Ⅲ型登革病毒感染THP-1细胞及其介导的抗体依赖性增强感染中差异性LncRNA的CeRNA网络构建  

Construction of CeRNA network of differential LncRNAs in THP-1 cells infected by Dengue virus typeⅢand its antibody dependent enhancement

作　　者：龙明望 王涵 张丽 贾凡 刘燕会 宁雪磊 陈俊英[1,2] 潘玥 孙强明[1,2] LONG Mingwang;WANG Han;ZHANG Li;JIA Fan;LIU Yanhui;NING Xuelei;CHEN Junying;PAN Yue;SUN Qiangming(Institute of Medical Biology,Chinese Academy of Medical Sciences&Peking Union Medical College,Kunming 650118,Yunnan Province,China)

机构地区：[1]中国医学科学院北京协和医学院医学生物学研究所,云南昆明650118 [2]云南省重大传染病疫苗研发重点实验室,云南昆明650118 [3]昆明医科大学医学生物学研究所,云南昆明650500 [4]云南大学生命科学学院,云南昆明650091

出　　处：《中国生物制品学杂志》2023年第9期1039-1046,1053,共9页Chinese Journal of Biologicals

基　　金：国家自然科学基金面上项目(31970868);云南省重大专项和重点研发计划(2019ZF004);云南省高层次科技人才及创新团队选拔专项——创新团队项目(202105AE160020);云南省医学领军人才项目(L-2019030)。

摘　　要：目的在急性单核细胞白血病细胞(THP-1)水平建立Ⅲ型登革病毒(Dengue virus typeⅢ,DENV-3,DV-3)感染和抗体依赖性增强(antibody dependent enhancement,ADE)感染模型,探讨胞内长链非编码RNA(long non-coding RNA,LncRNA)的差异性表达,绘制竞争性内源RNA(competitive endogenous RNA,CeRNA)调控网络并进行LncRNA翻译功能预测。方法DENV-3感染C6/36细胞6 d后,收获培养上清,采用CCID50法测定病毒滴度,并通过PCR进行型别以及基因组全长扩增鉴定;扩增DENV-3标准质粒,PCR鉴定,绘制标准曲线;将THP-1细胞分为阴性对照(THP-1)、直接感染(DV-3)、ADE及空白对照[1640(-)]组,感染48 h后提取胞内总RNA,测定病毒拷贝数;通过全转录组测序技术,对THP-1 vs DENV-3、THP-1 vs ADE、DENV-3 vs ADE各组中上调和下调前5个的LncRNA进行CeRNA调控网络构建,并分析其编码蛋白的功能。结果DENV-3感染C6/36细胞3 d后有明显的细胞融合、空泡和脱落;病毒滴度约为1.0×10^(4.64)PFU/mL,PCR特异引物鉴定为DENV-3,获得病毒完整的基因序列;ADE组胞内病毒核酸拷贝数明显高于DV-3组和空白对照组;在THP-1 vs DENV-3中,预测到人细胞黏附蛋白相互作用蛋白(cytohesin interacting protein,CYTIP)的表达量出现上调;在THP-1 vs ADE中,预测到驱动蛋白家族成员5A(kinesin family 5A,KIF5A)的表达量下调;在DENV-3 vs ADE中,预测到簇分化抗原9(cluster differentiation antigen 9,CD9)和胰岛素样生长因子2(insulin like growth factor 2,IGF2)的表达量上调。这些差异性的LncRNA均具有开放阅读框(open reading frame,ORF),除了Lnc-SH3BP1和Lnc-RPL41以外,其余的LncRNA均具有内部核糖体结合位点(internal ribosome binding site,IRES)。结论在DENV-3感染THP-1细胞及其介导的ADE感染中,LncRNA的表达发生了明显的差异性改变,且可能通过多种生物学功能调控感染的进程,有助于更深层次理解ADE感染的发生机制。Objective To establish models of Dengue virus typeⅢ(DENV-3,DV-3)infection and antibody dependent enhancement(ADE)infection at the acute monocytic leukemia cells(THP-1),investigate the differential expression of long non-coding RNAs(LncRNAs),map the competitive endogenous RNA(CeRNA)regulatory network and predict the translation function of LncRNAs.MethodsThe culture supernatant was harvested 6 d after C6/36 cells were infected with DENV-3,the virus titer was determined by CCID50,and the type and full-length genome amplification were identified by PCR;The DENV-3 standard plasmid was amplified,identified by PCR,and the standard curve was drawn;THP-1 cells were divided into negative control group(THP-1),direct infection group(DV-3),ADE group and blank control group[1640(-)].After 48 h of infection,the total RNA was extracted and the copy number of intracellular virus nucleic acid was measured;Through the whole transcriptome sequencing technology,the CeRNA regulatory network was constructed for the top five up-regulated and down-regulated LncRNAs in THP-1 vs DENV3,THP-1 vs ADE,DENV3 vs ADE groups,and the functions of their coding proteins were analyzed.ResultsC6/36 cells infected with DENV-3 for 3 d showed obvious cell fusion,vacuoles and abscission;The virus had a titer of about 1.0×10^(4.64)PFU/mL and was identified as DENV-3 by PCR specific primers,of which the complete gene sequence was obtained;The number of viral nucleic acid copies in ADE group was significantly higher than those in DV-3 group and blank control group;In THP-1 vs DENV-3,the expression of cytohesin interacting protein(CYTIP)was predicted to be up-regulated;In THP-1 vs ADE,the expression of kinesin family5A(KIF5A)was predicted to be down-regulated;In DENV-3 vs ADE,the expression of cluster differentiation antigen 9(CD9)and insulin like growth factor 2(IGF2)was predicted to be up-regulated.All of these differential LncRNAs had open reading frames(ORFs).Except Lnc-SH3BP1 and Lnc-RPL41,all of the other LncRNAs had internal ribosome binding site(IR

关 键 词：登革病毒 抗体依赖性增强 竞争性内源RNA 全转录组测序技术 长链非编码RNA 

分 类 号：Q936[生物学—微生物学]