miRNA-155在ALV-J与REV共感染中对肌动蛋白细胞骨架通路的影响
|
摘要
该研究探讨了miRNA-155的表达在J亚群禽白血病病毒(subgroup J avian leucosis virus, ALV-J)和网状内皮组织增生症病毒(reticuloendotheliosis virus, REV)共感染中对肌动蛋白细胞骨架通路的影响。通过蛋白质组学和转录组学检测技术对ALV-J、REV和ALV-J+REV感染DF-1细胞分泌的外泌体进行蛋白质和miRNA定量差异表达综合分析,筛选共感染组与单独感染组相比共同变化的因子及其参与的信号通路。结果显示, ALV-J和REV的共感染对彼此具有协同效应, REV对ALV-J的协同起主导作用; GTP结合蛋白J(GTP-binding protein J, RhoJ)、NCK相关蛋白1(NCKassociation protein 1, NCKAP1)、肌动蛋白相关2/3复合体亚基5(actin-related 2/3 complex subunit5, ARPC5)和miRNA-155的靶蛋白整合蛋白(integrin, ITG)共同参与肌动蛋白细胞骨架通路。体外转染促进或抑制miRNA-155表达,采用RT-PCR检测其对病毒复制以及ITG、RhoJ、NCKAP1和ARPC5表达的影响。结果显示,促进miRNA-155的表达时,有利于病毒的复制, RhoJ和NCKAP1的表达上升,而ITG和ARPC5表达下降;抑制mi RNA-155的表达时,则抑制病毒复制, RhoJ和NCKAP1的表达下降, ITG和ARPC5表达上升;且共感染组中变化更为显著,这与前期病毒感染引起的相关蛋白变化结果相吻合。该研究结果表明, ALV-J与REV共感染时的协同促进作用可能与miRNA-155调节肌动蛋白细胞骨架通路中4种蛋白的表达变化密切相关, miRNA-155是两种病毒协同作用的关键调节因子。
This study was aimed to investigate the effects of miRNA-155 expression on actin cytoskeletal pathways in ALV-J and REV co-infection. The exosomes from ALV-J, REV and ALV-J+REV infected DF-1 cells were used to quantity the differentially expressed proteins and miRNAs by proteomic and transcriptome detection technology. Then, the common changed factor and their participation in the signal pathway in co-infected group compared with single infected group were screened. The results showed that ALV-J and REV had synergistic effects on each other, and REV played a leading role in synergism on ALV-J. RhoJ, NACKP1 and ARPC5, and the miRNA-155 target protein ITG were involved in the actin cytoskeleton pathway. After transfection of miRNA-155 inhibitors or mimics, RT-PCR was used to detect the effects of miRNA-155 expression on the virus replication, and the expression of ITG, RhoJ, NCKAP1 and ARPC5 as well. The results demonstrated that the virus replication and the expression of RhoJ and NCKAP1 were promoted, whereas, ITG and ARPC5 expression was decreased, when the miRNA-155 expression was increased. Inhibition of miRNA-155 expression inhibited virus replication, blocked the expression of RhoJ and NCKAP1, and increased the expression of ITG and ARPC5. These changes were more significant in the co-infected group, which was consistent with the previous results that the changes of associated proteins induced by virus infection. Our results suggested that the synergistic effect of ALV-J and REV co-infection might be associated with the miRNA-155 regulation the 4 proteins in actin cytoskeleton pathway. The miRNA-155 is a key factor regulating the synergistic effects of the two viruses.
