REV对SPF雏鸡细胞凋亡相关miRNA及其靶基因表达的影响
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摘要
为探究禽网状内皮组织增生病病毒(Reticuloendotheliosis virus,REV)对SPF雏鸡细胞凋亡相关信号通路的影响,通过REV感染1日龄雏鸡后,对其临床症状、大体病理学变化进行观察,并应用高通量测序及实时荧光定量PCR方法,检测并验证其法氏囊中与细胞凋亡信号通路相关的miRNA及其靶基因表达情况。结果显示:SPF雏鸡感染REV后第21天法氏囊萎缩最显著,法氏囊中gga-mir-18a-5p、gga-mir-155和gga-mir-222b-5p表达量显著下调,其靶基因分别为Caspase-6、Caspase-7和p27,上述靶基因表达量显著上调;同时还发现gga-mir-29b-3p表达量上调,其靶基因Mcl-1表达量下调。证明REV通过下调gga-mir-18a-5p、gga-mir-155和gga-mir-222b-5p以及上调gga-mir-29b-3p的表达量,来降低或增加对其靶基因的抑制,从而使促凋亡因子Caspase-6、Caspase-7和p27的表达量显著增加,同时抑凋亡因子Mcl-1表达量显著减少来促进细胞凋亡的发生。研究结果为REV致病机理的相关研究提供了理论依据。
To investigate the effect of avian reticuloendotheliosis virus(REV) on apoptosis-related signal pathway in SPF chicken, high-throughput sequencing and real-time fluorescence quantitative PCR were used to detect and verify the expression of miRNAs and their target genes related to apoptosis signal pathway in bursa of Fabricius. The results showed that the atrophy of bursa of Fabricius in SPF chicken was the most significant on the 21 st day after infection with REV. The expressions of gga-mir-18 a-5 p, gga-mir-155 and gga-mir-222 b-5 p in bursa of Fabricius were significantly down-regulated, and the target genes were Caspase-6, Caspase-7 and p27, respectively. The expression levels of these target genes were significantly up-regulated. At the same time, it was found that the expression of gga-mir-29 b-3 p was up-regulated. The Mcl-1 expression was down-regulated. It was suggested that REV could decrease or increase the inhibition of its target gene by down-regulating the expression of gga-mir-18 a-5 pngga-mir-155 and gga-mir-222 b-5 p and up-regulating the expression of gga-mir-29 b-3 p, thus increasing the expression of Caspase-6, Caspase-7 and p27. At the same time, the expression of anti-apoptotic factor Mcl-1 was significantly reduced. REV promoted apoptosis in these ways. This study provided a theoretical basis for the study of pathogenesis of REV in the future.
To investigate the effect of avian reticuloendotheliosis virus(REV) on apoptosis-related signal pathway in SPF chicken, high-throughput sequencing and real-time fluorescence quantitative PCR were used to detect and verify the expression of miRNAs and their target genes related to apoptosis signal pathway in bursa of Fabricius. The results showed that the atrophy of bursa of Fabricius in SPF chicken was the most significant on the 21 st day after infection with REV. The expressions of gga-mir-18 a-5 p, gga-mir-155 and gga-mir-222 b-5 p in bursa of Fabricius were significantly down-regulated, and the target genes were Caspase-6, Caspase-7 and p27, respectively. The expression levels of these target genes were significantly up-regulated. At the same time, it was found that the expression of gga-mir-29 b-3 p was up-regulated. The Mcl-1 expression was down-regulated. It was suggested that REV could decrease or increase the inhibition of its target gene by down-regulating the expression of gga-mir-18 a-5 pngga-mir-155 and gga-mir-222 b-5 p and up-regulating the expression of gga-mir-29 b-3 p, thus increasing the expression of Caspase-6, Caspase-7 and p27. At the same time, the expression of anti-apoptotic factor Mcl-1 was significantly reduced. REV promoted apoptosis in these ways. This study provided a theoretical basis for the study of pathogenesis of REV in the future.
