PK-15细胞中与CSFV感染相关的microRNAs筛选及miR-214的功能研究
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摘要
【目的】利用制作的猪的miRNA表达谱芯片筛选猪瘟病毒(classical swine fever virus,CSFV)感染PK-15细胞后表达有差异的miRNA,并进一步探讨其中表达差异较明显的miRNA的作用和功能,从miRNA的角度探究CSFV的致病机制,为猪瘟(classical swine fever,CSF)的防控提供新依据。【方法】为了研究CSFV感染PK-15细胞后miRNA表达的变化情况,根据miRBase version 19.0数据库中326条猪的miRNA合成探针,采用原位合成技术制作表达谱芯片,筛选得到CSFV感染PK-15细胞后表达有差异的miRNA。挑选CSFV感染PK-15细胞后表达差异最明显的miR-214作为进一步研究对象,研究miR-214在CSFV感染过程中的功能和作用。CSFV感染PK-15细胞后,用荧光定量PCR检测miR-214的mRNA表达水平。为了进一步研究miR-214对CSFV感染的影响,合成miR-214模拟物及抑制物,并分别转染PK-15细胞,转染后24 h感染CSFV,感染后48h用荧光定量PCR检测CSFV的基因拷贝数,用间接免疫荧光方法检测CSFV的病毒滴度。为了进一步探究miR-214参与调控CSFV复制的机制,通过生物信息学软件预测miR-214参与调控CSFV复制的靶蛋白,并用荧光素酶报告基因系统进一步确证miR-214能够靶向作用于凋亡通路中重要分子TNFR1相关的死亡区域蛋白(TNF Receptor-Associated Death Domain,TRADD),因此猜测miR-214通过影响靶蛋白TRADD的表达水平从而影响PK-15细胞凋亡,将miR-214模拟物及抑制物分别转染PK-15细胞,转染后24 h感染CSFV,同步设立不感染CSFV的细胞对照,感染后48 h用荧光定量PCR和Western blot分别检测TRADD的mRNA和蛋白表达量的变化,同时用流式细胞术检测miR-214对CSFV感染的PK-15细胞凋亡的影响。【结果】CSFV感染PK-15细胞后,通过表达谱芯片技术筛选得到69条表达有差异的miRNA,其中miR-214表达量上调且差异最明显。qRT-PCR结果显示,CSFV感染PK-15细胞后miR-214的mRNA表达量上调,验证了表达谱芯片的结果。将miR-214模拟物转染PK-15细胞后再感染CSFV, CSFV的基因拷贝数及病毒滴度均显著下降;转染miR-214抑制物后再感染CSFV,CSFV的基因拷贝数及病毒滴度均显著上升,表明miR-214促进了CSFV的复制。为了进一步探究miR-214促进CSFV复制的机制,用生物信息学软件预测TRADD为miR-214的靶蛋白,荧光素酶报告基因系统验证了miR-214能够靶向作用于TRADD。转染miR-214模拟物后,PK-15细胞中TRADD的mRNA和蛋白表达量均显著上升,而转染miR-214抑制物后,PK-15细胞中TRADD的mRNA和蛋白表达量均显著下降,表明miR-214抑制TRADD的表达。通过流式细胞术,验证了CSFV感染PK-15抑制细胞凋亡,miR-214抑制CSFV感染的PK-15细胞凋亡。【结论】CSFV感染PK-15后,上调细胞内miR-214的表达。miR-214能通过靶向抑制TRADD蛋白的表达,从而抑制PK-15细胞凋亡,促进CSFV在细胞内的复制。
【Objective】 In this study, differential expression of miRNAs in CSFV-infected PK-15 cells were determined by miRNA expression array, and further explore the function of miRNAs in the pathogenic of Classical swine fever virus(CSFV), and provide some new basis for the prevention and control of Classical swine fever(CSF).【Method】 In order to investigate the changes of the miRNAs expression in CSFV-infected PK-15 cells, we synthesized probes of 326 miRNAs of pig according to the miRBase database version 19.0, and screening of differential expression of miRNAs in CSFV-infected PK-15 cells using miRNA expression array. Then, miR-214, the most obvious difference in expression of CSFV-infected PK-15 cells, was selected as the further study object to investigate the function of miR-214 in the infection process of CSFV. We detected mRNA expression of miR-214 in CSFV-infected PK-15 cells using qRT-PCR. In order to further study the effect of miR-214 of CSFV infection, we synthesized miR-214 analog and inhibitor and transfected into PK-15 cells respectively, follow with CSFV infection at 24 h post-transfection, and then detected CSFV titers and quantity of CSFV genomic copies. In order to further explore the mechanism of miR-214 participate in the regulation of CSFV replication, we predicted the target protein of miR-214 using bioinformatics software and confirmed it by luciferase reporter gene system. Given TRADD can specific interacts with TNFR1 intracellular dead zones and participate in the programmed cell death, we assume that miR-214 influencing apoptosis of PK 15 cells by influencing expression level of target protein TRADD. PK 15 cells transfected with miR-214 and inhibitor respectively, follow with CSFV infection at 24 h post-transfection. At 48 h post-infection, the expression levels of TRADD were detected, and the effect of miR-214 on the apoptosis of CSFV-infected PK-15 cells was detected by flow cytometry. 