Clonorchiasis, caused by Clonorchis sinensis (C. sinensis), is an emerging zoonotic parasitic disease worldwide, distributed mainly in China, Korea, and northern Vietnam (Han et al., 2012; Lai et al., 2016). C. sinensis, belonging to family Opisthorchiidae (Ovchinnikov et al., 2015), has been classified by the International Agency for Research on Cancer (IARC) as a group 1 biocarcinogen in humans in 2009 (Bouvard et al., 2009). Humans become infected by eating freshwater fish with C. sinensis metacercariae. Metacercariae exist in the small intestine and then migrate via the ampulla of Vater to the biliary ducts (Lun et al., 2005). The adult fluke inhabits the biliary passages of humans and mammals and could cause epithelial hyperplasia of the biliary mucosa and even periductal fibrosis (Chung et al., 2004). Clinical manifestations of clonorchiasis vary from asymptomatic infections to severe morbidity and mortality, including epithelial hyperplasia, cholecystitis, periductal fibrosis, hepatic fibrosis, and cholangiocarcinoma (Keiser and Utzinger, 2005). Previously, we found that hepatic apoptosis and iron overload were involved in clonorchiasis (Han et al., 2017). In addition, an aberrant expression of hepatic microRNA has been associated with clonorchiasis (Han et al., 2016). However, the mechanism of liver/biliary injury, and whether other regulatory mechanisms or other small molecules are involved, remains unclear. Therefore, exploring the molecular mechanisms underlying clonorchiasis may contribute to the development of prevention measures and targeted drugs.

The majority of long non-coding RNAs (lncRNAs) are transcribed by RNA polymerase II as the product of alternative cleavage and splicing, with sizes greater than 200 nt in length (Quinn and Chang, 2016; Yamamura et al., 2018). LncRNAs are broadly classified into those that regulate gene expression in cis and in trans (Kopp and Mendell, 2018). Recently, lncRNAs are identified to bind with proteins, RNA, DNA, or their combination to play their functions (Fasolo et al., 2019; Hu et al., 2019). Acting as miRNA sponge, decoys, scaffolds, guides, and posttranslation regulators, lncRNAs could participate in various biological processes (Rinn and Chang, 2012; Mumtaz et al., 2017; Fernandes et al., 2019). Accumulating studies showed that lncRNAs were involved in inflammation, cancer, and autoimmunity (Fitzgerald and Caffrey, 2014; Marques-Rocha et al., 2015; Renganathan and Felley-Bosco, 2017).

Recent studies found that parasite infection changed the expression profiles of lncRNAs in the host. For example, numerous lncRNAs were differentially expressed in S. japonicum-infected liver and spleen compared to control (Xia et al., 2020). Furthermore, differentially expressed lncRNAs and their predicted target mRNAs were found associated with immunology and liver pathology (Xia et al., 2020). Besides, lncRNA WNT5b and H19 were found downregulated to regulate the metabolic reprogramming and differentiation of adipocytes in the protoscoleces of Echinococcus granulosus-infected mice (Lu et al., 2020). LncRNAs were also identified as important regulatory factors in regulating immunological stress during cystic echinococcosis (Yu et al., 2018). However, knowledge of lncRNAs and their biological functions in clonorchiasis remains limited.

This study used microarray analysis to assess the expression of lncRNAs and mRNAs in the liver tissue of mice infected with C. sinensis. Next, we analyzed the possible biological processes and pathways associated with C. sinensis infections. Our findings support this powerful new approach for the elucidation of the molecular mechanisms of clonorchiasis.

Previous studies on the interactions between parasites and their hosts have focused mainly on protein-coding genes (Melo et al., 2013; He et al., 2016). Recently, it was reported that lncRNAs play important roles in gene imprinting, cell differentiation, and tumorigenesis and are also involved in host–pathogen interactions (Jathar et al., 2017). For instance, RNA-Seq revealed that differentially expressed lncRNAs of Schistosoma mansoni were mainly involved in RNA-dependent DNA replication and G-protein coupled receptor (GPCR) signaling pathway/GPCR activity (Vasconcelos et al., 2017). However, the expression profiles of lncRNAs specific to C. sinensis infection were unclear. In this study, the expression profiles of lncRNAs and mRNAs in the liver of C. sinensis-infected mice at 4 wpi were fully explored using microarray analysis.

In this study, a total of 388 lncRNAs and 1,172 mRNAs were dysregulated in C. sinensis-infected group. As the most upregulated lncRNA, Gm6135 is primarily localized to the cytoplasm and participates in posttranslational modification (Rashid et al., 2016; Ji et al., 2019). LncRNA Gm6135 could increase the expression of toll-like receptor 4 (TLR4), by competitive binding to miR-203-3p, further activating the secretion of pro-inflammatory cytokines in mice (Ji et al., 2019; Loganathan et al., 2020). A previous study showed that TLR4 was upregulated in activated hematopoietic stem cells (HSCs) and myofibroblasts infected with C. sinensis (Yan et al., 2015). Thus, differentially expressed lncRNA Gm6135 may regulate the expression of TLR4 to induce inflammatory response in clonorchiasis. However, the role of 4930581F22Rik (the most downregulated lncRNA) has not been described clearly. The FMO3 protein encoded by Fmo3 (the most upregulated mRNA) is found associated with gallstone formation in mice (Chen et al., 2019). FMO3 protein and its metabolite (TMAO) participate in the TAF–FMO3–TMAO pathway, which could regulate lipid metabolism (Schugar and Brown, 2015; Warrier et al., 2015), while the downregulated mRNA Dmbt1 could encode the DMBT1 protein, which is related to liver injury and repair mechanisms in liver diseases (Bisgaard et al., 2002; Deng et al., 2012). Therefore, we speculate that mRNA Fmo3 and Dmbt1 might contribute to lipid metabolism and liver injury in clonorchiasis.

