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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2018, Vol. 38 Issue (5): 1-9    DOI: 10.13523/j.cb.20180501
研究报告     
结核分枝杆菌H37Rv刺激巨噬细胞后差异表达lncRNA分析及鉴定 *
谭杨,刘胜,罗凤玲,章晓联()
武汉大学基础医学院 病毒学国家重点实验室 湖北省过敏及免疫相关疾病重点实验室和医学研究院 武汉 430071
Analysis of Differential lncRNA Expression Profile in the Macrophages after Mycobacterium tuberculosis Stimulation
Yang TAN,Sheng LIU,Feng-ling LUO,Xiao-lian ZHANG()
Department of Immunology, State Key Laboratory of Virology, Hubei Province Key Laboratory of Allergy and Immunology and Institute of Medical Research, Wuhan University School of Medicine,Wuhan 430071, China
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摘要：

分析毒性结核分枝杆菌(Mycobacterium tuberculosis, M.tb)H37Rv刺激RAW264.7巨噬细胞的表达谱芯片以及筛选和鉴定M.tb H37Rv感染巨噬细胞RAW264.7后差异表达的lncRNA。首先,分析热灭活的H37Rv刺激RAW264.7细胞24h后lncRNA表达谱芯片,并对差异表达的lncRNA和mRNA进行生物信息分析,然后采用实时定量聚合酶链反应对16条在芯片中差异表达的lncRNA进行细胞水平验证;进一步在H37Rv感染小鼠的脾脏和肺脏中检测差异表达的lncRNA。结果显示,表达谱芯片中4 730条lncRNA 的表达水平上调, 9 558条lncRNA 的表达水平下调。生物信息分析筛选的16条lncRNA,其染色体定位于附近蛋白质编码基因的基因间区或者与外显子区域有重叠,mRNA 功能注释显示差异表达的mRNA 主要集中于转录调节、磷酸化、凋亡等生化过程以及丝裂原活化蛋白激酶(MAPK)等抗结核的信号通路中。在H37Rv作用下的细胞水平和动物感染模型的组织中RT-qPCR验证出与芯片结果相同的4条lncRNA,其中上调的有3条,下调的有1条。研究中异常表达的lncRNA可为巨噬细胞在结核分枝杆菌感染中的功能紊乱提供线索,为后续的研究奠定基础,进一步的研究将集中于发掘lncRNA在宿主调控中发挥的功能。
关键词： 结核分枝杆菌(M.tb);  巨噬细胞;  长链非编码RNA;  mRNA;  芯片分析    
Abstract:

To screen for differentially lncRNAs expression profile and predict their functional roles in the macrophage after Mycobacterium tuberculosis (M.tb) stimulation. Firstly, microarray and bioinformatics analysis of lncRNA and mRNA expression profiles in RAW264.7 macrophage after stimulation with M.tb for 24h.Then, 16 differentially expressed lncRNAs from microarray analysis were further verified by RT-qPCR using H37Rv infected macrophages and mouse model. The results shown that the expression levels of 4 730 lncRNAs was up-regulated, and the expression levels of 9 558 lncRNAs was down-regulated. 16 differentially expressed lncRNAs from microarray screening were associated with protein coding genes in adjacent locations. The mRNA function annotation analysis revealed that the mRNAs of differential expression were mainly concentrated in the biochemical process of transcriptional regulation, phosphorylation, apoptosis and MAPK signaling pathway which participating in the biochemical process of anti-tuberculosis. The expression trend of 4 lncRNAs in the iH37Rv stimulated RAW264.7 and mouse infection model was vertified as the same in both microarray and RT-qPCR analysis. Three of these 4 lncRNAs was up-regulated and one of them was down-regulated.The abnormal expression of lncRNAs may provide clues to the dysfunction of macrophages with M.tb infection, and further research will focus on the investigation of the function and regulation mechanism of lncRNA in M.tb infected macrophage.
Key words: Mycobacterium tuberculosis (M.tb)    Macrophage    Long non-coding RNA (lncRNA)    mRNA    Microarray analysis
收稿日期: 2018-01-19 出版日期: 2018-06-05
ZTFLH:  Q819  
基金资助: * “十二五”国家重大传染病专项(2012ZX10003002-015);“十三五”国家重大传染病专项资助项目(2017ZX10201301-006)
通讯作者: 章晓联     E-mail: zhangxiaolian@whu.edu.cn
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引用本文:

谭杨,刘胜,罗凤玲,章晓联. 结核分枝杆菌H37Rv刺激巨噬细胞后差异表达lncRNA分析及鉴定 *[J]. 中国生物工程杂志, 2018, 38(5): 1-9.

