Identification and bioinformatics analysis of differentially expressed miRNAs in splenic lymphocytes in Echinococcus multilocularis-infected mice

doi:10.12140/j.issn.1000-7423.2022.03.003

Abstract

Abstract:

Objective To screen and identify the differentially expressed microRNAs (miRNAs) in spleen lymphocytes of mice infected with Echinococcus multilocularis, analyze the possible biological processes involved and signalling pathways, and provide experimental basis for further study of the role of miRNAs in parasite infection. Methods Twelve mice were randomly divided into two groups with 6 mice in each group. Each mouse in the experimental group was intraperitoneally injected with 600 protoscoleces, while the control group was injected with the same amount of PBS. On the 90th day after infection, the spleen lymphocytes of the mice in each group were separated by density gradient centrifugation, and the total RNA of the spleen lymphocytes was extracted by the TRIzol method. The differentially expressed miRNAs were identified and screened by high-throughput sequencing. Five differentially expressed miRNAs were randomly selected for real-time quantitative PCR (qRT-PCR) validation. MiRanda and RNAhybrid databases were used to predict the target genes of differentially expressed miRNAs, and gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis was performed. Cytoscape software was used to construct miRNA-mRNA interaction network diagrams. Results In the high-throughput sequencing, 12 104 631 and 10 856 249 clean reads were obtained in the control group and the experimental group, respectively. The sequences of clean reads were abundant in 20~24 nt in length. A total of 69 differentially expressed miRNAs (fold change> 2) were screened by high-throughput sequencing technology, of 40 were up-regulated, and 29 were down-regulated. The qRT-PCR results showed that the expression of miR-150-5p, miR-181a-5p, miR-467a-5p, and miR-467b-5p was down-regulated while the expression of miR-223-3p was up-regulated. GO enrichment results showed that the targets of 69 differentially expressed miRNAs were involved in mucosal immunity, stress response, and bacterial defence response. KEGG pathway analysis revealed that these genes were involved in the mitogen-activated protein kinase (MAPK) signalling pathway, phosphatidylinositol 3-kinase/protein kinase B (PI3K-Akt) signaling pathway, adenosine monophosphate-activated protein kinase (AMPK) signalling pathway, and other related pathways. The miR-150-5p, miR-181a-5p, miR-467a-5p, and miR-467b-5p and mRNA interaction network diagram shows that a miRNA interacted with multiple mRNAs, and a mRNA can also be regulated by multiple miRNAs. Conclusion The miRNA expression in mice spleen lymphocytes was significantly elevated during E. multilocularis infection, while the differentially expressed miRNAs were mainly enriched in some immune-related signalling pathways, which may play a role in the immune responses against E. multilocularis.

Key words: Echinococcus multilocularis, miRNA, Splenic lymphocytes, High-throughput sequencing, Bioinformatics

CLC Number:

R383.33

ZHONG Shun-hu, SUN Yue, GUO Xiao-la, ZHENG Ya-dong, CHEN Yi-xia. Identification and bioinformatics analysis of differentially expressed miRNAs in splenic lymphocytes in Echinococcus multilocularis-infected mice[J]. CHINESE JOURNAL OF PARASITOLOGY AND PARASITIC DISEASES, 2022, 40(3): 288-294.

Figures/Tables 9

Table 1

Table 2

Fig. 1

Fig. 2

Table 3

The top 20 up- and down-regulated miRNAs in spleen lymphocytes RNA from mice infected by E. multilocularis

