林麝不同组织miRNA表达谱

doi:10.16829/j.slxb.150744

摘要/Abstract

摘要： 林麝(Moschus berezovskii)是药用经济动物，其雄麝分泌的麝香是中成药和香水的重要成分，具有极高的药用价值和经济价值。miRNAs在动物细胞发育、分化、凋亡及疾病发生发展过程中起着至关重要的作用，并且在不同组织中miRNAs选择性表达。本研究利用RNA-seq技术在Hi-seq 2500测序平台对林麝的心脏、肝脏、脾脏、肺脏、肾脏和肌肉组织进行高通量测序，运用生物统计学和生物信息学方法分析这6种器官和组织中miRNAs的表达情况，采用RT-qPCR对测序结果进行验证。结果表明，林麝的心脏、肝脏、脾脏、肺脏、肾脏和肌肉组织中共表达的miRNAs有1 650个，特异表达的miRNAs分别有86个、54个、44个、68个、83个和50个(P < 0.01)，共表达的miRNAs在不同器官和组织中表达水平存在显著差异(P < 0.05)，林麝心脏中部分特异miRNAs的表达水平远远高于其他5种器官和组织中特异miRNAs的表达水平。KEGG分析表明：林麝肝脏特异表达miR-NA靶基因主要涉及Wnt信号通路(ko04310)、cAMP信号通路(ko04024)、ECM受体相互作用(ko04512)、Notch信号途径(ko04330)、AMPK信号通路(ko04152)等(P < 0.01)。随机选择9个miRNAs，利用RT-qPCR验证其在健康和化脓性林麝肝脏中的表达情况，结果表明其中9个miRNAs的表达趋势与测序结果一致。

关键词: microRNA, 表达谱, 表达分析, 荧光定量PCR, 林麝

Abstract: Forest musk deer (Moschus berezovskii) is a medicinal economic species and the musk secreted by the male musk deer is an important component of Chinese patent medicine and perfume, which has high medicinal and economic value. miRNAs play an important role in animal cell development, differentiation, apoptosis, and disease development, which are selectively expressed in different tissues. The heart, liver, spleen, lung, kidney, and muscle tissues of forest musk deer were high-throughput sequenced by using RNA-seq technology on the Hi-seq 2500 sequencing platform. The microRNA expression of these 6 tissues was analyzed by using biostatistics and bioinformatics methods and RT-qPCR was used to verify the sequencing results. The results showed that 1 650 miRNAs co-expressed in the heart, liver, spleen, lung, kidney, and muscle tissues of forest musk deer, of which there were 86, 54, 44, 68, 83, and 50 specific miRNAs, respectively (P < 0.01). The expression levels of co-expressed miRNAs were significantly different in these six types of tissues (P < 0.05) and the expression levels of some specific miRNAs in the heart were much higher than that in other tissues. KEGG analysis showed that the target genes of specific miRNAs in the liver of forest musk deer were closely related to the Wnt signaling pathway (ko04310), cAMP signaling pathway (ko04024), ECM-receptor interaction (ko04512), Notch signaling pathway (ko04330), and AMPK signaling pathway (ko04152) (P < 0.01). Nine miRNAs were randomly selected for verification with RT-qPCR in liver tissue of health and abscess forest musk deer and the results were consistent with the sequencing results.

Key words: MicroRNA, Expression profile, Expression analysis, Fluorescence quantitative PCR, Forest musk deer

中图分类号:

Q752

余苗杰, 徐忠鲜, 蒋雪梅, 王春花, 赵婵娟, 戚文华, 竭航. 林麝不同组织miRNA表达谱[J]. 兽类学报, 2023, 43(6): 723-733.

YU Miaojie, XU Zhongxian, JIANG Xuemei, WANG Chunhua, ZHAO Chanjuan, QI Wenhua, JIE Hang. The expression profile of miRNA in the different tissues of forest musk deer (Moschus berezovskii)[J]. ACTA THERIOLOGICA SINICA, 2023, 43(6): 723-733.