【Result】 69 miRNAs with different expressions were screened by miRNA expression array in CSFV-infected PK-15 cells. Among which the expression changes of miR-214 were most obvious and up-regulated, and confirmed it by qRT-PCR. After transfected with miR-214 to PK-15 cells, CSFV titers and quantity of CSFV genomic copies decreased significantly, while transfected with miR-214 inhibitor, CSFV titers and quantity of CSFV genomic copies were increased significantly, which suggested that miR-214 promoted the replication of CSFV. In order to further explore the mechanism of miR-214 promoting CSFV replication, we confirmed TRADD is the target protein of miR-214. After transfected miR-214 to PK-15 cells, mRNA and protein expression of TRADD were increased significantly, while transfected with miR-214 inhibitor, that were decreased significantly, suggesting that miR-214.inhibits the expression of TRADD. And then, we verified that CSFV infection inhibits apoptosis of PK-15 cells, and miR-214 inhibits apoptosis of CSFV-infected PK-15 cells. 【Conclusion】 The expression of miR-214 in cells was up-regulated after CSFV infected PK-15. miR-214 inhibits the apoptosis of PK-15 cells and promote the replication of CSFV in cells by targeting inhibits the expression of TRADD protein.
【Objective】 In this study, differential expression of miRNAs in CSFV-infected PK-15 cells were determined by miRNA expression array, and further explore the function of miRNAs in the pathogenic of Classical swine fever virus(CSFV), and provide some new basis for the prevention and control of Classical swine fever(CSF).【Method】 In order to investigate the changes of the miRNAs expression in CSFV-infected PK-15 cells, we synthesized probes of 326 miRNAs of pig according to the miRBase database version 19.0, and screening of differential expression of miRNAs in CSFV-infected PK-15 cells using miRNA expression array. Then, miR-214, the most obvious difference in expression of CSFV-infected PK-15 cells, was selected as the further study object to investigate the function of miR-214 in the infection process of CSFV. We detected mRNA expression of miR-214 in CSFV-infected PK-15 cells using qRT-PCR. In order to further study the effect of miR-214 of CSFV infection, we synthesized miR-214 analog and inhibitor and transfected into PK-15 cells respectively, follow with CSFV infection at 24 h post-transfection, and then detected CSFV titers and quantity of CSFV genomic copies. In order to further explore the mechanism of miR-214 participate in the regulation of CSFV replication, we predicted the target protein of miR-214 using bioinformatics software and confirmed it by luciferase reporter gene system. Given TRADD can specific interacts with TNFR1 intracellular dead zones and participate in the programmed cell death, we assume that miR-214 influencing apoptosis of PK 15 cells by influencing expression level of target protein TRADD. PK 15 cells transfected with miR-214 and inhibitor respectively, follow with CSFV infection at 24 h post-transfection. At 48 h post-infection, the expression levels of TRADD were detected, and the effect of miR-214 on the apoptosis of CSFV-infected PK-15 cells was detected by flow cytometry. 