This study was aimed to investigate the effects of miRNA-155 expression on actin cytoskeletal pathways in ALV-J and REV co-infection. The exosomes from ALV-J, REV and ALV-J+REV infected DF-1 cells were used to quantity the differentially expressed proteins and miRNAs by proteomic and transcriptome detection technology. Then, the common changed factor and their participation in the signal pathway in co-infected group compared with single infected group were screened. The results showed that ALV-J and REV had synergistic effects on each other, and REV played a leading role in synergism on ALV-J. RhoJ, NACKP1 and ARPC5, and the miRNA-155 target protein ITG were involved in the actin cytoskeleton pathway. After transfection of miRNA-155 inhibitors or mimics, RT-PCR was used to detect the effects of miRNA-155 expression on the virus replication, and the expression of ITG, RhoJ, NCKAP1 and ARPC5 as well. The results demonstrated that the virus replication and the expression of RhoJ and NCKAP1 were promoted, whereas, ITG and ARPC5 expression was decreased, when the miRNA-155 expression was increased. Inhibition of miRNA-155 expression inhibited virus replication, blocked the expression of RhoJ and NCKAP1, and increased the expression of ITG and ARPC5. These changes were more significant in the co-infected group, which was consistent with the previous results that the changes of associated proteins induced by virus infection. Our results suggested that the synergistic effect of ALV-J and REV co-infection might be associated with the miRNA-155 regulation the 4 proteins in actin cytoskeleton pathway. The miRNA-155 is a key factor regulating the synergistic effects of the two viruses.
引文
1 Payne L,Brown S,Bumstead N,Howes K,Frazier JA,Thouless ME.A novel subgroup of exogenous avian leukosis virus in chickens.J Gen Virol 1991;72(Pt4):801-7.
2 Zavala G,Cheng S,Barbosa T,Haefele H.Enzootic reticuloendotheliosis in the endangered attwater’s and greater prairie chickens.Avian Dis 2006;50(4):520-5.
3 Dong X,Ju S,Zhao P,Li Y,Meng F,Sun P,et al.Synergetic effects of subgroup J avian leukosis virus and reticuloendotheliosis virus co-infection on growth retardation and immunosuppression in SPF chickens.Vet Microbiol 2014;172(3/4):425-31.
4 Cui Z,Sun S,Zhang Z,Meng S.Simultaneous endemic infections with subgroup J avian leukosis virus and reticuloendotheliosis virus in commercial and local breeds of chickens.Avian Pathol2009;38(6):443-8.
5王小满,庄萍萍,孙霞,杜旭升,王晓宇,成子强,等.REV感染DF-1细胞分泌外体的生物信息学分析.中国细胞生物学学报(Wang Xiaoman,Zhuang Pingping,Sun Xia,Du Xusheng,Wang Xiaoyu,Cheng Ziqiang,et al.Bioinformatics analyses of exosome from REV-infected DF-1 cells.Chinese Journal of Cell Biology)2017;39(12):1565-72.
6 Wang G,Wang Z,Zhuang P,Zhao X,Cheng Z.Exosomes carring gag/env of ALV-J possess negative effect on immunocytes.Microb Pathog 2017;112(10):142-7.
7 O’Connell RM,Rao DS,Baltimore D.microRNA regulation of inflammatory responses.Annu Rev Immunol 2012;30(1):295-312.
8 Bala S,Macros M,Kodys K,Csak T,Catalano D,Mandrekar P,et al.Up-regulation of microRNA-155 in macrophages contributes to increased tumor necrosis factor-α(TNF-α)production via increased mRNA half-life in alcoholic liver disease.J Bio Chem2011;286(2):1436-44.
9 Bala S,Petrasek J,Mundkur S,Catalano D,Levin I,Ward J,et al.Circulating microRNAs in exosomes indicate hepatocyte injury and inflammation in alcoholic,drug-induced and inflammatory liver diseases.Hepatology 2012;56(5):1946-57.
10 Ahmadvand M,Eskandari M,Pashaiefar H,Yaghmaie M,Manoocheharbadi S,Khspour G,et al.Over expression of circulating miRNA-155 predicts prognosis in diffuse large B-cell lymphoma.Leuk Res 2018;70(5):45-8.
11 Momen-Heravi F,Bala S,Bukong T,Szabo G.Exosomemediated delivery of functionally active miRNA-155 inhibitor to macrophages.Nanom Edicine 2014;10(7):1517-27.
12 Ho H,Hopkin AS,Kapadia R,Vasudeva P,Schilling J,Ganesan AK.RhoJ modulates melanoma invasion by altering actin cytoskeletal dynamics.Pigment Cell Melanoma Res 2015;26(2):218-25.
