HSV-2-encoded miRNA-H4 Regulates Cell Cycle Progression and Act-Dinduced Apoptosis in HeLa Cells by Targeting CDKL2 and CDKN2A
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摘要
MicroRNAs(miRNAs) encoded by latency-associated transcript are associated with both latent and acute stages of herpes simplex virus 2(HSV-2) infection. In this study, miRNA-H4-5 p and miRNA-H4-3 p were ectopically expressed in HeLa cells to explore potential cellular targets of viral miRNAs and demonstrate their potential biological functions. The results showed that miRNA-H4-5 p could reverse apoptosis induced by actinomycin D(Act-D) and promote cell cycle progression,but miRNA-H4-3 p had no such obvious functions. Bioinformatics analysis, luciferase report assay, quantitative reverse transcription polymerase chain reaction(qRT-PCR), and Western blotting demonstrated that miRNA-H4-5 p could bind to the 30-untranslated region(UTR) of cyclin-dependent kinase inhibitor 2 A(CDKN2 A) and cyclin-dependent kinase-like 2(CDKL2) to negatively regulate their expression. We verified that these two targeted genes were associated with cell apoptosis and cell cycle. Furthermore, in HeLa cells infected with HSV-2, we detected significantly reduced expression of CDKN2 A and CDKL2 and demonstrated the negative regulation effect of miRNA-H4-5 p on these two target genes. Our findings show that viral miRNAs play a vital role in regulating the expression of the host's cellular genes that participate in cell apoptosis and progression to reshape the cellular environment in response to HSV-2 infection, providing further information on the roles of encoded herpesvirus miRNAs in pathogen–host interaction.
MicroRNAs(miRNAs) encoded by latency-associated transcript are associated with both latent and acute stages of herpes simplex virus 2(HSV-2) infection. In this study, miRNA-H4-5 p and miRNA-H4-3 p were ectopically expressed in HeLa cells to explore potential cellular targets of viral miRNAs and demonstrate their potential biological functions. The results showed that miRNA-H4-5 p could reverse apoptosis induced by actinomycin D(Act-D) and promote cell cycle progression,but miRNA-H4-3 p had no such obvious functions. Bioinformatics analysis, luciferase report assay, quantitative reverse transcription polymerase chain reaction(qRT-PCR), and Western blotting demonstrated that miRNA-H4-5 p could bind to the 30-untranslated region(UTR) of cyclin-dependent kinase inhibitor 2 A(CDKN2 A) and cyclin-dependent kinase-like 2(CDKL2) to negatively regulate their expression. We verified that these two targeted genes were associated with cell apoptosis and cell cycle. Furthermore, in HeLa cells infected with HSV-2, we detected significantly reduced expression of CDKN2 A and CDKL2 and demonstrated the negative regulation effect of miRNA-H4-5 p on these two target genes. Our findings show that viral miRNAs play a vital role in regulating the expression of the host's cellular genes that participate in cell apoptosis and progression to reshape the cellular environment in response to HSV-2 infection, providing further information on the roles of encoded herpesvirus miRNAs in pathogen–host interaction.
MicroRNAs(miRNAs) encoded by latency-associated transcript are associated with both latent and acute stages of herpes simplex virus 2(HSV-2) infection. In this study, miRNA-H4-5 p and miRNA-H4-3 p were ectopically expressed in HeLa cells to explore potential cellular targets of viral miRNAs and demonstrate their potential biological functions. The results showed that miRNA-H4-5 p could reverse apoptosis induced by actinomycin D(Act-D) and promote cell cycle progression,but miRNA-H4-3 p had no such obvious functions. Bioinformatics analysis, luciferase report assay, quantitative reverse transcription polymerase chain reaction(qRT-PCR), and Western blotting demonstrated that miRNA-H4-5 p could bind to the 30-untranslated region(UTR) of cyclin-dependent kinase inhibitor 2 A(CDKN2 A) and cyclin-dependent kinase-like 2(CDKL2) to negatively regulate their expression. We verified that these two targeted genes were associated with cell apoptosis and cell cycle. Furthermore, in HeLa cells infected with HSV-2, we detected significantly reduced expression of CDKN2 A and CDKL2 and demonstrated the negative regulation effect of miRNA-H4-5 p on these two target genes. Our findings show that viral miRNAs play a vital role in regulating the expression of the host's cellular genes that participate in cell apoptosis and progression to reshape the cellular environment in response to HSV-2 infection, providing further information on the roles of encoded herpesvirus miRNAs in pathogen–host interaction.
