摘要
绵羊肺腺瘤病(OPA)是由绵羊肺腺瘤病毒(JSRV)引起的绵羊传染性肺肿瘤疾病。为探究JSRV对绵羊肺脏的致瘤机制,本研究从自然感染JSRV的羊(OPA患羊)肺肿瘤组织和健康羊肺组织中提取总RNA,构建二者的cDNA文库后采用Illumina Hi Seq 4000高通量测序平台进行转录组学测序(RNA-Seq),采用DESeqR筛选健康绵羊与患病绵羊肺组织中的转录差异基因,并以P<0.05和log2(Fold change)≥1筛选转录显著差异基因。通过GO和KEGG数据库对转录显著差异基因进行GO功能和KEGG信号通路的富集分析,并采用RT-qPCR对随机选择的10个转录显著差异基因进行验证。结果显示,与对照组相比,OPA羊肺肿瘤组织中共筛选到1 360个转录上调基因和783个转录下调基因,其中154个转录显著上调基因,212个转录显著下调基因。GO功能分析显示,转录显著差异基因显著富集在178个GO条目中,包括114个生物过程(BP)、19个细胞成分(CC)和45个分子功能(MF),主要涉及生长因子活性、复制后修复、NAD^(+)二磷酸酶活性、核苷代谢过程和鸟苷核苷酸交换因子活性等生物学功能。KEGG分析显示转录显著差异基因主要富集在细胞增殖、分化和肿瘤生成,如PI3K/Akt、MAPK和Hippo等信号通路。RT-qPCR结果显示,10个转录显著差异基因与RNA-Seq筛选结果均一致。本研究进一步通过RT-qPCR、western blot检测Hippo信号通路核心蛋白哺乳动物STE20样蛋白激酶1/2 (MST1/2)、大肿瘤抑制因子1/2 (LATS1/2)、YES相关蛋白1/2 (YAP1)在两组绵羊肺组织样品中的转录及表达水平和YAP1的磷酸化水平(p-YAP1);采用免疫组化试验(IHC)检测Hippo信号通路中上述核心蛋白在两组绵羊肺组织中的表达及定位。RT-qPCR结果显示,与对照组相比,OPA羊肺组织中MST1和YAP1 mRNA的转录水平均极显著上调(P<0.01、P<0.001),LATS1 mRNA的转录水平显著上调(P<0.05)。Western blot结果显示,MST1/2蛋白在59 ku、YAP1和p-YAP1蛋白在65 ku、LATS1/2蛋白在140 ku处分别出现特异性条带;与对照组相比,OPA绵羊肺肿瘤组织中MST1/2和p-YAP1的表达水平显著上调(P<0.05),LATS1/2和YAP1的表达水平极显著上调(P<0.01)。IHC结果显示,在OPA组和对照组羊肺组织中MST1/2、LATS1/2及p-YAP1均定位于肺组织细胞质中,且与对照组相比,OPA组羊肺组织中上述分子均呈强阳性表达。YAP1主要定位于对照组羊肺组织细胞的胞质中,而在OPA组中YAP1主要定位于细胞核中(少量定位于细胞质中)。上述结果表明,OPA组绵羊肺组织中存在转录显著差异基因,主要参与细胞增殖、肿瘤发生和转移等过程,且主要富集在PI3K/Akt、MAPK、Hippo等信号通路,尤其是Hippo信号通路中的YAP蛋白可能通过核异位促进肿瘤的发生与发展。本研究为JSRV致瘤机制提供了新的研究基础与研究思路和方向。
Ovine pulmonary adenomatosis(OPA) is an infectious lung tumor disease in sheep caused by Jaagsiekte sheep retrovirus(JSRV).To investigate the tumorigenetic mechanism of JSRV in sheep,total RNA was extracted from lung tumor tissues of sheep naturally infected with JSRV(OPA sheep) and lung tissues of healthy sheep.The Illumina Hi Seq 4000 high-throughput sequencing platform was used for transcriptome sequencing(RNA-Seq).DESeqR was used to screen the differentially transcribed genes in the lung tissue of healthy and OPA sheep.P<0.05 and log2(Fold change) ≥1 were used to screen the significantly differentially transcribed genes.The enrichment analysis of GO function and KEGG signaling pathway of the significantly differentially transcribed genes was performed by GO and KEGG databases,and 10 randomly selected significantly differentially transcribed genes were verified by RT-qPCR.Compared with the control group,1360 up-regulated genes and 783 down-regulated genes were screened,among which 154 significantly up-regulated genes and 212 significantly down-regulated genes.GO functional and KEGG analysis showed that the significantly differentially transcribed genes were significantly enriched in 178 GO terms,including 114 biological process(BP),19 cellular component(CC) and 45 molecular function(MF).They are mainly involved in biological functions such as growth factor activity,post-replication repair,NAD~+diphosphatase activity,nucleoside metabolic process and guanosine nucleotide exchange factor activity and mainly enriched in cell proliferation,differentiation