13 Kim C,Yang H,Park I,Chon HJ,Kim JH,Kwon WS,et al.Rho GTPase RhoJ is associated with gastric cancer progression and metastasis.J Cancer 2016;7(11):1550-56.
14 Kim C,Yang H,Fukushima Y,Saw PE,Lee J,Park JS,et al.Vascular RhoJ is an effective and selective target for tumor angiogenesis and vascular disruption.Cancer Cell 2014;25(1):102-17.
15 Bledzka K,Schiemann B,Schiemann WP,Fox P,Plow EF,Sossey-Alaoui K.The WAVE3-YB1 interaction regulates cancer stem cells activity in breast cancer.Oncotarget 2017;8(61):104072-89.
16 Lu J,Wang SL,Wang YC,Wu YN,Yu X Zhao WZ,et al.High WAVE3 expression correlates with proliferation,migration and invasion in human ovarian cancer.Oncotarget 2017;8(25):41189-201.
17 Ko HS,Kim JS,Cho SM,Lee HJ,Ahn KS,Kim SH,et al.Urokinase-type plasminogen activator expression and Rac1/WAVE-2/Arp2/3 pathway are blocked by pterostilbene to suppress cell migration and invasion in MDA-MB-231 cells.Bioorg Med Chem Lett 2014;24(4):1176-9.
18 Tang H,Li A,Bi J,Veltman DM,Zech T,Spence HJ,et al.Loss of Scar/WAVE complex promotes N-WASP-and FAK-dependent invasion.Curr Biol 2012;23(2):107-17.
19 Millard TH,Behrendt B,Launay S,Fütterer K,Machesky LM.Identification and characterisation of a novel human isoform of Arp2/3 complex subunit p16-ARC/ARPC5.Cell Motil Cytoskeleton 2003;54(1):81-90.
20 Kinoshita T,Nohata N,Watanabe-Takano H,Yoshino H Hidaka H,Fujimura L,et al.Actin-related protein 2/3 complex subunit5(ARPC5)contributes to cell migration and invasion and is directly regulated by tumor-suppressive microRNA-133a in head and neck squamous cell carcinoma.Int J Oncol 2012;40(6):1770-8.
21庄萍萍,王小满,孟薇,李根,成子强,王桂花.网状内皮增生症病毒感染改变外泌体蛋白质组成和免疫调节功能.中国细胞生物学学报(Zhuang Pingping,Wang Xiaoman,Meng Wei,Li Gen,Cheng Ziqiang,Wang Guihua.The infection of reticuloendotheliosis virus changed the protein composition and immunomodulation of exosome.Chinese Journal of Cell Biology)2016;38(6):682-90.
2 Zavala G,Cheng S,Barbosa T,Haefele H.Enzootic reticuloendotheliosis in the endangered attwater’s and greater prairie chickens.Avian Dis 2006;50(4):520-5.
3 Dong X,Ju S,Zhao P,Li Y,Meng F,Sun P,et al.Synergetic effects of subgroup J avian leukosis virus and reticuloendotheliosis virus co-infection on growth retardation and immunosuppression in SPF chickens.Vet Microbiol 2014;172(3/4):425-31.
4 Cui Z,Sun S,Zhang Z,Meng S.Simultaneous endemic infections with subgroup J avian leukosis virus and reticuloendotheliosis virus in commercial and local breeds of chickens.Avian Pathol2009;38(6):443-8.
5王小满,庄萍萍,孙霞,杜旭升,王晓宇,成子强,等.REV感染DF-1细胞分泌外体的生物信息学分析.中国细胞生物学学报(Wang Xiaoman,Zhuang Pingping,Sun Xia,Du Xusheng,Wang Xiaoyu,Cheng Ziqiang,et al.Bioinformatics analyses of exosome from REV-infected DF-1 cells.Chinese Journal of Cell Biology)2017;39(12):1565-72.
6 Wang G,Wang Z,Zhuang P,Zhao X,Cheng Z.Exosomes carring gag/env of ALV-J possess negative effect on immunocytes.Microb Pathog 2017;112(10):142-7.