Yang TAN,Sheng LIU,Feng-ling LUO,Xiao-lian ZHANG. Analysis of Differential lncRNA Expression Profile in the Macrophages after Mycobacterium tuberculosis Stimulation. China Biotechnology, 2018, 38(5): 1-9.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20180501    或    https://manu60.magtech.com.cn/biotech/CN/Y2018/V38/I5/1

Number ACC.No.1) Sense-primer(5'-3') Anti-sense-primer(5'-3')
1 FR394054 TGTTTATCTGTTTGGGTTTG CCTGGCATCAAATAACCT
2 FR327109 CACTAAAGCATAAAATGAAAGG ACATGCAGGCAAAACAGC
3 FR155939 GATGGGAACGCCGAGTAAA CCTGGAAGGACATTGGGT
4 FR009074 TATCAGGTGGTAGCAGGAA TGTGAAGTTGGGATGGTG
5 FR321982 GGAGGCAGCAGAACACCA AACAGGGACAGCCAGAGT
6 FR245513 TCCTAGTAAAAATCCCCTAA TTTGCTTGGATAAAGGCAT
7 FR009059 AAGCAAAGACAGGTGGAT GGAGAAAGTTCAAGAGGC
8 n291603 GACTGCTGCGTGTCACCTAA CCCTGCTGTCAAAGACCTCC
9 chr4:3437474-3438228 GGACTTTCCAATGTGACTAT ATATGTAGGGGTATTGAACC
10 chr19:5834117-5835940 ACACTATCCTTTGAATCTCG GTCAGGTTAGGTTAAGCCAC
11 chr19:5795690-5797464 CTCTGGCCCCTTGAATAGAT GCTGTGCTGCCTTAGGTAAA
12 chr19:5798135-5800557 TTTCGTTTGCCTCAGACAGG AGCACATAATGATCCCTTTC
13 chr8:19692532-19694101 GTTCAATTCCCAGCAACCAC CATAGCCCAGCACTTCTTCC
14 chr1:134883350-134883624 TGAACACTAAGGAATTTGAG CTCCATCTTCAAGGCAGTCA
15 n270524 AGAACCCAAGTCAACCAG TCCCTACAGCAGTATCCC
16 chr16:4592919-4594230 CGCCAGAGCCACATCAAGAC TCAAACCGAAGCACCCATCA
表1  RT-qPCR验证16 条差异表达lnRNA 的引物
图1  iH37Rv刺激巨噬细胞RAW264.7细胞 24h后200条 lncRNA 差异表达谱
Number Acc. No.1) Chr
strand		 FC.2) Relationship Nearby coding
gene		 Chr
strand		 FC.2)
1 FR394054 15(+) +72.9 Exon-sense overlapping Mtdh 15((+) +8.6
2 FR327109 19(-) +40.2 Exon-sense overlapping LF201763 19(-) n.a.
3 FR155939 2(+) +28.1 Exon-sense overlapping AK037119 2(+) n.a.
4 FR009074 12(-) +46.2 Intergenic Rps6ka5 12(-) -3.3
5 FR321982 2(+) +31.0 Exon-sense overlapping KF703539 2(+) n.a.
6 FR245513 10(-) +26.2 Exon-sense overlapping Cpsf6 10(-) n.a.
7 FR009059 X(+) +23.6 Exon-sense overlapping Zrsr2 X(-) +10.2
8 n291603 7(+) +96.4 Unknow n.a.
9 chr4:3437474-3438228 4(-) +58.1 Unknow n.a.
10 chr19:5834117-5835940 19(-) +40.7 Unknow n.a.
11 chr19:5795690-5797464 19(+) +34.7 Exon-sense overlapping AY722410 19(-) +2.0
12 chr19:5798135-5800557 19(-) +31.3 Intergenic Gm20417 19(+) n.a.
13 chr8:19692532-19694101 8(-) +30.1 Intergenic Gm6483 8(+) n.a.
14 chr1:134883350-134883624 1(-) +29.6 Intergenic Ppp1r12b 1(-) +5.1
15 n270524 2(-) -56.2 unknow n.a.
16 chr16:4592919-4594230 16(-) -25.7 Exon-sense overlapping Ak021285 16(-) -46.6
表2  筛选的16条差异表达 lncRNA 的染色体定位以及与附近蛋白质编码基因的关系
图2  差异表达的mRNAs功能注释
图3  RT-qPCR验证iH37Rv刺激RAW264.7细胞24h后16条lncRNA的表达水平
图4  RT-qPCR检测H37Rv感染小鼠7天后肺脏和脾脏组织中16条lncRNA 的表达水平
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