引文
Afonso-Grunz F,Muller S(2015)Principles of miRNA-mRNAinteractions:beyond sequence complementarity.Cell Mol Life Sci 72:3127-3141
Bartel DP(2009)microRNAs:target recognition and regulatory functions.Cell 136:215-233
Cullen BR(2009)Viral and cellular messenger RNA targets of viral microRNAs.Nature 457:421-425
Cullen BR(2011)Herpesvirus microRNAs:phenotypes and functions.Curr Opin Virol 1:211-215
Dalmay T(2013)Mechanism of miRNA-mediated repression of m RNA translation.Essays Biochem 54:29-38
Du T,Han Z,Zhou G,Roizman B(2015)Patterns of accumulation of miRNAs encoded by herpes simplex virus during productive infection,latency,and on reactivation.Proc Natl Acad Sci USA112:E49-E55
Duan F,Liao J,Huang Q,Nie Y,Wu K(2012)HSV-1 miR-H6inhibits HSV-1 replication and IL-6 expression in human corneal epithelial cells in vitro.Clin Dev Immunol 2012:192791
Everett RD(2000)ICP0,a regulator of herpes simplex virus during lytic and latent infection.BioEssays 22:761-770
Goldberg IH,Rabinowitz M(1962)Actionmycin D inhibition of deoxyribonucleic acid-dependent synthesis of ribonucleic acid.Science 136:315-316
Gomi H,Sassa T,Thompson RF,Itohara S(2010)Involvement of cyclin-dependent kinase-like 2 in cognitive function required for contextual and spatial learning in mice.Front Behav Neurosci4:17
Gottwein E,Cullen BR(2008)Viral and cellular microRNAs as determinants in pathogenesis and immunity.Cell Host Microbe3:375-387
Grey F(2015)Role of microRNAs in herpesvirus latency and persistence.J Gen Virol 96:739-751
Gupta A,Gartner JJ,Sethupathy P,Hatzigeorgiou AG,Fraser NW(2006)Anti-apoptotic function of a microRNA encoded by the HSV-1 latency-associated transcript.Nature 442:82-85
Hooykaas MJ,Kruse E,Wiertz EJ,Lebbink RJ(2016)Comprehensive profiling of functional Epstein-Barr virus miRNA expression in human cell lines.BMC Genom 17:644
Journey LJ,Goldstein MN(1961)Electron microscope studies on HeLa cell lines sensitive and resistant to actinomycin D.Cancer Res 21:929-932
Jurak I,Kramer MF,Mellor JC,van Lint AL,Roth FP,Knipe DM,Coen DM(2010)Numerous conserved and divergent microRNAs expressed by herpes simplex viruses 1 and 2.J Virol84:4659-4672
Kleeff J,Kornmann M,Sawhney H,Korc M(2000)Actinomycin Dinduces apoptosis and inhibits growth of pancreatic cancer cells.Int J Cancer 86:399-407
Li L,Liu C,Amato RJ,Chang JT,Du G,Li W(2014)CDKL2promotes epithelial-mesenchymal transition and breast cancer progression.Oncotarget 5:10840-10853
Liu Y,Yang HL,Zhong FF,Fan JY(2016)Anti-apoptotic function of herpes simplex virus-2 latency-associated transcript RL1sequence and screening of its encoded microRNAs.Clin Exp Dermatol 41:782-791
Livak KJ,Schmittgen TD(2001)Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method.Methods 25:402-408
Looker KJ,Elmes JAR,Gottlieb SL,Schiffer JT,Vickerman P,Turner KME,Boily MC(2017a)Effect of HSV-2 infection on subsequent HIV acquisition:an updated systematic review and meta-analysis.Lancet Infect 17:1303-1316
Looker KJ,Magaret AS,May MT,Turner KME,Vickerman P,Newman LM,Gottlieb SL(2017b)First estimates of the global and regional incidence of neonatal herpes infection.Lancet Glob Health 5:e300-e309
