PHEV诱发宿主细胞miR-142a-3p的表达变化及其靶基因Tmem59的验证
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摘要
为了确定宿主miR-142a-3p是否参与猪血凝性脑脊髓炎病毒(PHEV)感染致神经损伤的过程,本研究利用实时荧光定量PCR方法对不同时间点感染PHEV的N2a细胞和BALB/c小鼠脑组织中miR-142a-3p的表达情况进行了检测。结果显示,在PHEV的体内外感染模型中,miR-142a-3p的表达量均明显上调。进而通过生物信息学软件预测分析,筛选出跨膜蛋白59(Tmem59)可能是miR-142a-3p的靶基因,并利用双荧光素酶报告系统对该靶基因进行验证。结果表明,宿主感染PHEV后,体内的miR-142a-3p表达明显上调,并筛选出与机制相关的靶基因Tmem59。
In order to determine whether host miR-142a-3p was involved in the process of nerve damage caused by PHEV(porcine hemagglutinating encephalomyelitis virus) infection,this study used quantitative real-time PCR to detect the expression of miR-142a-3p in N2 a cells and BALB/c mouse brain tissues infected with PHEV at different time. The results showed that the expression level of miR-142a-3p was significantly up-regulated after PHEV infection in vivo and in vitro.Furthermore,based on the prediction and analysis of bioinformatics software,it was found that transmembrane protein 59(Tmem59) might be a target gene of miR-142a-3p. Dual-Luciferase reporter assay identified that miR-142a-3p was directly bound to Tmem59 mRNA. The results of this study demonstrated that the expression of miR-142a-3p was up-regulated in host infected with PHEV,and screened its mechanism-related target gene-Tmem59.
In order to determine whether host miR-142a-3p was involved in the process of nerve damage caused by PHEV(porcine hemagglutinating encephalomyelitis virus) infection,this study used quantitative real-time PCR to detect the expression of miR-142a-3p in N2 a cells and BALB/c mouse brain tissues infected with PHEV at different time. The results showed that the expression level of miR-142a-3p was significantly up-regulated after PHEV infection in vivo and in vitro.Furthermore,based on the prediction and analysis of bioinformatics software,it was found that transmembrane protein 59(Tmem59) might be a target gene of miR-142a-3p. Dual-Luciferase reporter assay identified that miR-142a-3p was directly bound to Tmem59 mRNA. The results of this study demonstrated that the expression of miR-142a-3p was up-regulated in host infected with PHEV,and screened its mechanism-related target gene-Tmem59.
引文
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[13] Li X,Feng R L,Huang C,et al.MicroRNA-351 regulates TMEM59 (DCF1) expression and mediates neural stem cell morphogenesis[J].Rna Biology,2012,9(3):292-301.
[14] Hong Y,Zhao T,Li X J,et al.Mutant Huntingtin Impairs BDNF Release from Astrocytes by Disrupting Conversion of Rab3a-GTP into Rab3a-GDP[J].Journal of Neuroscience,2016,36(34):8790-8801.
[15] Zhang L W,Ju X C,Cheng Y M,et al.Identifying Tmem59 related gene regulatory network of mouse neural stem cell from a compendium of expression profiles[J].Bmc Systems Biology,2011,5(1):152-161.
[2] Gao W,Zhao K,Zhao C B,et al.Vomiting and wasting disease associated with Hemagglutinating Encephalomyelitis Viruses infection in piglets in Jilin, China[J].Virology Journal,2011,8(1):1-7.
[3] Hara Y,Hasebe R,Sunden Y,et al.Propagation of Swine Hemagglutinating Encephalomyelitis Virus and Pseudorabies Virus in Dorsal Root Ganglia Cells[J].Journal of Veterinary Medical Science,2009,71(5):595-601.
[4] Lan Y G,Zhao K,Zhao J K,et al.Gene-expression patterns in the cerebral cortex of mice infected with porcine haemagglutinating encephalomyelitis virus detected using microarray[J].Journal of General Virology,2014,95(10):2192-2203.
[5] Shrestha A,Carraro G,El Agha E,et al.Generation and Validation of miR-142 Knock Out Mice[J].Plos One,2015,10(9):1932-1944.
[6] Li Z,Lan Y G,Zhao K,et al.miR-142-5p Disrupts Neuronal Morphogenesis Underlying Porcine Hemagglutinating Encephalomyelitis Virus Infection by Targeting Ulk1[J].Frontiers in Cellular and Infection Microbiology,2017,12(7):2235-2244.
[7] Lv X,Zhao K,Lan Y,et al.miR-21a-5p Contributes to Porcine Hemagglutinating Encephalomyelitis Virus Proliferation via Targeting CASK-Interactive Protein1 In vivo and vitro[J].Front Microbiol,2017,8(1):304-311.
[8] Greig A S,Mitchell D,Corner A H,et al.A Hemagglutinating Virus Producing Encephalomyelitis in Baby Pigs[J].Can J Comp Med Vet Sci,1962,26(3):49-56.
[9] Li Y C,Bai W Z,Hirano N,et al.Neurotropic virus tracing suggests a membranous-coating-mediated mechanism for transsynaptic communication[J].Journal of Comparative Neurology,2013,521(1):203-212.
[10] Lan Y G,Zhao K,Wang G L,et al.Porcine hemagglutinating encephalomyelitis virus induces apoptosis in a porcine kidney cell line via caspase-dependent pathways[J].Virus Research,2013,176(1-2):292-297.
[11] Han B, Zhang Y,Zhang Y H,et al. Novel insight into circular RNA HECTD1 in astrocyte activation via autophagy by targeting MIR142-TIPARP: implications for cerebral ischemic stroke[J].Autophagy,2018,14(7):1164-1184.
[12] Wan T Q,Gu P,Che F X.Discovery of two novel functional genes from differentiation of neural stem cells in the striatum of the fetal rat[J].Neuroscience Letters,2002,329(1):101-105.
[13] Li X,Feng R L,Huang C,et al.MicroRNA-351 regulates TMEM59 (DCF1) expression and mediates neural stem cell morphogenesis[J].Rna Biology,2012,9(3):292-301.
[14] Hong Y,Zhao T,Li X J,et al.Mutant Huntingtin Impairs BDNF Release from Astrocytes by Disrupting Conversion of Rab3a-GTP into Rab3a-GDP[J].Journal of Neuroscience,2016,36(34):8790-8801.
[15] Zhang L W,Ju X C,Cheng Y M,et al.Identifying Tmem59 related gene regulatory network of mouse neural stem cell from a compendium of expression profiles[J].Bmc Systems Biology,2011,5(1):152-161.