miRNA名称 miRNA name	log₂（差异倍数） log₂(Fold change)	P值 P value	上调/下调 Up/Down
mmu-miR-146b-5p	1.933 5	0	上调Up
mmu-miR-148a-3p	2.174 6	0	上调Up
mmu-miR-223-3p	3.626 6	0	上调Up
mmu-miR-451a	1.510 9	0	上调Up
mmu-miR-143-3p	2.027 5	3.36E-79	上调Up
mmu-miR-30a-5p	1.026 8	5.47E-79	上调Up
mmu-miR-223-5p	3.131 8	3.15E-60	上调Up
mmu-miR-144-3p	2.097 3	5.82E-54	上调Up
mmu-miR-486a-3p	1.627 7	1.51E-51	上调Up
mmu-miR-486a-5p	1.628 6	5.71E-51	上调Up
mmu-miR-486b-3p	1.631 7	7.76E-51	上调Up
mmu-miR-152-3p	2.264 8	6.81E-31	上调Up
mmu-miR-221-3p	1.540 8	2.21E-26	上调Up
mmu-miR-122-5p	2.540 3	2.00E-23	上调Up
mmu-miR-122b-3p	2.540 3	2.00E-23	上调Up
mmu-miR-148a-5p	2.455 4	3.85E-22	上调Up
mmu-miR-147-3p	3.554 5	4.85E-21	上调Up
mmu-miR-652-3p	1.093 6	7.16E-18	上调Up
mmu-miR-99a-5p	1.655 5	9.39E-16	上调Up
mmu-miR-126a-3p	1.822 3	1.06E-12	上调Up
mmu-miR-150-5p	-1.159 3	0	下调Down
mmu-miR-181a-5p	-1.344 0	0	下调Down
mmu-miR-467a-5p	-1.099 9	2.20E-42	下调Down
mmu-miR-467b-5p	-1.099 9	2.20E-42	下调Down
mmu-miR-669a-3p	-1.225 8	5.35E-24	下调Down
mmu-miR-467e-5p	-1.209 5	1.51E-21	下调Down
mmu-miR-467c-5p	-1.354 8	9.30E-18	下调Down
mmu-miR-20b-5p	-1.075 0	2.80E-16	下调Down
mmu-miR-467a-3p	-1.097 2	1.18E-12	下调Down
mmu-miR-467d-3p	-1.097 2	1.18E-12	下调Down
mmu-miR-466a-3p	-1.176 1	6.21E-11	下调Down
mmu-miR-466b-3p	-1.167 6	7.84E-11	下调Down
mmu-miR-466d-3p	-1.361 8	8.03E-09	下调Down
mmu-miR-34c-5p	-2.290 3	2.37E-08	下调Down
mmu-miR-184-3p	-3.596 7	4.32E-08	下调Down
mmu-miR-1a-3p	-3.574 3	1.01E-06	下调Down
mmu-miR-1b-5p	-3.574 3	1.01E-06	下调Down
mmu-miR-455-5p	-1.306 9	4.94E-06	下调Down
mmu-miR-100-5p	-1.161 6	7.96E-06	下调Down
mmu-miR-297b-3p	-1.260 0	1.79E-05	下调Down