Lu DF,Wang YS,Li C,Wei GJ,Chen R,Dong DM,Yao M(2015)Actinomycin D inhibits cell proliferations and promotes apoptosis in osteosarcoma cells.Int J Clin Exp Med 8:1904-1911
Niederacher D,Yan HY,An HX,Bender HG,Beckmann MW(1999)CDKN2A gene inactivation in epithelial sporadic ovarian cancer.Br J Cancer 80:1920-1926
Pal A,Potjer TP,Thomsen SK,Ng HJ,Barrett A,Scharfmann R,James TJ,Bishop DT,Karpe F,Godsland IF,Vasen HF,Newton-Bishop J,Pijl H,McCarthy MI,Gloyn AL(2016)Lossof-function mutations in the cell-cycle control gene CDKN2Aimpact on glucose homeostasis in humans.Diabetes 65:527-533
Pfeffer S,Zavolan M,Grasser FA,Chien M,Russo JJ,Ju J,John B,Enright AJ,Marks D,Sander C,Tuschl T(2004)Identification of virus-encoded microRNAs.Science 304:734-736
Piedade D,Azevedo-Pereira JM(2016)The role of microRNAs in the pathogenesis of herpesvirus infection.Viruses 8:E156
Salic A,Mitchison TJ(2008)A chemical method for fast and sensitive detection of DNA synthesis in vivo.Proc Natl Acad Sci USA 105:2415-2420
Skalsky RL,Cullen BR(2010)Viruses,microRNAs,and host interactions.Annu Rev Microbiol 64:123-141
Tang S,Bertke AS,Patel A,Wang K,Cohen JI,Krause PR(2008)An acutely and latently expressed herpes simplex virus 2 viral microRNA inhibits expression of ICP34.5,a viral neurovirulence factor.Proc Natl Acad Sci USA 105:10931-10936
Tang S,Patel A,Krause PR(2009)Novel less-abundant viral microRNAs encoded by herpes simplex virus 2 latencyassociated transcript and their roles in regulating ICP34.5 and ICP0 mRNAs.J Virol 83:1433-1442
Tang S,Bertke AS,Patel A,Margolis TP,Krause PR(2011)Herpes simplex virus 2 microRNA miR-H6 is a novel latency-associated transcript-associated microRNA,but reduction of its expression does not influence the establishment of viral latency or the recurrence phenotype.J Virol 85:4501-4509
Tang S,Bosch-Marce M,Patel A,Margolis TP,Krause PR(2015)Characterization of herpes simplex virus 2 primary microRNAtranscript regulation.J Virol 89:4837-4848
Umbach JL,Kramer MF,Jurak I,Karnowski HW,Coen DM,Cullen BR(2008)microRNAs expressed by herpes simplex virus 1during latent infection regulate viral mRNAs.Nature454:780-783
Umbach JL,Wang K,Tang S,Krause PR,Mont EK,Cohen JI,Cullen BR(2010)Identification of viral microRNAs expressed in human sacral ganglia latently infected with herpes simplex virus2.J Virol 84:1189-1192
Whitley RJ(2015)Herpes simplex virus infections of the central nervous system.Continuum(Minneap Minn)21:1704-1713
Wu W,Guo Z,Zhang X,Guo L,Liu L,Liao Y,Wang J,Wang L,Li Q(2013)A microRNA encoded by HSV-1 inhibits a cellular transcriptional repressor of viral immediate early and early genes.Sci China Life Sci 56:373-383
Yamada M,Banno Y,Takuwa Y,Koda M,Hara A,Nozawa Y(2004)Overexpression of phospholipase D prevents actinomycin D-induced apoptosis through potentiation of phosphoinositide 3-kinase signalling pathways in Chinese-hamster ovary cells.Biochem J 378:649-656
