microRNA在TGEV感染PK-15细胞过程中的作用与调控机制

发布时间：2018-01-12 01:29

本文关键词：microRNA在TGEV感染PK-15细胞过程中的作用与调控机制　出处：《西北农林科技大学》2016年博士论文　论文类型：学位论文

更多相关文章： TGEV microRNA PK-15细胞 细胞凋亡

【摘要】：microRNA(miRNA)是一类长18~23个核苷酸的非编码RNA,在生长、发育、病毒复制、线粒体损伤和细胞凋亡等很多生物学过程中发挥重要作用。猪传染性胃肠炎病毒(transmissible gastroenteritis virus,TGEV)感染PK-15细胞导致线粒体损伤,并通过死亡受体通路和线粒体通路诱导PK-15细胞凋亡,然而,在TGEV感染PK-15细胞过程中miRNA是否参与了调控TGEV复制、线粒体损伤和细胞凋亡,目前尚不清楚。本论文拟用TGEV感染PK-15细胞,检测TGEV感染后细胞miRNA表达谱的变化,筛选差异表达较大的miRNA,鉴定miRNA的靶基因,研究mi RNA及靶基因对TGEV复制、线粒体损伤和细胞凋亡的作用。研究取得如下结果。1.TGEV感染对miRNA表达谱的影响。利用miRNA芯片检测TGEV感染PK-15细胞后miRNA表达谱的变化,得到21个差异表达显著的miRNA(fold change1.5,p0.01),其中13个miRNA表达量上调,8个miRNA表达量下调。Real-time PCR验证这21个miRNA表达量变化,验证发现与miRNA芯片检测结果一致。采用TargetScan和miRanda算法预测21个差异表达miRNA的靶基因,得到7 844个靶基因。对预测到的靶基因进行GO和KEGG注释和聚类分析,发现靶基因参与了Toll样受体、RIG-I样受体和MAPK等信号通路,以及蛋白结合、锌离子结合、蛋白质磷酸化和蛋白激酶活性等生物学过程。2.miR-4331抑制TGEV gene 7转录。将miR-4331 mimics或inhibtors(各自相对应的control)分别转染PK-15细胞,再用TGEV感染细胞,real-time PCR检测miR-4331对TGEV转录的影响,结果发现,miR-4331抑制TGEV gene 7的转录(p0.01),对TGEV基因组和其他亚基因组的转录没有影响(p0.05)。筛选与病毒转录相关的16个靶基因,构建含靶基因3′UTR的重组双荧光素酶质粒,检测miR-4331与靶基因3′UTR的结合活性,结果发现,过表达miR-4331后只有含CDCA7 3′UTR重组质粒的细胞双荧光素酶活性显著降低(p0.01)。将miR-4331 mimics或mimics control转染至PK-15细胞,western blotting检测结果发现,过表达miR-4331后CDCA7蛋白水平明显降低。设计合成CDCA7的siRNA,构建重组CDCA7真核表达质粒,将CDCA7的siRNA或重组CDCA7真核表达质粒转染至PK-15细胞,检测TGEV gene 7转录水平,结果发现,过表达CDCA7抑制TGEV gene 7的转录(p0.01),抑制CDCA7表达则促进TGEV gene7转录(p0.01)。3.miR-27b抑制TGEV诱导的细胞凋亡。将miR-27b mimics或inhibtors(及其相对应的control)分别转染PK-15细胞,再用TGEV感染细胞,检测细胞凋亡率、caspase-3、caspase-9和caspase-8活性。结果发现,miR-27b mimics(过表达miR-27b)显著抑制PK-15细胞凋亡率(p0.05)、caspase-3(p0.05)和caspase-9活性(p0.05),对caspase-8活性没有影响(p0.05);miR-27 inhibitors(抑制miR-27b表达)增加TGEV诱导的细胞凋亡率(p0.05)、caspase-3(p0.05)和caspase-9的活性(p0.05),对caspase-8没有影响(p0.05)。筛选与细胞凋亡相关的10个靶基因,构建含靶基因3′UTR的重组双荧光素酶质粒,检测miR-27b与靶基因3′UTR的结合活性,发现只有miR-27b mimics显著降低RUNX1重组质粒的海肾荧光素酶表达(p0.01)。western blotting检测发现miR-27b mimics明显降低RUNX1蛋白水平。设计合成RUNX1的siRNA,构建重组RUNX1真核表达质粒,检测在TGEV感染PK-15细胞过程中,RUNX1对线粒体通路的调控作用。过表达RUNX1可以促进Bax的表达,增加caspase-9(p0.05)和caspase-3的活性(p0.05),siRNA干扰RUNX1的表达导致Bax表达量下调,抑制caspase-9(p0.05)和caspase-3的活性(p0.05)。4.miR-222抑制TGEV诱导细胞线粒体损伤。将mi R-222 mimics或者mimics control转染至PK-15细胞,然后感染TGEV,利用JC1和Rhod-2分别检测线粒体膜电位和钙离子浓度的变化,结果发现,过表达miR-222则线粒体膜电位增加了1倍(p0.01)、钙离子浓度降低了38%(p0.01)。iTRAQ检测miR-222过表达后TGEV感染PK-15细胞中蛋白表达谱变化,发现100个差异表达蛋白(蛋白量大于1.2倍或小于0.83倍为差异表达),其中57个蛋白表达量上调,43个蛋白表达量下调。构建9个含靶基因3′UTR的重组双荧光素酶质粒,检测miR-222与靶基因3′UTR的结合活性,发现只有miR-222mimics显著降低THBS1重组质粒的海肾荧光素酶活性(p0.01)。western blotting检测发现miR-222 mimics明显降低THBS1蛋白水平。检测在TGEV感染PK-15细胞过程中,THBS1 siRNA对线粒体损伤的调控作用。干扰THBS1表达(THBS1 siRNA)线粒体膜电位增加了1倍(p0.01),钙离子浓度降低了60%(p0.01)。本研究发现TGEV诱导PK-15细胞mi RNA表达谱发生了改变,进一步研究差异表达的miRNA在TGEV感染PK-15细胞中的作用,发现miR-4331抑制TGEV gene 7转录,miR-27b抑制TGEV诱导的细胞凋亡,miR-222抑制TGEV诱导细胞线粒体损伤。研究结果阐明了差异表达的miRNA在TGEV感染PK-15细胞过程中的作用与调控机制,为进一步研究miRNA与TGEV的相互作用提供理论依据。