【Result】 69 miRNAs with different expressions were screened by miRNA expression array in CSFV-infected PK-15 cells. Among which the expression changes of miR-214 were most obvious and up-regulated, and confirmed it by qRT-PCR. After transfected with miR-214 to PK-15 cells, CSFV titers and quantity of CSFV genomic copies decreased significantly, while transfected with miR-214 inhibitor, CSFV titers and quantity of CSFV genomic copies were increased significantly, which suggested that miR-214 promoted the replication of CSFV. In order to further explore the mechanism of miR-214 promoting CSFV replication, we confirmed TRADD is the target protein of miR-214. After transfected miR-214 to PK-15 cells, mRNA and protein expression of TRADD were increased significantly, while transfected with miR-214 inhibitor, that were decreased significantly, suggesting that miR-214.inhibits the expression of TRADD. And then, we verified that CSFV infection inhibits apoptosis of PK-15 cells, and miR-214 inhibits apoptosis of CSFV-infected PK-15 cells. 【Conclusion】 The expression of miR-214 in cells was up-regulated after CSFV infected PK-15. miR-214 inhibits the apoptosis of PK-15 cells and promote the replication of CSFV in cells by targeting inhibits the expression of TRADD protein.
引文
[1]MOENNIG V.Introduction to classical swine fever:virus,disease and control policy.Veterinary Microbiology,2000,73(2/3):93-102.
[2]STEGEMAN A,ELBERS A,DE SMIT H.The 1997-1998 epidemic of classical swine fever in the Netherlands.Veterinary Microbiology,2000,73(2/3):183-196.
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[5]MICHAEL M Z,O'C S,VAN HOLST P N,YOUNG G P,JAMES RJ.Reduced accumulation of specific microRNAs in colorectal neoplasia.Molecular Cancer Research,2003,1(12):882-891.
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[7]ZHAO H,WEN G,HUANG Y,YU X,CHEN Q,AFZAL T A,LUONG LE A,ZHU J,YE S,ZHANG L,XIAO Q.Microrna-22regulates smooth muscle cell differentiation from stem cells by targeting methyl cpg-binding protein 2.Arteriosclerosis,Thrombosis,and Vascular Biology,2015,35(4):918-929.
[8]YANG M,LIU R,LI X,LIAO J,PU Y,PAN E,YIN L,WANG Y.Mirna-183 suppresses apoptosis and promotes proliferation in esophageal cancer by targeting pdcd4.Molecular Cells,2014,37(12):873-880.
[9]XU P,VERNOOY S Y,GUO M,HAY B A.The drosophila microRNAmir-14 suppresses cell death and is required for normal fat metabolism.Current Biology,2003,13(9):790-795.
[10]BOUTLA A,DELIDAKIS C,TABLER M.Developmental defects by antisense-mediated inactivation of micro-RNAs 2 and 13 in drosophila and the identification of putative target genes.Nucleic Acids Research,2003,31(17):4973-4980.
[11]NEILSON J R,ZHENG G X,BURGE C B,SHARP P A.Dynamic regulation of miRNA expression in ordered stages of cellular development.Genes Development,2007,21(5):578-589.
[12]LI Q J,CHAU J,EBERT P J,SYLVESTER G,MIN H,LIU G,BRAICH R,MANOHARAN M,SOUTSCHEK J,SKARE P,KLEINL O,DAVIS M M,CHEN C Z.Mir-181a is an intrinsic modulator of t cell sensitivity and selection.Cell,2007,129(1):147-161.
[13]VIGORITO E,PERKS K L,ABREU-GOODGER C,BUNTING S,XIANG Z,KOHLHAAS S,DAS PP,MISKA E A,RODRIGUEZ A,BRADLEY A,SMITH KG,RADA C,ENRIGHT A J,TOELLNERK M,MACLENNAN I C,TURNER M.Microrna-155 regulates the generation of immunoglobulin class-switched plasma cells.Immunity,2007,27(6):847-859.
[14]KUMAR A.RNA interference:a multifaceted innate antiviral defense.Retrovirology,2008,5:17.