Zheng SQ,Li YX,Zhang Y,Li X,Tang H(2011)miR-101 regulates HSV-1 replication by targeting ATP5B.Antivir Res 89:219-226
Bartel DP(2009)microRNAs:target recognition and regulatory functions.Cell 136:215-233
Cullen BR(2009)Viral and cellular messenger RNA targets of viral microRNAs.Nature 457:421-425
Cullen BR(2011)Herpesvirus microRNAs:phenotypes and functions.Curr Opin Virol 1:211-215
Dalmay T(2013)Mechanism of miRNA-mediated repression of m RNA translation.Essays Biochem 54:29-38
Du T,Han Z,Zhou G,Roizman B(2015)Patterns of accumulation of miRNAs encoded by herpes simplex virus during productive infection,latency,and on reactivation.Proc Natl Acad Sci USA112:E49-E55
Duan F,Liao J,Huang Q,Nie Y,Wu K(2012)HSV-1 miR-H6inhibits HSV-1 replication and IL-6 expression in human corneal epithelial cells in vitro.Clin Dev Immunol 2012:192791
Everett RD(2000)ICP0,a regulator of herpes simplex virus during lytic and latent infection.BioEssays 22:761-770
Goldberg IH,Rabinowitz M(1962)Actionmycin D inhibition of deoxyribonucleic acid-dependent synthesis of ribonucleic acid.Science 136:315-316
Gomi H,Sassa T,Thompson RF,Itohara S(2010)Involvement of cyclin-dependent kinase-like 2 in cognitive function required for contextual and spatial learning in mice.Front Behav Neurosci4:17
Gottwein E,Cullen BR(2008)Viral and cellular microRNAs as determinants in pathogenesis and immunity.Cell Host Microbe3:375-387
Grey F(2015)Role of microRNAs in herpesvirus latency and persistence.J Gen Virol 96:739-751
Gupta A,Gartner JJ,Sethupathy P,Hatzigeorgiou AG,Fraser NW(2006)Anti-apoptotic function of a microRNA encoded by the HSV-1 latency-associated transcript.Nature 442:82-85
Hooykaas MJ,Kruse E,Wiertz EJ,Lebbink RJ(2016)Comprehensive profiling of functional Epstein-Barr virus miRNA expression in human cell lines.BMC Genom 17:644
Journey LJ,Goldstein MN(1961)Electron microscope studies on HeLa cell lines sensitive and resistant to actinomycin D.Cancer Res 21:929-932
Jurak I,Kramer MF,Mellor JC,van Lint AL,Roth FP,Knipe DM,Coen DM(2010)Numerous conserved and divergent microRNAs expressed by herpes simplex viruses 1 and 2.J Virol84:4659-4672
Kleeff J,Kornmann M,Sawhney H,Korc M(2000)Actinomycin Dinduces apoptosis and inhibits growth of pancreatic cancer cells.Int J Cancer 86:399-407
Li L,Liu C,Amato RJ,Chang JT,Du G,Li W(2014)CDKL2promotes epithelial-mesenchymal transition and breast cancer progression.Oncotarget 5:10840-10853
Liu Y,Yang HL,Zhong FF,Fan JY(2016)Anti-apoptotic function of herpes simplex virus-2 latency-associated transcript RL1sequence and screening of its encoded microRNAs.Clin Exp Dermatol 41:782-791
Livak KJ,Schmittgen TD(2001)Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T))method.Methods 25:402-408
Looker KJ,Elmes JAR,Gottlieb SL,Schiffer JT,Vickerman P,Turner KME,Boily MC(2017a)Effect of HSV-2 infection on subsequent HIV acquisition:an updated systematic review and meta-analysis.Lancet Infect 17:1303-1316
Looker KJ,Magaret AS,May MT,Turner KME,Vickerman P,Newman LM,Gottlieb SL(2017b)First estimates of the global and regional incidence of neonatal herpes infection.Lancet Glob Health 5:e300-e309