KEGG enrichment analysis showed that the top 20 predicted pathways mainly participated in “FOXO signaling pathway”, “Wnt signaling pathway”, and “AMPK signaling pathway”. For instance, mRNA Irs2 and Crebbp were rich in “FOXO signaling pathway”, which was involved in cellular differentiation, apoptosis, cell proliferation, DNA damage and repair, and oxidative stress (Gomes et al., 2008; Vurusaner et al., 2012). Nk1d1 and Crebbp mRNA were rich in “Wnt signaling pathway”, which played critical roles in precancerous lesions, malignant transformation of liver cells, and malignant expansion of cancer cells (Zhang et al., 2017). Nr1d1 mRNA was also involved in “negative regulation of TLR4 signaling pathway” in biological processes. TLR contributes to host defense by regulating host innate and adaptive immune responses (Iwasaki and Medzhitov, 2004). TLR4 was involved in immune responses, including increased levels of tumor necrosis factor alpha and interferon gamma during clonorchiasis (Yan et al., 2015). Interestingly, we also found that Irs2 and Gpam mRNA participated in “response to glucose” in biological processes. Moreover, “regulation of lipid metabolic process”, “negative regulation of fatty acid oxidation”, “Glucagon signaling pathway”, and “Fatty acid biosynthesis” demonstrated to be involved in C. sinensis infection. Therefore, we speculated that C. sinensis infection affected the cell function as well as lipid and glucose metabolism of the host.

Based on our previous miRNA expression profiling data ( Additional File: Table S1 ) and differentially expressed lncRNAs and mRNAs, a ceRNA network was constructed, in order to explore the functions of lncRNAs. In ceRNA networks, mRNA Gpam and Tnfrsf19 were negatively correlated with lncRNA Gm8801 and A_30_P01020438, indicating an upregulation of Gpam and Tnfrsf19 mRNA in the C. sinensis-infected group. Gpam mRNA is correlated with T lymphocyte proliferation, lipid metabolism, defense against viruses, and IL-2 secretion (Brockmoller et al., 2012; Warrier et al., 2015). In addition, Tnfrsf19 mRNA belongs to the tumor necrosis factor receptor superfamily, which commonly transduces cytokine signals and is associated with poor prognosis in various types of cancer (Spanjaard et al., 2007; Fafilek et al., 2013; Loftus et al., 2013; Schon et al., 2014). Otherwise, Irs2 mRNA was positively correlated with Gm8801 and A_30_P01020438 lncRNA, indicating a downregulation of Irs2 in the C. sinensis-infected group. Irs2 plays requisite roles in cell proliferation, apoptosis, migration, and tumor invasion (Lea et al., 2014; Zhao et al., 2014). Moreover, Irs2 mRNA is also important in hepatic nutrient homeostasis, as it mediates the anabolic effects of insulin through the PI3K-Akt cascade (Rother et al., 1998; Andersson et al., 2004) and suppresses gluconeogenesis (Valverde et al., 2003). Therefore, we hypothesized that lncRNA Gm8801 and A_30_P01020438 might play important roles in host metabolism and liver injury in clonorchiasis by regulating its co-expressed mRNA.

In this study, a microarray approach was used to explore the differential expression profiles of lncRNAs and mRNAs in clonorchiasis for the first time. However, there were some limitations that should be addressed. We only analyzed the expression profiles of lncRNAs and mRNAs at 4 weeks after being infected with C. sinensis, which corresponds to the early stage. However, C. sinensis infection is a dynamic process; the expression profiles of lncRNAs and mRNAs are also needed for detection at the late stages of infection. Moreover, the potential interaction between LncRNA and mRNA is needed to validate in the future.

In this study, we investigated the differential expression profiles of lncRNAs (388) and mRNAs (1,172) in the livers of mice infected with C. sinensis. Our results provided valuable insights into the lncRNAs and mRNAs involved in clonorchiasis, which may be useful for future control strategies. In the future, the functions of the differentially expressed lncRNAs and mRNAs in the pathogenesis of C. sinensis infection need to be further verified.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found below: https://www.ncbi.nlm.nih.gov/geo/, accession ID: GSE188841.

The animal study was reviewed and approved by the Care and Use of Laboratory Animals of the Ministry of Science and Technology, China.

SH and Xi-LZ offered advice for the article framework and conceived the conception and design of the trial. SH and Xi-LZ drafted the first manuscript and participated in trial communication and monitoring. Xu-LZ, XJ, XL, JD, JZ, SD, RC, BS, YH and NG modified the manuscript the carried out statistical calculations. All authors participated in revision of the manuscript and approved the final version.

This work was supported by the National Natural Science Foundation of China (81971958, 81601785, and 81401684), Heilongjiang Provincial Natural Science Foundation of China (LH2020H016), Post-doctoral Science Foundation (LBH-Q174), and Postgraduate Research & Practice Innovation Program of Harbin Medical University (YJSKYCX2019-67HYD). The funder had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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