miR-383调控血管生成因子VEGFA的表达并抑制血管生成
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摘要
目的探讨miR-383对血管生成的影响及分子机制。方法通过生物信息学数据库进行预测,寻找miR-383调控的靶基因,挑选与血管生成相关的靶基因作为候选基因,采用荧光素酶报告基因检测系统进行验证。利用实时荧光定量PCR(qPCR)和Western-blot等实验证实miR-383对候选基因表达的靶向调控;通过体外血管内皮细胞成管实验检测miR-383对血管生成的影响。结果生物信息学预测显示,miR-383靶向多个与血管生成相关的基因;荧光素酶报告基因实验证实,miR-383可通过结合血管内皮生成因子(VEGFA)基因3′-非翻译区(3′-UTR)而抑制其表达;过表达miR-383并不影响VEGFA的mRNA转录水平,而只是在蛋白水平下调VEGFA表达;体外血管生成实验表明,miR-383所介导的VEGFA表达下调可明显抑制血管生成。结论 miR-383可通过靶向结合VEGFA基因的3′-UTR,抑制其翻译效率而下调VEGFA的表达,并抑制内皮细胞血管生成能力。
Objective To explore the effect of microRNA-383 on angiogenesis and its molecular mechanism. Methods Bioinformatic prediction was performed to find the target genes of miR-383. Then the target genes associated with angiogenesis were selected as the candidate genes for luciferase assays. Fluorescence quantitative polymerase chain reaction(qPCR) were performed to measure the mRNA level of endogenous VEGFA expression in HUVEC cells affected by overexpression of miR-383. Protein level of endogenous VEGFA was tested by ELISA and Western-blot assays. In vitro angiogenesis assay was performed to verify the effects of miR-383 mediated downregulation of VEGFA on angiogenesis. Results Bioinformatics prediction showed that miR-383 targeted multiple genes related to angiogenesis, such as VEGFA, FGF9. The luciferase assay confirmed that miR-383 could inhibit VEGFA expression by binding to its 3'-UTR. qPCR, while ELISA and Western-blot assay revealed that overexpression of miR-383 reduced VEGFA expression only at protein levels. In vitro matrigel based angiogenesis assay showed that overexpression of miR-383 reduced angiogenesis by downregulation of VEGFA. Conclusion miR-383 can downregulates VEGFA expression and reduces angiogenesis.
Objective To explore the effect of microRNA-383 on angiogenesis and its molecular mechanism. Methods Bioinformatic prediction was performed to find the target genes of miR-383. Then the target genes associated with angiogenesis were selected as the candidate genes for luciferase assays. Fluorescence quantitative polymerase chain reaction(qPCR) were performed to measure the mRNA level of endogenous VEGFA expression in HUVEC cells affected by overexpression of miR-383. Protein level of endogenous VEGFA was tested by ELISA and Western-blot assays. In vitro angiogenesis assay was performed to verify the effects of miR-383 mediated downregulation of VEGFA on angiogenesis. Results Bioinformatics prediction showed that miR-383 targeted multiple genes related to angiogenesis, such as VEGFA, FGF9. The luciferase assay confirmed that miR-383 could inhibit VEGFA expression by binding to its 3'-UTR. qPCR, while ELISA and Western-blot assay revealed that overexpression of miR-383 reduced VEGFA expression only at protein levels. In vitro matrigel based angiogenesis assay showed that overexpression of miR-383 reduced angiogenesis by downregulation of VEGFA. Conclusion miR-383 can downregulates VEGFA expression and reduces angiogenesis.
引文
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[14] Cui Y,Chen L,Yao H,et al.Upregulation of microRNA-383 inhibits the proliferation,migration and invasion of colon cancer cells[J].Oncol Lett,2017,15(1):1184-1190.
[15] Teng P,Jiao Y,Hao M,et al.microRNA-383 suppresses the PI3K-AKT-MTOR signaling pathway to inhibit development of cervical cancer via down-regulating PARP2[J].J Cell Biochem,2018,119(7):5243-5252.
[16] Fang Z,He L,Jia H,et al.The miR-383-LDHA axis regulates cell proliferation,invasion and glycolysis in hepatocellular cancer[J].Iran J Basic Med Sci,2017,20(2):187-192.
[17] Yan F,Tu Z,Duan L,et al.MicroRNA-383 suppresses cell proliferation and invasion in colorectal cancer by directly targeting paired box 6[J].Mol Med Rep,2018,17(5):6893-6901.
[18] Malinda K M.In vivo matrigel migration and angiogenesis assay[M].Second edition.New Jersey:Humana Press,2009:287-294.
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[21] Kazerounian S,Lawler J.Integration of pro- and anti-angiogenic signals by endothelial cells[J].J Cell Commun Signal,2018,12(1):171-179.
[22] Failla C M,Carbo M,Morea V.Positive and negative regulation of angiogenesis by soluble vascular endothelial growth factor receptor-1[J].Int J Mol Sci,2018,19(5):1306.