[Abstract]:MicroRNA (miRNA) is a non encoding RNA, a class of 18~23 nucleotides in the growth, development, play an important role in virus replication, mitochondrial damage and apoptosis in many biological processes. Porcine transmissible gastroenteritis virus (transmissible gastroenteritis, virus, TGEV) infected PK-15 cells resulted in the injury of mitochondria, and by inducing PK-15 cell apoptosis and death the receptor pathway and the mitochondrial pathway. However, in TGEV infected PK-15 cells in the process of miRNA is involved in the regulation of TGEV replication, mitochondrial damage and apoptosis is unclear. This paper intends to use the TGEV infection of PK-15 cells, the detection of TGEV infection after cell miRNA expression, screening the differential expression of large miRNA, target gene identification miRNA the study of MI, RNA and target genes of TGEV replication, mitochondrial damage and apoptosis. Studies on the expression of miRNA spectrum of.1.TGEV infection are as follows . expression of miRNA using miRNA chip for detection of TGEV infected PK-15 cells, expression of miRNA 21 (fold change1.5, P0.01 difference), of which 13 miRNA up-regulated the expression of miRNA 8, PCR 21 to verify the down-regulation of.Real-time miRNA expression changes, and verify that miRNA chip test results using TargetScan and miRanda algorithm. The expression of target gene miRNA 21 difference prediction, 7844 target genes. Analysis of GO and KEGG of target gene annotation and clustering of predicted target genes, found in the Toll like receptor, RIG-I like receptor and MAPK signaling pathways, and protein binding, zinc ion binding. Protein phosphorylation and protein kinase activity in biological process of.2.miR-4331 inhibition of TGEV gene 7. MiR-4331 mimics or inhibtors transcription (corresponding to control) were transfected into PK-15 cells, and then infected with TGEV fine Effect of real-time cell, PCR detection of miR-4331 transcription of TGEV showed that miR-4331 inhibited the transcription of TGEV gene 7 (P0.01), had no effect on transcription of TGEV genome and other sub genome (P0.05). 16 genes related to screening and viral transcription, constructs containing the target gene 3 'UTR recombinant luciferase detection of plasmid, miR-4331 and target gene 3' UTR binding activity, found that over expression of miR-4331 after only 3 'CDCA7 containing recombinant plasmid UTR cell dual luciferase activity was significantly decreased (P0.01). The miR-4331 mimics or mimics control transfected into PK-15 cells, Western blotting test results found that overexpression of CDCA7 protein levels significantly decreased after miR-4331. SiRNA design and synthesis of CDCA7, construction of recombinant eukaryotic expression plasmid of CDCA7, CDCA7 siRNA or CDCA7 recombinant eukaryotic expression plasmid was transfected into PK-15 cells, detect TGEV gene transcription level 7,. The results showed that the overexpression of CDCA7 transcription inhibition of TGEV gene 7 (P0.01), the inhibition of CDCA7 expression by promoting TGEV transcription gene7 (P0.01).3.miR-27b inhibit the apoptosis induced by TGEV. The miR-27b mimics or inhibtors (and the control) were transfected into PK-15 cells, then TGEV infected cells, the cell apoptosis rate was detected by caspase-3. Caspase-9 and caspase-8 activity. The results showed that miR-27b mimics (expression of miR-27b) significantly inhibited the apoptosis rate of PK-15 cells (P0.05), caspase-3 (P0.05) and caspase-9 activity (P0.05), has no effect on the activity of caspase-8 (P0.05); miR-27 inhibitors (inhibiting miR-27b expression) increased TGEV induced apoptosis rate (P0.05). Caspase-3 (P0.05) and caspase-9 activity (P0.05), had no effect on caspase-8 (P0.05). 