参考文献

Adur M K, Hale B J, Ross J W. 2017. Detection of miRNA in mammalian oocytes and embryos. Methods in Molecular Biology, 1605: 63-81.
Boyerinas B, Park S M, Har A, Murmann A E, Peter M E. 2010. The role of let-7 in cell differentiation and cancer. Endocr Relat Cancer, 17 (1): F19-F36.
Cheng H X, Zhang J, Jia Z Z, Xu Y J, He R C, Fan G Q. 2017. Research progress of miRNA-10a in the molecular mechanism of digestive disease. China ＆ Foreign Medical Treatment, 36 (26):193-195. (in Chinese)
Chen L L, Wang H, Wang J K, Wang J B, Chai Z X, Chen Z H, Zhong J C. 2020. Comparative analysis of miRNA expression profiles in the hearts of Tibetan cattle and Xuanhan cattle. Scientia Agricultura Sinica, 53 (8): 1677-1687. (in Chinese)
Deo A, Carlsson J, Lindlöf A. 2011. How to choose a normalization strategy for miRNA quantitative real-time (qPCR) arrays. Journal of Bioinformatics and Computational Biology, 9 (6): 795-812.
Fichtlscherer S, De Rosa S, Fox H, Schwietz T, Fischer A, Liebetrau C, Weber M, Hamm C W, Röxe T, Müller-Ardogan M, Bonauer A, Zeiher A M, Dimmeler S. 2010. Circulating microRNAs in patients with coronary artery disease. Circulation Research, 107(5): 677-684.
Gong P, Wei P L, Wang X T, Chai T, Wu L, Wu W W. 2020. miRNA-150 and miRNA-143 expression spectrum in sheep.
Grass-Feeding Livestock, 2020 (3): 8-15. (in Chinese)
Guo Y T, Zhang X X, Huang W L, Miao X Y. 2017. Identification and characterization of differentially expressed miRNAs in subcutaneous adipose between Wagyu and Holstein cattle. Scientific Reports, 7: 44026.
Hede K. 2005. Studies define role of microRNA in cancer. Journal of the National Cancer Institute, 97 (15): 1114-1115.
Jiang H R, Xu H F. 2007. Rare and endangered resource animal: forest musk deer. Biology Bulletin, 42 (12): 22-23. (in Chinese)
Jiang Z G Ed. 2020. China's Red List of Biodiversity: Vertebrates. Volume I, Mammals. Beijing: China Science Press, 110-150. (in Chinese)
Jie H, Zhang P, Xu Z X, Mishra S K, Lei M Y, Zeng D J, Zhao G J, Li D Y. 2019. microRNA and other small RNA sequence profiling across six tissues of Chinese forest musk deer (Moschus berezovskii). BioMed Research International, 2019: 4370704.
Ji H, Wang H, Chai Z X, Wang J K, Wang J B, Luo X L, Ji Q M, Xin J W, Zhong J C. 2019. Differential expression profile and bioinformatics analysis of miRNAs in yak muscle tissue during development. Acta Veterooinaria et Zootechnica Sinica, 50 (5): 957-971. (in Chinese)
Katarova Z, Mugnaini E, Sekerková G, Mann J R, Aszódi A, Bösze Z, Greenspan R, Szabó G. 1998. Regulation of cell-type specific expression of lacZ by the 5'-flanking region of mouse GAD67 gene in the central nervous system of transgenic mice. European Journal of Neuroscience, 10 (3): 989-999.
Kato M, Slack F J. 2008. MicroRNAs: small molecules with big roles-C. elegans to human cancer. Biology of the Cell, 100 (2): 71-81.
Koralov S B, Muljo S A, Galler G R, Krek A, Chakraborty T, Kanellopoulou C, Jensen K, Cobb B S, Merkenschlager M, Rajewsky N, Rajewsky K. 2008. Dicer ablation affects antibody diversity and cell survival in the B lymphocyte lineage. Cell, 132 (5):860-874.
Kota J, Chivukula R R, O’ Donnell K A, Wentzel E A, Montgomery C L, Hwang H W, Chang T C, Vivekanandan P, Torbenson M, Clark K R, Mendell J R, Mendell J T. 2009. Therapeutic microRNA delivery suppresses tumorigenesis in a murine liver cancer model. Cell, 137 (6): 1005-1017.
Langmead B, Trapnell C, Pop M, Salzberg S L. 2009. Ultrafast and memory-efficient alignment of short DNA sequences to the human genome. Genome Biology, 10 (3): R25.
Lawrie C H. 2007. MicroRNAs and haematology: small molecules, big function. British Journal of Haematology, 137 (6): 503-512.
Lee Y S, Dutta A. 2009. MicroRNAs in cancer. Annual Review of Pathology, 4: 199-227.
Li Y, Kowdley K V. 2012. MicroRNAs in common human diseases. Genomics, Proteomics & Bioinformatics, 10 (5): 246-253.