and tumor formation,such as PI3K/Akt,MAPK and Hippo signaling pathways.The results of RT-qPCR were consistent with the results of RNA-Seq screening.In this study,the transcription and expression levels of mammalian Ste20-like protein kinase 1/2(MST1/2),large tumor suppressor 1/2(LATS1/2),Yes-associated protein 1(YAP1) and the phosphorylation level of YAP1(p-YAP1) in the lung tissue samples of sheep in the two groups were detected by RT-qPCR and western blot(WB).Immunohistochemistry(IHC)was used to detect the expression and localization of the core proteins of Hippo signaling pathway in the lung tissue of the two sheep groups.The results of RT-qPCR showed that,compared with the control group,the m RNA transcription levels of MST1 and YAP1 in the lung tissue of OPA sheep were extremely increased(P<0.01,P<0.001),and the m RNA transcription level of LATS1was significantly increased(P<0.05).WB results showed that MST1/2 had a specific band at 59ku,YAP1 and p-YAP1 had a specific band at 65ku,and LATS1/2 had a specific band at 140ku.Compared with the control group,the expression levels of MST1/2 and p-Yap1 in the lung tissue of sheep in the OPA group were significantly increased(P<0.05),and the expression levels of LATS1/2 and YAP1 were extremely increased(P<0.01).IHC results showed that MST1/2,LATS1/2 and p-YAP1 were located in the cytoplasm of lung tissue in both OPA group and control group.MST1/2,LATS1/2 and p-Yap1 were strongly expressed in lung tissue of OPA group.YAP1 was mainly localized in the cytoplasm of control group,while in the OPA group,YAP1 was mainly localized in the nucleus(with a small amount in the cytoplasm).These results indicated that there were significantly differentially transcribed genes in the lung tissue of sheep between OPA group and control group,which were mainly involved in cell proliferation,tumorigenesis and metastasis,and were mainly enriched in PI3K/Akt,MAPK,Hippo signaling pathways.In particular,YAP,one of the core proteins in the Hippo signaling pathway,may promote the occurrence and development of tumors through nuclear heterotopic.This study provides a new research basis and direction for the tumorigenesis mechanism of JSRV.
作者
段续接
杨惠
孙志伟
杜晓悦
张培
梁浚哲
刘淑英
DUAN Xu-jie;YANG Hui;SUN Zhi-wei;DU Xiao-yue;ZHANG Pei;LIANG Jun-zhe;LIU Shu-ying(College of Veterinary Medicine,Inner Mongolia Agricultural University,Hohhot 010018,China;Key Laboratory of Clinical Diagnosis and Treatment Technology of Animal Diseases,Ministry of Agriculture and Rural Areas,Hohhot 010018,China;Key Laboratory of Basic Veterinary Medicine,Inner Mongolia Autonomous Region,Hohhot 010018,China)
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2024年第2期185-194,共10页
Chinese Journal of Preventive Veterinary Medicine
基金
国家自然科学基金项目(32072819、32360863)
内蒙古科技重大专项计划资助项目(2021ZD0010)
内蒙古草原英才创新团队项目(20151031)
牛羊疫病防控与发育工程创新团队资助项目(BR22-13-08)
研究生科研创新资助项目(B20231072Z、B20210174Z)。