed to investigate th...BACKGROUND Exosomal miRNAs play crucial roles in many central nervous system diseases.Cerebral small vessel disease(CVSD)is a small vessel disease that is affected by various factors.This study aimed to investigate the role of exosomal miR-320e in the Wnt/β-catenin pathway stimulated by oxidative stress and assess its clinical correlation with psychiatric symptoms in patients with CVSD.AIM To explore whether exosomal miR-320e could suppress the Wnt/β-catenin pathway and play a protective role in CVSD progression,as well as examine its potential correlation with cognitive impairment and depression in patients with CVSD.METHODS Differentially expressed exosomal miRNAs were filtered by sequencing plasma exosomes from patients with CVSD and healthy controls.Bioinformatics and dual luciferase analyses were used to confirm the binding of miR-320e to Wnt2,and the mRNA and protein levels of downstream components in the Wnt/β-catenin pathway were evaluated when overexpressed or with knockdown of miR-320e under H2O2-induced oxidative stress.In addition,Wnt2-targeting siRNA was used to confirm the role of miR-320e in the Wnt2-mediated inhibition of the Wnt/β-catenin pathway.A retrospective analysis was conducted among patients with CVSD to confirm the correlation between miR-320e expression and the severity of cognitive impairment and depression,which were quantified using the Montreal Cognitive Assessment(MoCA)/Executive Function Assessment(EFA),and the Hamilton Depression Scale(HAMD)/Beck Depression Inventory(BDI),respectively.RESULTS High-throughput sequencing revealed that exosomal miR-320e was downregulated in patients with CVSD.Bioinformatics analysis and dual-luciferase reporter gene experiments showed that exosomal miR-320e inhibited the Wnt/β-catenin pathway in response to oxidative stress by targeting the 3'noncoding region of Wnt2.Uptake of exosomes carrying miR-320e into endothelial cells could also target Wnt2 and inhibit the Wnt2/β-catenin pathway.Elevated miR-320e expression may protect patients with CVSD from relatively severe cognitive impairment and depression,as it was found to have a positive correlation with the MoCA/EFA and HAMD/BDI scores.CONCLUSION Our results suggest that exosomal miR-320e suppresses the Wnt/β-catenin pathway and may play a protective role in CVSD progression.展开更多
miRNA-429是细胞内源性非编码类小RNA,在转录后调节目标基因翻译。现有广泛研究提示,miRNA-429影响结直肠癌细胞发生及发展过程。E3泛素蛋白连接酶-1(seven in absentia homolog-1,SIAH1)是由SIAH1基因编码的酶类,通过降解蛋白调节细胞...miRNA-429是细胞内源性非编码类小RNA,在转录后调节目标基因翻译。现有广泛研究提示,miRNA-429影响结直肠癌细胞发生及发展过程。E3泛素蛋白连接酶-1(seven in absentia homolog-1,SIAH1)是由SIAH1基因编码的酶类,通过降解蛋白调节细胞内蛋白平衡。有研究显示,SIAH1参与结直肠癌发病。在结直肠癌缺乏特异性诊断标记物及晚期缺乏有效治疗措施的现状下,本文针对miRNA-429及靶位点SIAH1在结直肠癌发生及发展中作用的研究进展进行概括总结,并探讨其在结直肠癌诊断及治疗中的可能性。展开更多
文摘目的构建稳定表达成熟miRNA-30a和miRNA-30e腺病毒表达载体。方法小鼠基因组中分别扩增出带有酶切位点的mmu-miR-30a、mmu-miR-30e目的基因,目的基因连接到穿梭质粒pSES-HUS,带有目的基因的穿梭质粒重组到骨架质粒AdEasy1上,重组质粒转染到Hek-293细胞中包装成腺病毒载体,反复扩增之后获得高滴度的pAd-mmu-miR-30a、pAd-mmu-miR-30e腺病毒。用目的腺病毒分3组(RFP对照组,miR-30a组,miR-30e组)感染小鼠Mefs细胞,用荧光定量PCR方法检测mmu-miR-30a、mmu-miR-30e表达情况。结果分别扩增出带有酶切位点的357 bp mmu-miR-30a,324 bp mmu-miR-30e,并成功连接到pSES-HUS上,进一步与AdEasy1重组,包装得到腺病毒表达载体,通过荧光定量PCR检测,Mefs细胞中mmu-miR-30a升高26.46±7.46倍,mmu-miR-30e升高2.76±0.25倍。结论成功构建了成熟miRNA的腺病毒表达载体。
文摘BACKGROUND Exosomal miRNAs play crucial roles in many central nervous system diseases.Cerebral small vessel disease(CVSD)is a small vessel disease that is affected by various factors.This study aimed to investigate the role of exosomal miR-320e in the Wnt/β-catenin pathway stimulated by oxidative stress and assess its clinical correlation with psychiatric symptoms in patients with CVSD.AIM To explore whether exosomal miR-320e could suppress the Wnt/β-catenin pathway and play a protective role in CVSD progression,as well as examine its potential correlation with cognitive impairment and depression in patients with CVSD.METHODS Differentially expressed exosomal miRNAs were filtered by sequencing plasma exosomes from patients with CVSD and healthy controls.Bioinformatics and dual luciferase analyses were used to confirm the binding of miR-320e to Wnt2,and the mRNA and protein levels of downstream components in the Wnt/β-catenin pathway were evaluated when overexpressed or with knockdown of miR-320e under H2O2-induced oxidative stress.In addition,Wnt2-targeting siRNA was used to confirm the role of miR-320e in the Wnt2-mediated inhibition of the Wnt/β-catenin pathway.A retrospective analysis was conducted among patients with CVSD to confirm the correlation between miR-320e expression and the severity of cognitive impairment and depression,which were quantified using the Montreal Cognitive Assessment(MoCA)/Executive Function Assessment(EFA),and the Hamilton Depression Scale(HAMD)/Beck Depression Inventory(BDI),respectively.RESULTS High-throughput sequencing revealed that exosomal miR-320e was downregulated in patients with CVSD.Bioinformatics analysis and dual-luciferase reporter gene experiments showed that exosomal miR-320e inhibited the Wnt/β-catenin pathway in response to oxidative stress by targeting the 3'noncoding region of Wnt2.Uptake of exosomes carrying miR-320e into endothelial cells could also target Wnt2 and inhibit the Wnt2/β-catenin pathway.Elevated miR-320e expression may protect patients with CVSD from relatively severe cognitive impairment and depression,as it was found to have a positive correlation with the MoCA/EFA and HAMD/BDI scores.CONCLUSION Our results suggest that exosomal miR-320e suppresses the Wnt/β-catenin pathway and may play a protective role in CVSD progression.
文摘miRNA-429是细胞内源性非编码类小RNA,在转录后调节目标基因翻译。现有广泛研究提示,miRNA-429影响结直肠癌细胞发生及发展过程。E3泛素蛋白连接酶-1(seven in absentia homolog-1,SIAH1)是由SIAH1基因编码的酶类,通过降解蛋白调节细胞内蛋白平衡。有研究显示,SIAH1参与结直肠癌发病。在结直肠癌缺乏特异性诊断标记物及晚期缺乏有效治疗措施的现状下,本文针对miRNA-429及靶位点SIAH1在结直肠癌发生及发展中作用的研究进展进行概括总结,并探讨其在结直肠癌诊断及治疗中的可能性。