Lü X H, Qiao J Y, Wu X M, Su L N. 2009. A review of mainly affected on musk-deer diseases: Purulent, respiratory system and parasitic diseases. Journal of Animal Ecology, 13 (2): 104-107.(in Chinese)
Ma L, Teruya-Feldsrtein J, Weinberg R A. 2007. Tumour invasion and metastasis initiated by microRNA-10b in breast cancer. Nature, 449 (7163): 682-688.
Mendell J T, Olson E N. 2012. MicroRNAs in stress signaling and human disease. Cell, 148 (6): 1172-1187.
Qi W H, Li J, Zhang X Y, Wang Z K, Li X X, Yang C Z, Fu W L, Yue B S. 2011. The reproductive performance of female forest musk deer (Moschus berezovskii) in captivity. Theriogenology, 76 (5):874-881.
Sheng H L. 1996. Current situation and protection measures of Chinese musk deer resources. Chinese Journal of Wildlife, 17 (3):10-12. (in Chinese)
Shin C, Nam J W, Farh K K H, Chiang H R, Shkumatava A, Bartel D P. 2010. Expanding the microRNA targeting code: Functional sites with centered pairing. Molecular Cell, 38 (6): 789-802.
Stark A, Brennecke J, Bushati N, Russell R B, Cohen S M. 2005. Animal microRNAs confer robustness to gene expression and have a significant impact on 3'UTR evolution. Cell, 123 (6): 1133-1146.
Shi B X, Ma C, Liu G L, Guo Y J. 2019. miR-106a directly targets LIMK1 to inhibit proliferation and EMT of oral carcinoma cells. Cellular & Molecular Biology Letters, 24: 1.
Sun J, Chen X P. 2011. The research progress of microRNAs in circulating system. Journal of Molecular Diagnostics and Therapy, 3(3): 192-199. (in Chinese)
Tijsen A J, Creemers E E, Moerland P D, de Windt L J, van der Wal A C, Kok W E, Pinto Y M. 2010. MiR423-5p as a circulating biomarker for heart failure. Circulation Research, 106 (6): 1035-1039.
Wen D X, Zhang P Y, Xu Y F, Wang F P, Suo L S Z, Niu J Q. 2020. Expression analyses of yak MBL2 gene mRNA and related miRNAs in different tissues. Chinese Veterinary Science, 50 (7): 886-897. (in Chinese)
Xu B, Liu J, Xiang X, Liu S H, Zhong P Y, Xie F, Mou T G, Lai L. 2018. Expression of miRNA-143 in pancreatic cancer and its clinical significance. Cancer Biotherapy and Radiopharmaceuticals, 33 (9): 373-379.
Xu Y, Li W X, Liu X Y, Ma H L, Tu Z G, Dai Y. 2013a. Analysis of microRNA expression profile by small RNA sequencing in down syndrome fetuses. International Journal of Molecular Medicine, 32 (5): 1115-1125.
Xu Y, Li W X, Liu X Y, Chen H, Tan K B, Chen Y Y, Tu Z G, Dai Y. 2013b. Identification of dysregulated microRNAs in lymphocytes from children with down syndrome. Gene, 530 (2): 278-286.
Zhang J, Du Y Y, Lin Y F, Chen Y T, Yang L, Wang H J, Hu J, Ma D. 2008. The cell growth suppressor, mir-126, targets IRS-1. Biochemical and Biophysical Research Communications, 377 (1):136-140.
Zhang Y, Zhao B, Roy S, Saha T T, Kokoza V A, Li M, Raikhel A S. 2016. microRNA-309 targets the Homeobox gene SIX4 and controls ovarian development in the mosquito Aedes aegypti. Proceedings of the National Academy of Sciences of the United States of America, 113 (33): E4828-E4836.
Zhou J W, Gu H J, Li W Y. 2000. The musk deer farming in Sichuan Province. Chinese Wildlife, 21 (4): 10. (in Chinese)
吕向辉, 乔继英, 吴晓民, 苏丽娜. 2009. 主要危害麝类的化脓性、呼吸系统疾病及寄生虫病研究. 经济动物学报, 13 (2):104-107.
纪会, 王会, 柴志欣, 王吉坤, 王嘉博, 罗晓林, 姬秋梅, 信金伟, 钟金城. 2019. 牦牛不同年龄肌肉组织 microRNA 表达谱及生物信息学分析. 畜牧兽医学报, 50 (5): 957-971.
孙吉, 陈小平. 2011. microRNA 与心血管疾病的关系. 分子诊断与治疗杂志, 3 (3): 192-199.
陈露露, 王会, 王吉坤, 王嘉博, 柴志欣, 陈智华, 钟金城. 2020. 藏黄牛与宣汉黄牛心脏 miRNA 表达谱比较. 中国农业科学, 53 (8):1677-1687.
国家重点保护野生动物名录. 2021. http://www.forestry.gov.cn.
周继武, 顾海军, 李维余. 2000. 四川省的麝类养殖业. 野生动物, 21 (4): 10.
宫平, 魏佩玲, 王晓涛, 柴婷, 乌兰, 吴伟伟. 2020. 绵羊中 miRNA-150和 miRNA-143 的组织表达谱研究. 草食家畜, 2020 (3): 8-15.
姜海瑞, 徐宏发. 2007. 珍稀濒危资源动物: 林麝. 生物学通报, 42(12): 22-23.
盛和林. 1996. 我国麝资源现状及救护措施. 野生动物, 17 (3):10-12.
蒋志刚主编. 2020. 中国生物多样性红色名录: 脊椎动物, 第一卷, 哺乳动物. 北京: 科学出版社, 110-150.
程海霞, 张静, 贾增增, 徐月姣, 何若冲, 范国权. 2017. miRNA-10a在消化系统疾病分子机制中的研究进展. 中外医疗, 36 (26):193-195.
温东旭, 张鹏宇, 徐业芬, 王福鹏, 索朗斯珠, 牛家强. 2020. 牦牛MBL2 基因 mRNA 及相关 miRNAs 在不同组织中的表达分析.中国兽医科学, 50 (7): 886-897.

