本文關(guān)鍵詞：microRNA在TGEV感染PK-15細(xì)胞過(guò)程中的作用與調(diào)控機(jī)制　出處：《西北農(nóng)林科技大學(xué)》2016年博士論文　論文類(lèi)型：學(xué)位論文

  更多相關(guān)文章： TGEV microRNA PK-15細(xì)胞 細(xì)胞凋亡

【摘要】：microRNA(miRNA)是一類(lèi)長(zhǎng)18~23個(gè)核苷酸的非編碼RNA,在生長(zhǎng)、發(fā)育、病毒復(fù)制、線粒體損傷和細(xì)胞凋亡等很多生物學(xué)過(guò)程中發(fā)揮重要作用。豬傳染性胃腸炎病毒(transmissible gastroenteritis virus,TGEV)感染PK-15細(xì)胞導(dǎo)致線粒體損傷,并通過(guò)死亡受體通路和線粒體通路誘導(dǎo)PK-15細(xì)胞凋亡,然而,在TGEV感染PK-15細(xì)胞過(guò)程中miRNA是否參與了調(diào)控TGEV復(fù)制、線粒體損傷和細(xì)胞凋亡,目前尚不清楚。本論文擬用TGEV感染PK-15細(xì)胞,檢測(cè)TGEV感染后細(xì)胞miRNA表達(dá)譜的變化,篩選差異表達(dá)較大的miRNA,鑒定miRNA的靶基因,研究mi RNA及靶基因?qū)GEV復(fù)制、線粒體損傷和細(xì)胞凋亡的作用。研究取得如下結(jié)果。1.TGEV感染對(duì)miRNA表達(dá)譜的影響。利用miRNA芯片檢測(cè)TGEV感染PK-15細(xì)胞后miRNA表達(dá)譜的變化,得到21個(gè)差異表達(dá)顯著的miRNA(fold change1.5,p0.01),其中13個(gè)miRNA表達(dá)量上調(diào),8個(gè)miRNA表達(dá)量下調(diào)。Real-time PCR驗(yàn)證這21個(gè)miRNA表達(dá)量變化,驗(yàn)證發(fā)現(xiàn)與miRNA芯片檢測(cè)結(jié)果一致。采用TargetScan和miRanda算法預(yù)測(cè)21個(gè)差異表達(dá)miRNA的靶基因,得到7 844個(gè)靶基因。對(duì)預(yù)測(cè)到的靶基因進(jìn)行GO和KEGG注釋和聚類(lèi)分析,發(fā)現(xiàn)靶基因參與了Toll樣受體、RIG-I樣受體和MAPK等信號(hào)通路,以及蛋白結(jié)合、鋅離子結(jié)合、蛋白質(zhì)磷酸化和蛋白激酶活性等生物學(xué)過(guò)程。2.miR-4331抑制TGEV gene 7轉(zhuǎn)錄。將miR-4331 mimics或inhibtors(各自相對(duì)應(yīng)的control)分別轉(zhuǎn)染PK-15細(xì)胞,再用TGEV感染細(xì)胞,real-time PCR檢測(cè)miR-4331對(duì)TGEV轉(zhuǎn)錄的影響,結(jié)果發(fā)現(xiàn),miR-4331抑制TGEV gene 7的轉(zhuǎn)錄(p0.01),對(duì)TGEV基因組和其他亞基因組的轉(zhuǎn)錄沒(méi)有影響(p0.05)。篩選與病毒轉(zhuǎn)錄相關(guān)的16個(gè)靶基因,構(gòu)建含靶基因3′UTR的重組雙熒光素酶質(zhì)粒,檢測(cè)miR-4331與靶基因3′UTR的結(jié)合活性,結(jié)果發(fā)現(xiàn),過(guò)表達(dá)miR-4331后只有含CDCA7 3′UTR重組質(zhì)粒的細(xì)胞雙熒光素酶活性顯著降低(p0.01)。將miR-4331 mimics或mimics control轉(zhuǎn)染至PK-15細(xì)胞,western blotting檢測(cè)結(jié)果發(fā)現(xiàn),過(guò)表達(dá)miR-4331后CDCA7蛋白水平明顯降低。設(shè)計(jì)合成CDCA7的siRNA,構(gòu)建重組CDCA7真核表達(dá)質(zhì)粒,將CDCA7的siRNA或重組CDCA7真核表達(dá)質(zhì)粒轉(zhuǎn)染至PK-15細(xì)胞,檢測(cè)TGEV gene 7轉(zhuǎn)錄水平,結(jié)果發(fā)現(xiàn),過(guò)表達(dá)CDCA7抑制TGEV gene 7的轉(zhuǎn)錄(p0.01),抑制CDCA7表達(dá)則促進(jìn)TGEV gene7轉(zhuǎn)錄(p0.01)。3.miR-27b抑制TGEV誘導(dǎo)的細(xì)胞凋亡。