引文
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[2]王玥,姜艳萍,于琳琳,等.蛋种鸡ALV-J与REV共感染病变新特点[J].畜牧兽医学报,2011,42(11):1643-1648.
[3]GUO H J,LI H M,CHENG Z Q,et al.Influence of REV and ALV-J co-infection on immunologic function of T lymphocytes and histopathology in broiler chickens[J].Agricultural Sciences in China,2010,9(11):1667-1676.
[4]LIANG M,ZHAO Q,LIU G,et al.Pathogenicity of Bordetella avium under immunosuppression induced by reticuloendotheliosis virus in specific-pathogen-free chickens[J].Microb Pathog,2013(54):40-45.
[5]佟美姣,高雪丽,李博韬,等.传染性法氏囊病毒对SPF雏鸡免疫器官细胞凋亡及bcl-2 mRNA表达的影响[J].畜牧兽医学报,2013,44(4):617-621.
[6]祁保民,陈晓燕,吴宝成,等.番鸭呼肠孤病毒诱导的细胞凋亡研究[C]//福建省畜牧兽医学会2009年学术年会论文集,2009.
[7]ICHIHARA A,WANG Z,JINNIN M,et al.Upregulation of miR-18a-5p contributes to epidermal necrolysis in severe drug eruptions[J].J Allergy Clin Immun,2014,133(4):1065-1074.
[8]玛依努尔·达吾提,胡尼其古丽·阿巴克,阿依努尔·玉苏普,等.过表达miR-18a-5p对结肠癌细胞增殖和凋亡的影响及其作用机制[J].山东医药,2018,58(8):10-13.
[9]ALI-VON L C,ZOSCHKE C,DO N,et al.Improving topical non-melanoma skin cancer treatment:In vitro efficacy of a novel guanosiwe-analog phosphonate[J].Skin Pharmacol Physiol,2014,27(4):173-180.
[10]朱国富.miR-155在冠心病患者中的表达及对小鼠巨噬细胞凋亡的影响[D].重庆:第三军医大学,2013.
[11]陈淑如,黄宇康,吴楚成,等.miR-155对乳腺癌细胞增殖、凋亡及耐药蛋白表达的调控作用研究[J].临床肿瘤学杂志,2015,20(2):108-111.
[12]LICHT V,NOACK K,SCHLOTT B,et al.Caspase-3and Caspase-6 cleave STAT1 in leukemic cells[J].Oncotarget,2014,5(8):2305-2317.
[13]江方方,赵玮,胡逢春,等.反义miR-222靶向上调PUMA基因表达促进口腔鳞状细胞癌凋亡[J].中华口腔医学研究杂志(电子版),2013,7(3):201-206.
[14]KATAYOSE Y,MIM K,AMOL N S,et al.Promoting apoptosis,a novel activity associated with the cyclin-dependent kinase inhibitor p27[J].Cancer Res,1997,57(24):5441-5445.
[15]李亚娟.miR-29b在慢性粒细胞白血病细胞增殖及凋亡中的作用及其机制研究[D].重庆:重庆医科大学,2013.
[16]付莹.miR-29b靶向作用于HER-2对乳腺癌细胞生物学功能的影响[D].南昌:南昌大学,2016.
[17]SUBRAMANIAN S,STEER C J.MicroRNAs as gatekeepers of apoptosis[J].J Cell Physiol,2010(223):289-298.
[18]CUCONATI A,MUKHERJEE C,PEREZ D,et al.DNAdamage response and MCL-1 destruction initiate apoptosis in adenovirus-infected cells[J].Genes Dev,2003(17):2922-2932.
[19]MOTT J L,KOBAYASHI S,BRONK S F,et al.Mir-29regulates Mcl-1 protein expression and apoptosis[J].Oncogene,2007(26):6133-6140.