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Wanzhong Jia
Lanzhou Veterinary Research Institute
Corresponding Author
ORCiD: https://orcid.org/0000-0002-0801-7478
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This work is licensed under a CC BY 4.0 License. Read Full License
Background: Echinococcus multilocularis (Em) infection and the growth and proliferation of its metacestode within the internal organs of hosts are related to complex host–parasite interactions at the molecular level. Previous studies reported the profiles of long non-coding RNAs (lncRNAs) and mRNAs in Echinococcus granulosus-infected mice or cells, suggesting the potential role of lncRNAs in regulating host-parasite interplay. However, the profiles of lncRNAs and mRNAs of mice in response to Em are poorly understood.
Methods: Numerous differentially expressed lncRNAs (DELs) and mRNAs (DEMs) in the mouse liver at eight time points after Em infection were identified by microarray. Functional Annotation of dysregulated DEMs was conducted by gene ontology (GO) classification and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The potential function of DELs was predicted by constructing lncRNA-mRNA co-expression network and Transcription factor (TF)-lncRNA-mRNA Ternary Network. Additionally, qRT-PCR and western blotting were used to validate the upregulated DEMs at 30 days post-infection (dpi), which were enriched in Toll-like and RIG-I-like receptor signaling pathways. Cytokines and chemokines involved in these two pathways were determined by ELISA.
Results: Thirty-one DEMs and 68 DELs were found continuously dysregulated. These DEMs were notably enriched in the “antigen processing and presentation,” “Th1 and Th2 cell differentiation” and “Th17 cell differentiation” pathways. The potential function prediction of DELs revealed that most DELs might influence the differentiation of Th17 cell and TGF-β/Smad pathway through trans regulating the SMAD3, STAT1, and early growth response (EGR) genes. Additionally, the validated results by qRT-PCR and western blotting showed that the mRNA expression levels of these genes increased while the corresponding protein expression levels were unaltered except c-Jun amino-terminal kinase (JNK). Regardless, phospho-nuclear factor Kappa B (p-NF-κB) downstream of these two pathways was induced at 15 and 30 dpi, which led to the elevated levels of IL-1 beta and IL-6 in the serum.
Conclusion: Our data provide novel clues in understanding the roles of lncRNAs in the host–Em interplay and the influence of Em infection on host innate immunity.
Keywords
Echinococcus multilocularis, Alveolar echinococcosis, Long non-coding RNA, mRNA, Microarray, Innate immunity
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This is a list of supplementary files associated with this preprint. Click to download.
- Table1.tif
- 1AdditionalFigureS1avolcanoplotofDELs.tif
Additional Figure S1 Volcano plot and hierarchical clustering plot of differentially expressed lncRNAs and mRNAs in Echinococcus multilocularis-infected mice liver at eight time points a. Volcano plot of differentially expressed lncRNAs in Echinococcus multilocularis-infected mice liver at 2 dpi (ⅰ), 4 dpi (ⅱ), 8 dpi (ⅲ), 15 dpi (ⅳ), 30 dpi (ⅴ), 60 dpi (ⅵ)，90 dpi (ⅶ), 150 dpi (ⅷ). The significantly up- and downregulated mRNAs are presented as red and blue dots, respectively (fold change > 2 and p < 0.05). The expression of mRNAs with | fold change | < 2 is presented as green dots (p < 0.05) and the expression of mRNAs not significantly differentially expressed is presented as gray dots (p > 0.05) b. Volcano plot of differentially expressed mRNAs in Echinococcus multilocularis-infected mice liver at 2 dpi (ⅰ), 4 dpi (ⅱ), 8 dpi (ⅲ), 15 dpi (ⅳ), 30 dpi (ⅴ), 60 dpi (ⅵ)，90 dpi (ⅶ), 150 dpi (ⅷ). The significantly up- and downregulated mRNAs are presented as red and blue dots, respectively (fold change > 2 and p < 0.05). The expression of mRNAs with | fold change | < 2 is presented as green dots (p < 0.05) and the expression of mRNAs not significantly differentially expressed is presented as gray dots (p > 0.05) c. Hierarchical clustering plot of differentially expressed lncRNAs and mRNAs in Echinococcus multilocularis-infected mice liver at eight time points d. Hierarchical clustering plot of differentially expressed mRNAs in Echinococcus multilocularis-infected mice liver at eight time points
- 10AdditionalfigureS3Microarrayresultsof6genes.tif
Additional Figure S3 Expression levels of IRAK4, IKKβ, MKK, JNK, IFN-α, and NLRX1 in Echinococcus multilocularis-infected mouse liver at 15 and 30 dpi from microarray data. The y-axis indicates the log2 FC by microarray. Bars present as mean ± standard error of three biological replicates per time point. * p < 0.05, ** p < 0.01, *** p < 0.001
