mmu-miR-146a-5p对小鼠巨噬细胞的免疫调节作用
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摘要
为了探究mmu-miR-146a-5p(小鼠源miR-146a-5p)对小鼠巨噬细胞分泌细胞因子的影响,本研究利用mmu-miR-146a-5p模拟物和抑制物分别转染小鼠巨噬细胞系RAW264.7,通过qPCR定量分析巨噬细胞中细胞因子和LPS/TLR4信号传导途径中关键基因的表达情况,通过Griess试剂测定巨噬细胞培养上清中的亚硝酸盐浓度来评价NO的分泌情况。检测结果显示,与阴性对照组相比,在模拟物转染组,LPS/TLR4信号传导途径的几个关键基因相对表达量显著上调,如TLR4和CD14(P<0.05),而促炎因子(IL-1α、IL-1β、IL-12β)的相对表达量显著下调(P<0.05),而在转染后第24和36小时NO的含量出现上调。在抑制剂转染组中,TLR4、CD14的表达水平下降,促炎因子IL-1α、IL-12β和TNF-α的表达水平上调,i NOS基因表达和NO产生在第24和36小时显著下降(P<0.05)。结果表明,mmu-miR-146a-5p对小鼠巨噬细胞分泌细胞因子具有免疫调节作用。
To investigate the effect of mmu-miR-146 a-5 p(murine miR-146-5 p) on the secretion of cytokines by murine macrophages line RAW264.7,the cells were transfected with mmu-miR-146 a-5 p mimics and inhibitors,respectively.The expression of cytokines and key genes in LPS/TLR4 signaling pathways in macrophages were quantitatively analyzed by q PCR,and the secretion level of NO were determined by measuring the nitrite concentration in macrophage culture supernatants by Griess reagent.The results showed that the relative expression levels of several key genes in the LPS/TLR4 signaling pathway were significantly up-regulated in the mimics-transfected group compared with the negative control group,such as TLR4 and CD14(P<0.05),and pro-inflammatory factors IL-1α,IL-1β and IL-12β were significantly down-regulated(P<0.05).The content of NO was up-regulated at 24 and 36 hours after transfection.In the inhibitors-transfected group,the expression of TLR4 and CD14 were decreased,while the expression of pro-inflammatory factors IL-1α,IL-12β and TNF-α were up-regulated,and i NOS gene expression and NO production were significantly decreased at 24 and 36 hours(P<0.05).The above results indicated that mmu-miR-146 a-5 p had an immunomodulatory effect on the secretion of cytokines by murine macrophages.
To investigate the effect of mmu-miR-146 a-5 p(murine miR-146-5 p) on the secretion of cytokines by murine macrophages line RAW264.7,the cells were transfected with mmu-miR-146 a-5 p mimics and inhibitors,respectively.The expression of cytokines and key genes in LPS/TLR4 signaling pathways in macrophages were quantitatively analyzed by q PCR,and the secretion level of NO were determined by measuring the nitrite concentration in macrophage culture supernatants by Griess reagent.The results showed that the relative expression levels of several key genes in the LPS/TLR4 signaling pathway were significantly up-regulated in the mimics-transfected group compared with the negative control group,such as TLR4 and CD14(P<0.05),and pro-inflammatory factors IL-1α,IL-1β and IL-12β were significantly down-regulated(P<0.05).The content of NO was up-regulated at 24 and 36 hours after transfection.In the inhibitors-transfected group,the expression of TLR4 and CD14 were decreased,while the expression of pro-inflammatory factors IL-1α,IL-12β and TNF-α were up-regulated,and i NOS gene expression and NO production were significantly decreased at 24 and 36 hours(P<0.05).The above results indicated that mmu-miR-146 a-5 p had an immunomodulatory effect on the secretion of cytokines by murine macrophages.
引文
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[23] SHIN HJ,LEE H,PARK JD,et al.Kinetics of binding of LPS to recombinant CD14,TLR4,and MD-2 proteins[J].Molecular Cells,2007,24:119-124.
[24] LIN YS,ZHOU H,FORREST RH,et al.Association between variation in faecal egg count for a natural mixed field-challenge of nematode parasites and TLR4 variation[J].Veterinary Parasitology,2016,218:5-9.
[2] TORGERSON P R,DEVLEESSCHAUWER B,PRAET N,et al.World health organization estimates of the global and regional disease burden of 11 foodborne parasitic diseases,2010,a data synthesis[J].PLo S Medicine,2015,12:e1001920.
[3] CUCHER M,MACCHIAROLI N,KAMENETZKY L,et al.Highthroughput characterization of Echinococcus spp.metacestode mi RNomes[J].International Journal for Parasitology,2015,45:253-267.
