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

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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