- 11Additionaldocumentformethods.docx
- 12AdditionaltableformethodsprimersofDEMandDEL.xlsx
- 2AdditionalFigureS1bvolcanoplotDEMs.tif
- 3AdditionalFigureS1cDELshierarchicalclusteringplot.tif
- 4AdditionalFigureS1dDEMshierarchicalclusteringplot.tif
- 5AdditionalFigureS2aTLRpathwaymmu04620.tif
Additional Figure S2 KEGG pathway analysis of up- and downregulated differentially expressed mRNAs in Echinococcus multilocularis-infected mice liver. a. Toll-like receptor signaling pathways enriched by upregulated differentially expressed mRNAs (DEMs) in Echinococcus multilocularis-infected mice liver at 30 dpi. The upregulated genes are presented as red rectangles. b. RIG-I-like receptor signaling pathways enriched by upregulated differentially expressed mRNAs (DEMs) in Echinococcus multilocularis-infected mice liver at 30 dpi. The upregulated genes are presented as red rectangles. c. Ubiquitin-mediated proteolysis pathway enriched by downregulated differentially expressed mRNAs (DEMs) in Echinococcus multilocularis-infected mice liver at 15 dpi. The downregulated genes are presented as green rectangles.
- 6AdditionalFigureS2bRLRpathwaymmu04622.tif
- 7AdditionalFigureS2cubiquitinat15dpi.png
- 8AdditionaltableS1aVenn31mRNA.xlsx
- 9AdditionaltableS1bVenn68lncRNA.xlsx
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A new preprint design is coming!
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This is a preprint, a preliminary version of a manuscript that has not completed peer review at a journal. Research Square does not conduct peer review prior to posting preprints. The posting of a preprint on this server should not be interpreted as an endorsement of its validity or suitability for dissemination as established information or for guiding clinical practice.
Research
Wanzhong Jia
Lanzhou Veterinary Research Institute
Corresponding Author
ORCiD: https://orcid.org/0000-0002-0801-7478
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Complete author information
Appropriate declaration statements
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CURRENT STATUS: Posted
Version 1
Posted 10 Jun, 2021
No community comments so far
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Comments: 0
PDF Downloads: ...
HTML Views: ...
Subject Areas
General Cell Biology & Physiology
License:
This work is licensed under a CC BY 4.0 License. Read Full License
Background: Echinococcus multilocularis (Em) infection and the growth and proliferation of its metacestode within the internal organs of hosts are related to complex host–parasite interactions at the molecular level. Previous studies reported the profiles of long non-coding RNAs (lncRNAs) and mRNAs in Echinococcus granulosus-infected mice or cells, suggesting the potential role of lncRNAs in regulating host-parasite interplay. However, the profiles of lncRNAs and mRNAs of mice in response to Em are poorly understood.
Methods: Numerous differentially expressed lncRNAs (DELs) and mRNAs (DEMs) in the mouse liver at eight time points after Em infection were identified by microarray. Functional Annotation of dysregulated DEMs was conducted by gene ontology (GO) classification and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. The potential function of DELs was predicted by constructing lncRNA-mRNA co-expression network and Transcription factor (TF)-lncRNA-mRNA Ternary Network. Additionally, qRT-PCR and western blotting were used to validate the upregulated DEMs at 30 days post-infection (dpi), which were enriched in Toll-like and RIG-I-like receptor signaling pathways. Cytokines and chemokines involved in these two pathways were determined by ELISA.
Results: Thirty-one DEMs and 68 DELs were found continuously dysregulated. These DEMs were notably enriched in the “antigen processing and presentation,” “Th1 and Th2 cell differentiation” and “Th17 cell differentiation” pathways. The potential function prediction of DELs revealed that most DELs might influence the differentiation of Th17 cell and TGF-β/Smad pathway through trans regulating the SMAD3, STAT1, and early growth response (EGR) genes. Additionally, the validated results by qRT-PCR and western blotting showed that the mRNA expression levels of these genes increased while the corresponding protein expression levels were unaltered except c-Jun amino-terminal kinase (JNK). Regardless, phospho-nuclear factor Kappa B (p-NF-κB) downstream of these two pathways was induced at 15 and 30 dpi, which led to the elevated levels of IL-1 beta and IL-6 in the serum.