7 O’Connell RM,Rao DS,Baltimore D.microRNA regulation of inflammatory responses.Annu Rev Immunol 2012;30(1):295-312.
8 Bala S,Macros M,Kodys K,Csak T,Catalano D,Mandrekar P,et al.Up-regulation of microRNA-155 in macrophages contributes to increased tumor necrosis factor-α(TNF-α)production via increased mRNA half-life in alcoholic liver disease.J Bio Chem2011;286(2):1436-44.
9 Bala S,Petrasek J,Mundkur S,Catalano D,Levin I,Ward J,et al.Circulating microRNAs in exosomes indicate hepatocyte injury and inflammation in alcoholic,drug-induced and inflammatory liver diseases.Hepatology 2012;56(5):1946-57.
10 Ahmadvand M,Eskandari M,Pashaiefar H,Yaghmaie M,Manoocheharbadi S,Khspour G,et al.Over expression of circulating miRNA-155 predicts prognosis in diffuse large B-cell lymphoma.Leuk Res 2018;70(5):45-8.
11 Momen-Heravi F,Bala S,Bukong T,Szabo G.Exosomemediated delivery of functionally active miRNA-155 inhibitor to macrophages.Nanom Edicine 2014;10(7):1517-27.
12 Ho H,Hopkin AS,Kapadia R,Vasudeva P,Schilling J,Ganesan AK.RhoJ modulates melanoma invasion by altering actin cytoskeletal dynamics.Pigment Cell Melanoma Res 2015;26(2):218-25.
13 Kim C,Yang H,Park I,Chon HJ,Kim JH,Kwon WS,et al.Rho GTPase RhoJ is associated with gastric cancer progression and metastasis.J Cancer 2016;7(11):1550-56.
14 Kim C,Yang H,Fukushima Y,Saw PE,Lee J,Park JS,et al.Vascular RhoJ is an effective and selective target for tumor angiogenesis and vascular disruption.Cancer Cell 2014;25(1):102-17.
15 Bledzka K,Schiemann B,Schiemann WP,Fox P,Plow EF,Sossey-Alaoui K.The WAVE3-YB1 interaction regulates cancer stem cells activity in breast cancer.Oncotarget 2017;8(61):104072-89.
16 Lu J,Wang SL,Wang YC,Wu YN,Yu X Zhao WZ,et al.High WAVE3 expression correlates with proliferation,migration and invasion in human ovarian cancer.Oncotarget 2017;8(25):41189-201.
17 Ko HS,Kim JS,Cho SM,Lee HJ,Ahn KS,Kim SH,et al.Urokinase-type plasminogen activator expression and Rac1/WAVE-2/Arp2/3 pathway are blocked by pterostilbene to suppress cell migration and invasion in MDA-MB-231 cells.Bioorg Med Chem Lett 2014;24(4):1176-9.
18 Tang H,Li A,Bi J,Veltman DM,Zech T,Spence HJ,et al.Loss of Scar/WAVE complex promotes N-WASP-and FAK-dependent invasion.Curr Biol 2012;23(2):107-17.
19 Millard TH,Behrendt B,Launay S,Fütterer K,Machesky LM.Identification and characterisation of a novel human isoform of Arp2/3 complex subunit p16-ARC/ARPC5.Cell Motil Cytoskeleton 2003;54(1):81-90.
20 Kinoshita T,Nohata N,Watanabe-Takano H,Yoshino H Hidaka H,Fujimura L,et al.Actin-related protein 2/3 complex subunit5(ARPC5)contributes to cell migration and invasion and is directly regulated by tumor-suppressive microRNA-133a in head and neck squamous cell carcinoma.Int J Oncol 2012;40(6):1770-8.
21庄萍萍,王小满,孟薇,李根,成子强,王桂花.网状内皮增生症病毒感染改变外泌体蛋白质组成和免疫调节功能.中国细胞生物学学报(Zhuang Pingping,Wang Xiaoman,Meng Wei,Li Gen,Cheng Ziqiang,Wang Guihua.The infection of reticuloendotheliosis virus changed the protein composition and immunomodulation of exosome.Chinese Journal of Cell Biology)2016;38(6):682-90.