[15]LECELLIER C H,DUNOYER P,ARAR K,LEHMANN-CHE J,EYQUEM S,HIMBER C,SA?B A,VOINNET O.A Cellular microRNA mediates antiviral defense in human cells.Science,2005,308(5721):557-560.
[16]TILI E,MICHAILLE J J,CIMINO A,COSTINEAN S,DUMITRUC D,ADAIR B,FABBRI M,ALDER H,LIU C G,CALIN G A,CROCE C M.Modulation of Mir-155 and Mir-125B levels following lipopolysaccharide/Tnf-Alpha stimulation and their possible roles in regulating the response to endotoxin shock.Journal of Immunology,2007,179(8):5082-5089.
[17]TAGANOV K D,BOLDIN M P,CHANG K J,BALTIMORE D.Nf-kappab-dependent induction of microRNA mir-146,an inhibitor targeted to signaling proteins of innate immune responses.Proceedings of the National Academy of Sciences of the United States of America,2006,103(33):12481-12486.
[18]张旭,张彦明,张倩,程媛媛,谭晓妮.稳定表达猪Mirna Let-7C细胞株的建立及其对CSFV的调控作用.西北农林科技大学学报(自然科学版),2010(12):1-6.ZHANG X,ZHANG Y M,ZHANG Q,CHENG Y Y,TAN X N.Establishment of cell line expressing miRNA let-7c and regulation on classical swine fever virus.Journal of Northwest Agricultural and Forestry University(Natural Science Edition),2010(12):1-6.(in Chinese)
[19]CHEN Y,CHEN J,WANG H,SHI J,WU K,LIU S,LIU Y,WU J.Hcv-induced Mir-21 contributes to evasion of host immune system by targeting Myd88 and Irak1.The Public Library of Science Pathogens,2013,9(4):e1003248.
[20]JOPLING C L,NORMAN K L,SARNOW P.Positive and negative modulation of viral and cellular mRNAs by liver-specific microRNAMir-122.Cold Spring Harbor Symposia on Quantitative Biology,2006,71:369-376.
[21]BENSAUDE E,TURNER J L,WAKELEY P R M,SWEETMAN DA,PARDIEU C,DREW T W,WILEMAN T,POWELL P P.Classical swine fever virus induces proinflammatory cytokines and tissue factor expression and inhibits apoptosis and interferon synthesis during the establishment of long-term infection of porcine vascular endothelial cells.Journal of General Virology,2004,85(Pt 4):1029-1037.
[22]HSU H,XIONG J,GOEDDEL D V.The TNF Receptor 1-associated protein tradd signals cell death and Nf-Kappa B activation.Cell,1995,81(4):495-504.
[23]MICHEAU O,TSCHOPP J.Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes.Cell,2003,114(2):181-190.
[24]SONG L,LIU H,GAO S,JIANG W,HUANG W.Cellular microRNAs inhibit replication of the H1N1 influenza a virus in infected cells.Journal of Virology,2010,84(17):8849-8860.
[25]JOHNS H L,BENSAUDE E,LA ROCCA S A,SEAGO J,CHARLESTON B,STEINBACH F,DREW T W,CROOKE H,EVERETT H.Classical swine fever virus infection protects aortic endothelial cells from Pipc-Mediated apoptosis.Journal of General Virology,2010,91(Pt 4):1038-1046.
[26]WANG F,LIU M,LI X,TANG H.Mir-214 reduces cell survival and enhances cisplatin-induced cytotoxicity via down-regulation of Bcl2L2 in cervical cancer cells.The Federation of European Biochemical Societies Letters,2013,587(5):488-495.
[27]ZHANG Z C,LI Y Y,WANG H Y,FU S,WANG X P,ZENG M S,ZENG Y X,SHAO J Y.Knockdown of miR-214 promotes apoptosis and inhibits cell proliferation in nasopharyngeal carcinoma.The Public Library of Science One,2014,9(1):e86149.
[28]GRIFFIN D E,HARDWICK J M.Regulators of apoptosis on the road to persistent alphavirus infection.Annual Review of Microbiology,1997,51:565-592.
