MicroRNA-223通过靶向NLRP3调控VSV诱导的巨噬细胞Ⅰ型干扰素产生
发布时间：2019-04-13 17:04
【摘要】：病毒感染机体后,干扰素(Interferon, IFNs)大量产生,Ⅰ型干扰素的产生在天然免疫中起重要的作用,对宿主的防御起关键作用。MicroRNAs (miRNAs)是近年发现的一类内源性的非编码RNA,它除了对基因表达的转录调控作用外,今年来发现miRNAs介导的转录后调节在调节基因的表达也有重要的作用。MicroRNA-223(miR-223)是特异性表达在髓系细胞的miRNA。对于miR-223在巨噬细胞中Ⅰ型干扰素产生的调控作用,以及靶点都还未有研究。 我们发现,在小鼠巨噬细胞系RAW264.7或原代培养的巨噬细胞中,用VSV病毒感染,荧光定量PCR检测结果显示VSV病毒感染后巨噬细胞中miR-223的mRNA表达水平发生明显下调。用miR-223mimics进行功能性实验研究发现,miR-223过表达可明显促进VSV诱导的巨噬细胞IFN-α、IFN-βmRNA表达水平,而对TNF-α和IL-6的mRNA表达没有影响。在小鼠巨噬细胞系RAW264.7中转染miR-223mimics,用VSV-GFP病毒感染,运用共聚焦荧光显微镜和流式细胞术检测,发现过表达miR-223的细胞中荧光减少,说明miR-223在抗病毒感染中可能起重要的调节作用。由于miRNA发挥生物学功能主要通过调控其靶基因的表达,通过进一步的荧光素酶报告基因试验和荧光定量PCR等实验结果显示miR-223可以直接靶向抑制NLRP3的表达。同时,过表达NLRP3后,荧光素酶报告基因结果显示,IRF3和IFN-β的活性降低,说明NLRP3对Ⅰ型干扰素的产生可能有抑制作用。 综上所述,我们的实验结果初步发现,VSV感染后巨噬细胞中miR-223表达下调,miR-223可通过靶向抑制NLRP3的表达,促进巨噬细胞IFN-α、IFN-β的分泌。上述研究为揭示非编码RNA在巨噬细胞天然免疫功能的精细调控中发挥的作用提供了理论依据,并可能为感染性疾病的诊治提供潜在的靶点和思路。
[Abstract]:Interferon (Interferon, IFNs) is produced in large quantities after virus infection. Interferon I plays an important role in innate immunity and plays a key role in host defense. MicroRNAs (miRNAs) is a kind of endogenous non-coding RNA, discovered in recent years. In addition to its transcriptional regulation of gene expression, miRNAs-mediated post-transcriptional regulation is also found to play an important role in regulating gene expression this year. MicroRNA-223 (miR-223) is specifically expressed in the miRNA. of myeloid cells. The regulatory effects and targets of miR-223 on IFN-I production in macrophages have not been studied. We found that the expression level of miR-223 mRNA in murine macrophage cell line RAW264.7 or primary cultured macrophages was down-regulated by fluorescence quantitative PCR (FQ-PCR) after infection with VSV virus. The results showed that the expression level of VSV in macrophages was down-regulated after infection with VSV virus by fluorescence quantitative PCR (FQ-PCR). The results of functional experiment with miR-223mimics showed that the overexpression of miR-223 significantly promoted the expression of IFN- 伪 and IFN- 尾 in macrophages induced by VSV, but had no effect on the expression of mRNA in TNF- 伪 and IL-6. The transfection of miR-223mimics, into mouse macrophage cell line RAW264.7 was infected with VSV-GFP virus, and detected by confocal fluorescence microscope and flow cytometry, it was found that the fluorescence of over-expressed miR-223 cells decreased. These results suggest that miR-223 may play an important role in the regulation of anti-viral infection. Because miRNA plays a biological role by regulating the expression of its target gene, the results of luciferase reporter gene test and fluorescent quantitative PCR show that miR-223 can directly target the expression of NLRP3. At the same time, the results of luciferase reporter gene showed that the activities of IRF3 and IFN- 尾 decreased after overexpression of NLRP3, suggesting that NLRP3 may inhibit the production of interferon 鈪，
本文编号：2457770
[Abstract]:Interferon (Interferon, IFNs) is produced in large quantities after virus infection. Interferon I plays an important role in innate immunity and plays a key role in host defense. MicroRNAs (miRNAs) is a kind of endogenous non-coding RNA, discovered in recent years. In addition to its transcriptional regulation of gene expression, miRNAs-mediated post-transcriptional regulation is also found to play an important role in regulating gene expression this year. MicroRNA-223 (miR-223) is specifically expressed in the miRNA. of myeloid cells. The regulatory effects and targets of miR-223 on IFN-I production in macrophages have not been studied. We found that the expression level of miR-223 mRNA in murine macrophage cell line RAW264.7 or primary cultured macrophages was down-regulated by fluorescence quantitative PCR (FQ-PCR) after infection with VSV virus. The results showed that the expression level of VSV in macrophages was down-regulated after infection with VSV virus by fluorescence quantitative PCR (FQ-PCR). The results of functional experiment with miR-223mimics showed that the overexpression of miR-223 significantly promoted the expression of IFN- 伪 and IFN- 尾 in macrophages induced by VSV, but had no effect on the expression of mRNA in TNF- 伪 and IL-6. The transfection of miR-223mimics, into mouse macrophage cell line RAW264.7 was infected with VSV-GFP virus, and detected by confocal fluorescence microscope and flow cytometry, it was found that the fluorescence of over-expressed miR-223 cells decreased. These results suggest that miR-223 may play an important role in the regulation of anti-viral infection. Because miRNA plays a biological role by regulating the expression of its target gene, the results of luciferase reporter gene test and fluorescent quantitative PCR show that miR-223 can directly target the expression of NLRP3. At the same time, the results of luciferase reporter gene showed that the activities of IRF3 and IFN- 尾 decreased after overexpression of NLRP3, suggesting that NLRP3 may inhibit the production of interferon 鈪，
本文编号：2457770
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