Conclusion: Our data provide novel clues in understanding the roles of lncRNAs in the host–Em interplay and the influence of Em infection on host innate immunity.
Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7
Figure 8
This is a list of supplementary files associated with this preprint. Click to download.
- Table1.tif
- 1AdditionalFigureS1avolcanoplotofDELs.tif
Additional Figure S1 Volcano plot and hierarchical clustering plot of differentially expressed lncRNAs and mRNAs in Echinococcus multilocularis-infected mice liver at eight time points a. Volcano plot of differentially expressed lncRNAs in Echinococcus multilocularis-infected mice liver at 2 dpi (ⅰ), 4 dpi (ⅱ), 8 dpi (ⅲ), 15 dpi (ⅳ), 30 dpi (ⅴ), 60 dpi (ⅵ)，90 dpi (ⅶ), 150 dpi (ⅷ). The significantly up- and downregulated mRNAs are presented as red and blue dots, respectively (fold change > 2 and p < 0.05). The expression of mRNAs with | fold change | < 2 is presented as green dots (p < 0.05) and the expression of mRNAs not significantly differentially expressed is presented as gray dots (p > 0.05) b. Volcano plot of differentially expressed mRNAs in Echinococcus multilocularis-infected mice liver at 2 dpi (ⅰ), 4 dpi (ⅱ), 8 dpi (ⅲ), 15 dpi (ⅳ), 30 dpi (ⅴ), 60 dpi (ⅵ)，90 dpi (ⅶ), 150 dpi (ⅷ). The significantly up- and downregulated mRNAs are presented as red and blue dots, respectively (fold change > 2 and p < 0.05). The expression of mRNAs with | fold change | < 2 is presented as green dots (p < 0.05) and the expression of mRNAs not significantly differentially expressed is presented as gray dots (p > 0.05) c. Hierarchical clustering plot of differentially expressed lncRNAs and mRNAs in Echinococcus multilocularis-infected mice liver at eight time points d. Hierarchical clustering plot of differentially expressed mRNAs in Echinococcus multilocularis-infected mice liver at eight time points
- 10AdditionalfigureS3Microarrayresultsof6genes.tif
Additional Figure S3 Expression levels of IRAK4, IKKβ, MKK, JNK, IFN-α, and NLRX1 in Echinococcus multilocularis-infected mouse liver at 15 and 30 dpi from microarray data. The y-axis indicates the log2 FC by microarray. Bars present as mean ± standard error of three biological replicates per time point. * p < 0.05, ** p < 0.01, *** p < 0.001
- 11Additionaldocumentformethods.docx
- 12AdditionaltableformethodsprimersofDEMandDEL.xlsx
- 2AdditionalFigureS1bvolcanoplotDEMs.tif
- 3AdditionalFigureS1cDELshierarchicalclusteringplot.tif
- 4AdditionalFigureS1dDEMshierarchicalclusteringplot.tif
- 5AdditionalFigureS2aTLRpathwaymmu04620.tif
Additional Figure S2 KEGG pathway analysis of up- and downregulated differentially expressed mRNAs in Echinococcus multilocularis-infected mice liver. a. Toll-like receptor signaling pathways enriched by upregulated differentially expressed mRNAs (DEMs) in Echinococcus multilocularis-infected mice liver at 30 dpi. The upregulated genes are presented as red rectangles. b. RIG-I-like receptor signaling pathways enriched by upregulated differentially expressed mRNAs (DEMs) in Echinococcus multilocularis-infected mice liver at 30 dpi. The upregulated genes are presented as red rectangles. c. Ubiquitin-mediated proteolysis pathway enriched by downregulated differentially expressed mRNAs (DEMs) in Echinococcus multilocularis-infected mice liver at 15 dpi. The downregulated genes are presented as green rectangles.
- 6AdditionalFigureS2bRLRpathwaymmu04622.tif
- 7AdditionalFigureS2cubiquitinat15dpi.png
- 8AdditionaltableS1aVenn31mRNA.xlsx
- 9AdditionaltableS1bVenn68lncRNA.xlsx
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