[1]	江峰, 宋鹏飞, 张婧捷, 高红梅, 汪海静, 蔡振媛, 刘道鑫, 张同作. 不同养殖场林麝肠道微生物组成和功能的差异[J]. 兽类学报, 2023, 43(2): 129-140.
[2]	赵琪, 张琪, 李浩玲, 兰月, 鄢行安, 赵贵军, 戚文华. 林麝及其近缘物种编码区微卫星分布规律及功能分析[J]. 兽类学报, 2022, 42(6): 705-715.
[3]	王小琪, 郝文静, 张志超, 韩晶, 王儒敬, 段子渊. 腹泻柴达木马亚成体粪便微生物多样性分析及其生物标记物的筛选[J]. 兽类学报, 2022, 42(4): 432-441.
[4]	王静白瑞丹蔡永华黎勇程建国付文龙周密盛岩孟秀祥. 圈养林麝麝香分泌时间节律及影响因素[J]. 兽类学报, 2020, 40(5): 485-492.
[5]	刘嘉辉王艳边坤唐婕王伟峰郭林文王波方谷赵兰齐晓光. 重引入林麝的家域利用与个体迁移[J]. 兽类学报, 2020, 40(2): 109-119.
[6]	王豆许冠王洪永何森卜书海郑雪莉. 中国圈养林麝微卫星DNA多样性研究[J]. 兽类学报, 2019, 39(6): 599-607.
[7]	房璇孙太福蔡永华董霞黎勇周密孟秀祥. 圈养林麝的活跃性及与社会亲和度的关系[J]. 兽类学报, 2019, 39(5): 531-536.
[8]	赵贵军封孝兰朱吉彬郑程莉竭航曾德军张承露戚文华. 成年与未成年圈养林麝粪便菌群多样性的比较[J]. 兽类学报, 2019, 39(3): 266-275.
[9]	徐金会耿晓翠陈蕾薛慧良徐来祥. 生长抑素基因在不同性别和年龄黑线仓鼠哈氏腺的差异表达[J]. 兽类学报, 2016, 36(4): 429-437.
[10]	于永乐张传美杨海燕杨瑞梅张洪亮单虎. 貂肠炎病毒SYBR Green I 荧光定量PCR检测方法的建立[J]. 兽类学报, 2015, 35(1): 102-109.
[11]	薛程,孟秀祥,徐宏发,肖宇. 圈养林麝春季活动节律及时间分配[J]. , 2008, 28(2): 194-200.
[12]	徐正强徐玲李波徐宏发. 林麝幼体的换毛序和胎毛、夏毛、冬毛的形态[J]. , 2006, 26(1): 101-104.
[13]	邹方东　岳碧松　张义正　陈三. 林麝染色体制备方法及核型与G- 带带型研究[J]. , 2004, 24(2): 115-120.
[14]	徐正强　徐宏发. 饲养林麝的种群特征和幼麝的存活研究[J]. , 2003, 23(1): 17-20.
[15]	徐正强徐宏发. 圈养雌麝社会行为及其对后代的影响[J]. , 2002, 22(2): 155-158.