10 genes related to screening and apoptosis, constructs containing the target gene 3 'UTR recombinant plasmid miR-27b dual luciferase detection. Binding activity and target gene 3 'UTR, found that only miR-27b mimics significantly decreased the expression of recombinant plasmid RUNX1 of Renilla luciferase (P0.01).Western blotting assay showed that miR-27b mimics significantly decreased the protein level of RUNX1 siRNA. The design and synthesis of RUNX1, construction of recombinant eukaryotic expression plasmid of RUNX1, detected in TGEV infected PK-15 cells in the process of regulation RUNX1 on the mitochondrial pathway. The overexpression of RUNX1 can promote the expression of Bax, caspase-9 (P0.05) and caspase-3 activity (P0.05), the expression of siRNA by RUNX1 interference leads to down-regulation of Bax expression, inhibition of caspase-9 (P0.05) and the activity of Caspase-3 (P0.05).4.miR-222 inhibited TGEV cell mitochondrial damage induced by Mi R-222 mimics. Mimics or control was transfected into PK-15 cells, and TGEV infection, changes were detected in the mitochondrial membrane potential and calcium ion concentration using JC1 and Rhod-2 found that over the table MiR-222 is the mitochondrial membrane potential increased 1 times (P0.01), calcium concentration decreased by 38% (P0.01).ITRAQ detection miR-222 spectrum changes of PK-15 protein expression in cells infected with TGEV expression, 100 differentially expressed proteins found (protein content is 1.2 times greater than or less than 0.83 times the difference, of which 57 protein expression) up-regulated, 43 protein expression was down regulated. The construction of 9 target genes containing 3 'UTR recombinant dual luciferase reporter plasmid, detection of miR-222 binding activity and target gene 3' UTR, found in enzyme activity of Renilla miR-222mimics significantly decreased the fluorescence only the recombinant plasmid THBS1 (P0.01).Western blotting assay showed that miR-222 significantly decreased mimics the THBS1 protein level in TGEV infected PK-15 cells. The detection of THBS1, siRNA on regulation of mitochondrial injury. The expression of THBS1 (THBS1 siRNA) disturbance of mitochondrial membrane potential increased 1 times (P0.01), calcium ion concentration drop Low 60% (P0.01). The study found that the expression change of PK-15 cells induced by RNA mi TGEV, to further study the differential expression of miRNA in TGEV infected PK-15 cells in the role of TGEV gene 7 miR-4331 was found to inhibit the transcription inhibition of miR-27b induced apoptosis of TGEV cells, miR-222 inhibited TGEV cell mitochondrial damage induced by the results. The expression of miRNA in TGEV infected PK-15 cells in the process of the role and regulation mechanism, provide a theoretical basis for further research on the interaction of miRNA and TGEV.

【学位授予单位】：西北农林科技大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S855.3

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