Table 3

Fig. 3

Fig. 4

Fig. 5

Fig. 6

References 34

[1]	Moro P,, Schantz PM. Echinococcosis: a review[J]. Int J Infect Dis, 2009, 13(2): 125-133. doi: 10.1016/j.ijid.2008.03.037
[2]	Jenkins EJ,, Schurer JM,, Gesy KM. Old problems on a new playing field: helminth zoonoses transmitted among dogs, wildlife, and people in a changing northern climate[J]. Vet Parasitol, 2011, 182(1): 54-69. doi: 10.1016/j.vetpar.2011.07.015 pmid: 21802208
[3]	Jin XL,, Guo XL,, Zhu DQ, et al. miRNA profiling in the mice in response to Echinococcus multilocularis infection[J]. Acta Trop, 2017, 166: 39-44. doi: 10.1016/j.actatropica.2016.10.024
[4]	Liu HD,, Wang HB,, Fan HN, et al. Alveolar echinococcosis and immune evasion[J]. Chin J Parasitol Parasit Dis, 2018, 36(6): 655-660. (in Chinese)
	( 刘寒冬,, 王宏宾,, 樊海宁, 等. 多房棘球蚴病的免疫逃避机制[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(6): 655-660.)
[5]	Zhang MY,, Wu WP,, Guan YY, et al. Analysis on disease burden of hydatid disease in China[J]. Chin J Parasitol Parasit Dis, 2018, 36(1): 15-19, 25. (in Chinese)
	( 张梦媛,, 伍卫平,, 官亚宜, 等. 我国棘球蚴病疾病负担分析[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(1): 15-19, 25.)
[6]	He Z,, Yan TM,, Yuan Y, et al. miRNAs and lncRNAs in Echinococcus and echinococcosis[J]. Int J Mol Sci, 2020, 21(3): 730. doi: 10.3390/ijms21030730
[7]	Wen H,, Vuitton L,, Tuxun T, et al. Echinococcosis: advances in the 21st century[J]. Clin Microbiol Rev, 2019, 32(2): e00075-18.
[8]	Guo BP. Study on correlation between pathogenic differences and mitochondrial genetic markers in Echinococcus multilocularis[D]. Shihezi: Shihezi University, 2019: 1-5. (in Chinese)
	( 郭宝平. 多房棘球绦虫致病差异与线粒体遗传标志相关性的研究[D]. 石河子: 石河子大学, 2019: 1-5.)
[9]	Huang H,, Zhang SK. Research progress on mechanisms of infiltration and metastasis for the alveolar echinococcosis[J]. Chin J Zoonoses, 2016, 32(7): 670-673, 678. (in Chinese)
	( 黄红,, 张淑坤. 多房棘球蚴病的浸润和转移机制研究进展[J]. 中国人兽共患病学报, 2016, 32(7): 670-673, 678.)
[10]	Lou ZZ,, Li HM,, Yan HB, et al. Research advances in interplay of host immune mechanism and Echinococcus multilocularis metacestodes[J]. Chin J Parasitol Parasit Dis, 2012, 30(5): 401-405. (in Chinese)
	( 娄忠子,, 李宏民,, 闫鸿斌, 等. 多房棘球蚴感染与宿主相互作用的免疫学机制研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2012, 30(5): 401-405.)
[11]	Yang JQ,, Zhou YH,, Singh RR. Effects of invariant NKT cells on parasite infections and hygiene hypothesis[J]. J Immunol Res, 2016, 2016: 2395645.
[12]	Yang J,, Chen XQ,, Wu JE, et al. Immunoregulation of mmu-miR-146a-5p on mouse macrophages[J]. Chin Vet Sci, 2019, 49(4): 506-511. (in Chinese)
	( 杨静,, 陈晓倩,, 吴金恩, 等. mmu-miR-146a-5p对小鼠巨噬细胞的免疫调节作用[J]. 中国兽医科学, 2019, 49(4): 506-511.)
[13]	Rusca N,, Monticelli S. miR-146a in immunity and disease[J]. Mol Biol Int, 2011, 2011: 437301.
[14]	Zheng YD,, Cai XP,, Bradley JE. microRNAs in parasites and parasite infection[J]. RNA Biol, 2013, 10(3): 371-379. doi: 10.4161/rna.23716
[15]	Li LH,, Wang W,, Hou XL, et al. Affects of Echinococcus multilocularis metacestode infection on the natural killer T cells and their subsets in mouse spleen[J]. Chin J Parasitol Parasit Dis, 2021, 39(3): 311-317. (in Chinese)
	( 李玲慧,, 王伟,, 侯昕伶, 等. 多房棘球蚴感染对小鼠脾自然杀伤T细胞及其亚群的影响[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(3): 311-317.)
[16]	Li FR,, Shi YE,, Shi DZ, et al. Observation of lymphocyte from hosts with alveolar echinococcosis[J]. Chin J Zoonoses, 2003, 19(3): 91-94. (in Chinese)
	( 李富荣,, 石佑恩,, 史大中, 等. 泡状棘球蚴病宿主淋巴细胞的变化及意义[J]. 中国人兽共患病杂志, 2003, 19(3): 91-94.)
[17]	Vuitton DA,, Gottstein B. Echinococcus multilocularis and its intermediate host: a model of parasite-host interplay[J]. J Biomed Biotechnol, 2010, 2010: 923193.
[18]	Xu K,, Wang HJ,, Zhang L, et al. Research progress on the mechanisms underlying the impairment of host hepatocytes by Echinococcus multilocularis[J]. Chin J Parasitol Parasit Dis, 2021, 39(2): 256-260. (in Chinese)
	( 徐凯,, 王海久,, 张丽, 等. 多房棘球蚴对宿主肝细胞损害机制的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2021, 39(2): 256-260.)
[19]	Zhang N,, Zhang CS,, Li ZD, et al. The effects of Echinococcus multilocularis protoscoleces infection on lymphocytes and their subpopulations in mouse liver and spleen[J]. Chin J Parasitol Parasit Dis, 2020, 38(1): 30-35. (in Chinese)