將miR-27b mimics或inhibtors(及其相對(duì)應(yīng)的control)分別轉(zhuǎn)染PK-15細(xì)胞,再用TGEV感染細(xì)胞,檢測(cè)細(xì)胞凋亡率、caspase-3、caspase-9和caspase-8活性。結(jié)果發(fā)現(xiàn),miR-27b mimics(過(guò)表達(dá)miR-27b)顯著抑制PK-15細(xì)胞凋亡率(p0.05)、caspase-3(p0.05)和caspase-9活性(p0.05),對(duì)caspase-8活性沒(méi)有影響(p0.05);miR-27 inhibitors(抑制miR-27b表達(dá))增加TGEV誘導(dǎo)的細(xì)胞凋亡率(p0.05)、caspase-3(p0.05)和caspase-9的活性(p0.05),對(duì)caspase-8沒(méi)有影響(p0.05)。篩選與細(xì)胞凋亡相關(guān)的10個(gè)靶基因,構(gòu)建含靶基因3′UTR的重組雙熒光素酶質(zhì)粒,檢測(cè)miR-27b與靶基因3′UTR的結(jié)合活性,發(fā)現(xiàn)只有miR-27b mimics顯著降低RUNX1重組質(zhì)粒的海腎熒光素酶表達(dá)(p0.01)。western blotting檢測(cè)發(fā)現(xiàn)miR-27b mimics明顯降低RUNX1蛋白水平。設(shè)計(jì)合成RUNX1的siRNA,構(gòu)建重組RUNX1真核表達(dá)質(zhì)粒,檢測(cè)在TGEV感染PK-15細(xì)胞過(guò)程中,RUNX1對(duì)線粒體通路的調(diào)控作用。過(guò)表達(dá)RUNX1可以促進(jìn)Bax的表達(dá),增加caspase-9(p0.05)和caspase-3的活性(p0.05),siRNA干擾RUNX1的表達(dá)導(dǎo)致Bax表達(dá)量下調(diào),抑制caspase-9(p0.05)和caspase-3的活性(p0.05)。4.miR-222抑制TGEV誘導(dǎo)細(xì)胞線粒體損傷。將mi R-222 mimics或者mimics control轉(zhuǎn)染至PK-15細(xì)胞,然后感染TGEV,利用JC1和Rhod-2分別檢測(cè)線粒體膜電位和鈣離子濃度的變化,結(jié)果發(fā)現(xiàn),過(guò)表達(dá)miR-222則線粒體膜電位增加了1倍(p0.01)、鈣離子濃度降低了38%(p0.01)。iTRAQ檢測(cè)miR-222過(guò)表達(dá)后TGEV感染PK-15細(xì)胞中蛋白表達(dá)譜變化,發(fā)現(xiàn)100個(gè)差異表達(dá)蛋白(蛋白量大于1.2倍或小于0.83倍為差異表達(dá)),其中57個(gè)蛋白表達(dá)量上調(diào),43個(gè)蛋白表達(dá)量下調(diào)。構(gòu)建9個(gè)含靶基因3′UTR的重組雙熒光素酶質(zhì)粒,檢測(cè)miR-222與靶基因3′UTR的結(jié)合活性,發(fā)現(xiàn)只有miR-222mimics顯著降低THBS1重組質(zhì)粒的海腎熒光素酶活性(p0.01)。western blotting檢測(cè)發(fā)現(xiàn)miR-222 mimics明顯降低THBS1蛋白水平。檢測(cè)在TGEV感染PK-15細(xì)胞過(guò)程中,THBS1 siRNA對(duì)線粒體損傷的調(diào)控作用。干擾THBS1表達(dá)(THBS1 siRNA)線粒體膜電位增加了1倍(p0.01),鈣離子濃度降低了60%(p0.01)。本研究發(fā)現(xiàn)TGEV誘導(dǎo)PK-15細(xì)胞mi RNA表達(dá)譜發(fā)生了改變,進(jìn)一步研究差異表達(dá)的miRNA在TGEV感染PK-15細(xì)胞中的作用,發(fā)現(xiàn)miR-4331抑制TGEV gene 7轉(zhuǎn)錄,miR-27b抑制TGEV誘導(dǎo)的細(xì)胞凋亡,miR-222抑制TGEV誘導(dǎo)細(xì)胞線粒體損傷。研究結(jié)果闡明了差異表達(dá)的miRNA在TGEV感染PK-15細(xì)胞過(guò)程中的作用與調(diào)控機(jī)制,為進(jìn)一步研究miRNA與TGEV的相互作用提供理論依據(jù)。
[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.

【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類(lèi)號(hào)】：S855.3

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