Lu DF,Wang YS,Li C,Wei GJ,Chen R,Dong DM,Yao M(2015)Actinomycin D inhibits cell proliferations and promotes apoptosis in osteosarcoma cells.Int J Clin Exp Med 8:1904-1911
Niederacher D,Yan HY,An HX,Bender HG,Beckmann MW(1999)CDKN2A gene inactivation in epithelial sporadic ovarian cancer.Br J Cancer 80:1920-1926
Pal A,Potjer TP,Thomsen SK,Ng HJ,Barrett A,Scharfmann R,James TJ,Bishop DT,Karpe F,Godsland IF,Vasen HF,Newton-Bishop J,Pijl H,McCarthy MI,Gloyn AL(2016)Lossof-function mutations in the cell-cycle control gene CDKN2Aimpact on glucose homeostasis in humans.Diabetes 65:527-533
Pfeffer S,Zavolan M,Grasser FA,Chien M,Russo JJ,Ju J,John B,Enright AJ,Marks D,Sander C,Tuschl T(2004)Identification of virus-encoded microRNAs.Science 304:734-736
Piedade D,Azevedo-Pereira JM(2016)The role of microRNAs in the pathogenesis of herpesvirus infection.Viruses 8:E156
Salic A,Mitchison TJ(2008)A chemical method for fast and sensitive detection of DNA synthesis in vivo.Proc Natl Acad Sci USA 105:2415-2420
Skalsky RL,Cullen BR(2010)Viruses,microRNAs,and host interactions.Annu Rev Microbiol 64:123-141
Tang S,Bertke AS,Patel A,Wang K,Cohen JI,Krause PR(2008)An acutely and latently expressed herpes simplex virus 2 viral microRNA inhibits expression of ICP34.5,a viral neurovirulence factor.Proc Natl Acad Sci USA 105:10931-10936
Tang S,Patel A,Krause PR(2009)Novel less-abundant viral microRNAs encoded by herpes simplex virus 2 latencyassociated transcript and their roles in regulating ICP34.5 and ICP0 mRNAs.J Virol 83:1433-1442
Tang S,Bertke AS,Patel A,Margolis TP,Krause PR(2011)Herpes simplex virus 2 microRNA miR-H6 is a novel latency-associated transcript-associated microRNA,but reduction of its expression does not influence the establishment of viral latency or the recurrence phenotype.J Virol 85:4501-4509
Tang S,Bosch-Marce M,Patel A,Margolis TP,Krause PR(2015)Characterization of herpes simplex virus 2 primary microRNAtranscript regulation.J Virol 89:4837-4848
Umbach JL,Kramer MF,Jurak I,Karnowski HW,Coen DM,Cullen BR(2008)microRNAs expressed by herpes simplex virus 1during latent infection regulate viral mRNAs.Nature454:780-783
Umbach JL,Wang K,Tang S,Krause PR,Mont EK,Cohen JI,Cullen BR(2010)Identification of viral microRNAs expressed in human sacral ganglia latently infected with herpes simplex virus2.J Virol 84:1189-1192
Whitley RJ(2015)Herpes simplex virus infections of the central nervous system.Continuum(Minneap Minn)21:1704-1713
Wu W,Guo Z,Zhang X,Guo L,Liu L,Liao Y,Wang J,Wang L,Li Q(2013)A microRNA encoded by HSV-1 inhibits a cellular transcriptional repressor of viral immediate early and early genes.Sci China Life Sci 56:373-383
Yamada M,Banno Y,Takuwa Y,Koda M,Hara A,Nozawa Y(2004)Overexpression of phospholipase D prevents actinomycin D-induced apoptosis through potentiation of phosphoinositide 3-kinase signalling pathways in Chinese-hamster ovary cells.Biochem J 378:649-656
Zheng SQ,Li YX,Zhang Y,Li X,Tang H(2011)miR-101 regulates HSV-1 replication by targeting ATP5B.Antivir Res 89:219-226