	( 章宁,, 张传山,, 李智德, 等. 多房棘球蚴感染对小鼠肝脾淋巴细胞及其亚群的影响[J]. 中国寄生虫学与寄生虫病杂志, 2020, 38(1): 30-35.)
[20]	Zhang YE. Immunoregulatory effects of emu-miR-71 and its application in anti-E. multilocularis metacestode infection[D]. Beijing: Chinese Academy of Agricultural Sciences, 2021: 2-6). (in Chinese)
	张永娥. emu-miR-71的免疫调节作用及其在抗多房棘球蚴感染中的应用[D]. 北京: 中国农业科学院, 2021: 2-6).
[21]	Liu K,, Huang HB,, Yang GL. miRNA functions in parasite-related immune regulation in hosts[J]. Chin J Parasitol Parasit Dis, 2018, 36(4): 405-408. (in Chinese)
	( 刘可,, 黄海斌,, 杨桂连. miRNA在寄生虫宿主免疫调控中的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(4): 405-408.)
[22]	Ding JT,, He GT,, Wu JE, et al. miRNA-seq of Echinococcus multilocularis extracellular vesicles and immunomodulatory effects of miR-4989[J]. Front Microbiol, 2019, 10: 2707. doi: 10.3389/fmicb.2019.02707
[23]	Cai MT,, Ding JT,, Li YT, et al. Echinococcus multilocularis infection induces UBE2N suppression via exosomal emu-miR-4989[J]. Acta Trop, 2021, 223: 106087. doi: 10.1016/j.actatropica.2021.106087
[24]	Zhang CS,, Wang LM,, Ali T, et al. Hydatid cyst fluid promotes pericystic fibrosis in cystic echinococcosis by suppressing miR-19 expression[J]. Parasit Vectors, 2016, 9(1): 278. doi: 10.1186/s13071-016-1562-x
[25]	He X,, Tang R,, Sun Y, et al. MicroR-146 blocks the activation of M1 macrophage by targeting signal transducer and activator of transcription 1 in hepatic schistosomiasis[J]. eBio Medicine, 2016, 13: 339-347.
[26]	Li YM,, Yu JF,, Wang F, et al. miR-150-5p regulate T cell activation in severe aplastic Anemia by targeting Bach2[J]. Cell Tissue Res, 2021, 384(2): 423-434. doi: 10.1007/s00441-020-03373-9
[27]	Cao JP. The effect and mechanism of miR-150-5p on the IL-10, IL-1β via targeting IL-19 in macrophages[D]. Hengyang: University of South China, 2020: 16-18). (in Chinese)
	曹佳萍. miR-150-5p靶向作用IL-19影响巨噬细胞炎症因子IL-10和IL-1β的分泌[D]. 衡阳: 南华大学, 2020: 16-18).
[28]	Jiang F,, Li W,, Chen J, et al. Mechanism of down-regulation of mi R-150-5p inhibit apoptosis in diabetic nephropathy podocyte model in vitro through PI3K/AKT signaling pathway[J]. Chin J Integr Tradit West Nephrol, 2021, 22(3): 205-209. (in Chinese)
	( 江帆,, 李伟,, 陈娟, 等. 下调miR-150-5p调控PI3K/AKT信号通路抑制体外糖尿病肾病足细胞模型凋亡的机制研究[J]. 中国中西医结合肾病杂志, 2021, 22(3): 205-209.)
[29]	Leong JW,, Sullivan RP,, Fehniger TA. microRNA management of NK-cell developmental and functional programs[J]. Eur J Immunol, 2014, 44(10): 2862-2868. doi: 10.1002/eji.201444798
[30]	Lu J,, Li S,, Li XP, et al. Declined miR-181a-5p expression is associated with impaired natural killer cell development and function with aging[J]. Aging Cell, 2021, 20(5): e13353.
[31]	Li M,, He Y,, Zhou Z, et al. microRNA-223 ameliorates alcoholic liver injury by inhibiting the IL-6-p47 phox-oxidative stress pathway in neutrophils[J]. Gut, 2017, 66(4): 705-715. doi: 10.1136/gutjnl-2016-311861
[32]	Dorhoi A,, Iannaccone M,, Farinacci M, et al. microRNA-223 controls susceptibility to tuberculosis by regulating lung neutrophil recruitment[J]. J Clin Invest, 2013, 123(11): 4836-4848. pmid: 24084739
[33]	Gajeton J,, Krukovets I,, Yendamuri R, et al. miR-467 regulates inflammation and blood insulin and glucose[J]. J Cell Mol Med, 2021, 25(5): 2549-2562. doi: 10.1111/jcmm.16224
[34]	Liu K,, Huang HB,, Yang GL. miRNA functions in parasite-related immune regulation in hosts[J]. Chin J Parasitol Parasit Dis, 2018, 36(4): 405-408. (in Chinese)
	( 刘可,, 黄海斌,, 杨桂连. miRNA在寄生虫宿主免疫调控中的研究进展[J]. 中国寄生虫学与寄生虫病杂志, 2018, 36(4): 405-408.)

基因名称Gene name	引物序列（5′→3′） Primer sequence (5′→3′)
通用引物 Universal primer	GCGAGCACAGAATTAATACGAC
U6-F	GCTTCGGCAGCACATATACTAAAAT
U6-R	CGCTTCACGAATTTGCGTGTCAT
miR-150-5p	GTGACCATGTTCCCAACCCTCT
miR-181a-5p	TGAGTGGCTGTCGCAACTTACAA
miR-467a-5p	GCGTATATGTACGTCCGTGAAT
miR-467b-5p	GTATATGTACGTCCGTGAATG
miR-223-3p	ACCCCATAAACTGTTTGACTGT

样品 Sample	原始序列数/个 Total no. reads	纯净序列数/个 No. clean read	已知miRNA数/个 No. known miRNA	新miRNA数/个 No. novel miRNA
对照组 Control group	12 372 815	12 104 631	712	10
感染组Infected group	11 167 064	10 856 249	781	6