[29]KROEMER G,GALLUZZI L,BRENNER C.Mitochondrial membrane permeabilization in cell death.Physiological Reviews,2007,87(1):99-163.
[30]KUROKAWA M,KORNBLUTH S.Caspases and kinases in a death grip.Cell,2009,138(5):838-854.
[2]STEGEMAN A,ELBERS A,DE SMIT H.The 1997-1998 epidemic of classical swine fever in the Netherlands.Veterinary Microbiology,2000,73(2/3):183-196.
[3]BARTEL D P.MicroRNAs:Target recognition and regulatory functions.Cell,2009,136(2):215-233.
[4]FLYNT A S,LI N,THATCHER E J,SOLNICA-KREZEL L,PATTON J G.Zebrafish Mir-214 modulates hedgehog signaling to specify muscle cell fate.Nature Genetics,2007,39(2):259-263.
[5]MICHAEL M Z,O'C S,VAN HOLST P N,YOUNG G P,JAMES RJ.Reduced accumulation of specific microRNAs in colorectal neoplasia.Molecular Cancer Research,2003,1(12):882-891.
[6]CAMERON J E,YIN Q,FEWELL C,LACEY M,MCBRIDE J,WANG X,LIN Z,SCHAEFER B C,FLEMINGTON E K.Epsteinbarr virus latent membrane protein 1 induces cellular microRNAmir-146a,a modulator of lymphocyte signaling pathways.Journal of Virology,2008,82(4):1946-1958.
[7]ZHAO H,WEN G,HUANG Y,YU X,CHEN Q,AFZAL T A,LUONG LE A,ZHU J,YE S,ZHANG L,XIAO Q.Microrna-22regulates smooth muscle cell differentiation from stem cells by targeting methyl cpg-binding protein 2.Arteriosclerosis,Thrombosis,and Vascular Biology,2015,35(4):918-929.
[8]YANG M,LIU R,LI X,LIAO J,PU Y,PAN E,YIN L,WANG Y.Mirna-183 suppresses apoptosis and promotes proliferation in esophageal cancer by targeting pdcd4.Molecular Cells,2014,37(12):873-880.
[9]XU P,VERNOOY S Y,GUO M,HAY B A.The drosophila microRNAmir-14 suppresses cell death and is required for normal fat metabolism.Current Biology,2003,13(9):790-795.
[10]BOUTLA A,DELIDAKIS C,TABLER M.Developmental defects by antisense-mediated inactivation of micro-RNAs 2 and 13 in drosophila and the identification of putative target genes.Nucleic Acids Research,2003,31(17):4973-4980.
[11]NEILSON J R,ZHENG G X,BURGE C B,SHARP P A.Dynamic regulation of miRNA expression in ordered stages of cellular development.Genes Development,2007,21(5):578-589.
[12]LI Q J,CHAU J,EBERT P J,SYLVESTER G,MIN H,LIU G,BRAICH R,MANOHARAN M,SOUTSCHEK J,SKARE P,KLEINL O,DAVIS M M,CHEN C Z.Mir-181a is an intrinsic modulator of t cell sensitivity and selection.Cell,2007,129(1):147-161.
[13]VIGORITO E,PERKS K L,ABREU-GOODGER C,BUNTING S,XIANG Z,KOHLHAAS S,DAS PP,MISKA E A,RODRIGUEZ A,BRADLEY A,SMITH KG,RADA C,ENRIGHT A J,TOELLNERK M,MACLENNAN I C,TURNER M.Microrna-155 regulates the generation of immunoglobulin class-switched plasma cells.Immunity,2007,27(6):847-859.
[14]KUMAR A.RNA interference:a multifaceted innate antiviral defense.Retrovirology,2008,5:17.
[15]LECELLIER C H,DUNOYER P,ARAR K,LEHMANN-CHE J,EYQUEM S,HIMBER C,SA?B A,VOINNET O.A Cellular microRNA mediates antiviral defense in human cells.Science,2005,308(5721):557-560.