[4]邵忠伟,郭小腊,高泽乾,等.ova-miR-34a-5p在山羊痘病毒感染过程中的动态表达及其靶标基因的鉴定[J].中国兽医科学,2016.46(04):418-423.SHAO Zhong-wei,GUO Xiao-la,GAO Ze-qian,et al.Dynamic expression profile of ova-miR-34a-5p during goatpox virus infection and identification of its target[J].Chinese Veterinary Science,2016.46(04):418-423.(in Chinese)
[5]唐仪晓,沈元曦,洪炀,等.MicroRNA-181a对日本血吸虫童虫抗原刺激巨噬细胞的免疫应答起负调节作用[J].中国兽医科学,2018.48(02):204-210.TANG Yi-xiao,SHEN Yuan-xi,HONG Yang,et al.Micro RNA-181a plays a negative immune regulation role in macrophages stimulated with soluble schistosomula antigens of Schistosoma japonicum[J].Chinese Veterinary Science,2018.48(02):204-210.(in Chinese)
[6]钟纯燕,主性,李基棕,等.慢病毒介导的bta-miR-222稳定过表达MDBK细胞株的构建及鉴定[J].中国兽医科学,2019(01):50-57.ZHONG Chun-yan,ZHU Xing,LI Ji-zong,et al.Construction and identification of lentiviral-mediated MDBK cell line stably overexpressing bta-mi R-222[J].Chinese Veterinary Science,2019(01):50-57.(in Chinese)
[7] DAHIYA N,SARACHANA T,VU L,et al.Platelet microRNAs:An overview[J].Transfusion Medicine Reviews,2015,29:215-219.
[8] CHU PM,MA HI,CHEN LH,et al.Deregulated microRNAs identified in isolated glioblastoma stem cells:an overview[J].Cell Transplant,2013,22:741-753.
[9] BATISTELA MS,JOSVIAK ND,SULZBACH CD,et al.An overview of circulating cell-free microRNAs as putative biomarkers in Alzheimer's and Parkinson's Diseases[J].International Journal of Neuroscience,2017,127:547-558.
[10] YAN Y,ZHANG J,ZHANG Q,et al.The role of microRNAs in platelet biology during storage[J].Transfusion and Apheresis Science,2017,56:147-150.
[11] WANG J,LIEW OW,RICHARDS AM,et al.Overview of micro RNAs in cardiac hypertrophy,fibrosis,and apoptosis[J].International Journal of Molecular Sciences,2016,17(5).
[12] HARQUAIL J,BENZINA S,ROBICHAUD GA.MicroRNAs and breast cancer malignancy:an overview of miRNA-regulated cancer processes leading to metastasis[J].Cancer Biomark,2012,11:269-280.
[13] HAMMOND SM.An overview of microRNAs[J].Advanced Drug Delivery Reviews,2015,87:3-14.
[14] PLE H,LANDRY P,BENHAM A,et al.The repertoire and features of human platelet microRNAs[J].PLoS One,2012,7:e50746.
[15] MILITELLO KT,REFOUR P,COMEAUX CA,et al.Antisense RNA and RNAi in protozoan parasites:working hard or hardly working[J].Molecular and Biochemcal Parasitology,2008,157:117-126.
[16] ZHENG Y,CAI X,BRADLEY JE.microRNAs in parasites and parasite infection[J].RNA Biology,2013,10:371-379.
[17] SHOMALI N,MANSOORI B,MOHAMMADI A,et al.MiR-146a functions as a small silent player in gastric cancer[J].Biomed Pharmacother,2017,96:238-245.
[18] TAGANOV KD,BOLDIN MP,CHANG KJ,et al.NF-kappaB-dependent induction of microRNA miR-146,an inhibitor targeted to signaling proteins of innate immune responses[J].Proceedings National Academy of Sciences of USA,2006,103:12481-12486.
[19] CANNELLA D,BRENIER-PINCHART MP,BRAUN L,et al.miR-146a and miR-155 delineate a microRNA fingerprint associated with Toxoplasma persistence in the host brain[J].Cell Report,2014,6:928-937.
[20] GUO X,ZHENG Y.Expression profiling of circulating mi RNAs in mouse serum in response to Echinococcus multilocularis infection[J].Parasitology,2017,144:1079-1087.
[21] ZHENG Y,GUO X,HE W,et al.Effects of Echinococcus multilocularis mi R-71 mimics on murine macrophage RAW264.7 cells[J].International Immunopharmacology,2016,34:259-262.
[22] HE J,WANG S,ZENG Y,et al.Binding of CD14 to Mycoplasma genitalium-derived lipid-associated membrane proteins upregulates TNF-alpha[J].Inflammation,2014,37:322-330.
[23] SHIN HJ,LEE H,PARK JD,et al.Kinetics of binding of LPS to recombinant CD14,TLR4,and MD-2 proteins[J].Molecular Cells,2007,24:119-124.
[24] LIN YS,ZHOU H,FORREST RH,et al.Association between variation in faecal egg count for a natural mixed field-challenge of nematode parasites and TLR4 variation[J].Veterinary Parasitology,2016,218:5-9.