[16]TILI E,MICHAILLE J J,CIMINO A,COSTINEAN S,DUMITRUC D,ADAIR B,FABBRI M,ALDER H,LIU C G,CALIN G A,CROCE C M.Modulation of Mir-155 and Mir-125B levels following lipopolysaccharide/Tnf-Alpha stimulation and their possible roles in regulating the response to endotoxin shock.Journal of Immunology,2007,179(8):5082-5089.
[17]TAGANOV K D,BOLDIN M P,CHANG K J,BALTIMORE D.Nf-kappab-dependent induction of microRNA mir-146,an inhibitor targeted to signaling proteins of innate immune responses.Proceedings of the National Academy of Sciences of the United States of America,2006,103(33):12481-12486.
[18]张旭,张彦明,张倩,程媛媛,谭晓妮.稳定表达猪Mirna Let-7C细胞株的建立及其对CSFV的调控作用.西北农林科技大学学报(自然科学版),2010(12):1-6.ZHANG X,ZHANG Y M,ZHANG Q,CHENG Y Y,TAN X N.Establishment of cell line expressing miRNA let-7c and regulation on classical swine fever virus.Journal of Northwest Agricultural and Forestry University(Natural Science Edition),2010(12):1-6.(in Chinese)
[19]CHEN Y,CHEN J,WANG H,SHI J,WU K,LIU S,LIU Y,WU J.Hcv-induced Mir-21 contributes to evasion of host immune system by targeting Myd88 and Irak1.The Public Library of Science Pathogens,2013,9(4):e1003248.
[20]JOPLING C L,NORMAN K L,SARNOW P.Positive and negative modulation of viral and cellular mRNAs by liver-specific microRNAMir-122.Cold Spring Harbor Symposia on Quantitative Biology,2006,71:369-376.
[21]BENSAUDE E,TURNER J L,WAKELEY P R M,SWEETMAN DA,PARDIEU C,DREW T W,WILEMAN T,POWELL P P.Classical swine fever virus induces proinflammatory cytokines and tissue factor expression and inhibits apoptosis and interferon synthesis during the establishment of long-term infection of porcine vascular endothelial cells.Journal of General Virology,2004,85(Pt 4):1029-1037.
[22]HSU H,XIONG J,GOEDDEL D V.The TNF Receptor 1-associated protein tradd signals cell death and Nf-Kappa B activation.Cell,1995,81(4):495-504.
[23]MICHEAU O,TSCHOPP J.Induction of TNF receptor I-mediated apoptosis via two sequential signaling complexes.Cell,2003,114(2):181-190.
[24]SONG L,LIU H,GAO S,JIANG W,HUANG W.Cellular microRNAs inhibit replication of the H1N1 influenza a virus in infected cells.Journal of Virology,2010,84(17):8849-8860.
[25]JOHNS H L,BENSAUDE E,LA ROCCA S A,SEAGO J,CHARLESTON B,STEINBACH F,DREW T W,CROOKE H,EVERETT H.Classical swine fever virus infection protects aortic endothelial cells from Pipc-Mediated apoptosis.Journal of General Virology,2010,91(Pt 4):1038-1046.
[26]WANG F,LIU M,LI X,TANG H.Mir-214 reduces cell survival and enhances cisplatin-induced cytotoxicity via down-regulation of Bcl2L2 in cervical cancer cells.The Federation of European Biochemical Societies Letters,2013,587(5):488-495.
[27]ZHANG Z C,LI Y Y,WANG H Y,FU S,WANG X P,ZENG M S,ZENG Y X,SHAO J Y.Knockdown of miR-214 promotes apoptosis and inhibits cell proliferation in nasopharyngeal carcinoma.The Public Library of Science One,2014,9(1):e86149.
[28]GRIFFIN D E,HARDWICK J M.Regulators of apoptosis on the road to persistent alphavirus infection.Annual Review of Microbiology,1997,51:565-592.
[29]KROEMER G,GALLUZZI L,BRENNER C.Mitochondrial membrane permeabilization in cell death.Physiological Reviews,2007,87(1):99-163.
[30]KUROKAWA M,KORNBLUTH S.Caspases and kinases in a death grip.Cell,2009,138(5):838-854.