Identification of hydatidosis-related modules and key regulatory genes

Introduction

Echinococcosis, also known as hydatidosis, is a worldwide chronic zoonosis caused by larval of Echinococcus multilocularis (E.multilocularis) that mainly affects humans, livestock and wild mammals (Maldonado et al., 2017; Zhang et al., 2019). E.multilocularis is mainly distributed in holarctic regions, including Asia, North America, and Europe (Oksanen et al., 2016). In China, there are many Echinococcosis-prone areas, such as Qinghai, Gansu, and Sichuan (Massolo et al., 2014). The life-cycle of E.multilocularis involves small rodent intermediate hosts such as arvicolids and wild or domestic canid definitive hosts such as the foxes, wolves, dogs, and cats. The host is infected by accidentally eating the eggs of E.multilocularis, causing tumor-like lesions of the liver (Li et al., 2018b). Human can act as abnormal intermediate hosts and become infected when ingesting eggs released into vegetables and food by adult worms. Then, the parasite larvae travel to internal organs, mainly the liver (Conraths et al., 2017). Hydatidosis not only affects the world economy, but also seriously endangers the international public health problem (Paoletti et al., 2019). Echinococcosis has been reported as a prioritized neglected disease by the World Health Organisation (WHO) in 2012 (Maldonado et al., 2017). In 2015, according to WHO estimates, Echinococcosis was the cause of 19,300 deaths and approximately 871,000 disability-adjusted life years (DALYs) worldwide each year, and Echinococcosis-related treatment costs and livestock industry losses estimated at 3 billion dollars annually.

Therefore, it is urgent to study the pathogenesis and treatment of hydatidosis. Fortunately, many biologists and medical researchers have devoted themselves to the exploration of hydatidosis-related diseases and achieved great results. Research by Li et al. (2018a) shows that E.multilocularis is contagious in two stages: one is egg, the only stage for primarily infecting humans and intermediate host; and another stage is protoscolex, the only stage for infecting the definite hosts of the worms. Previous studies have shown that extracellular vesicles (EV) secreted by E.multilocularis participate in host-parasite interactions by exchanging biomolecules, and then play a role in the process of infection (Zheng et al., 2017). In addition, ECT 53, a homologous gene of p53, was found to play an important role in the regulation of antioxidant stress and apoptosis induced by oxidative stress in E.multilocularis (Cheng et al., 2015). EmSOX 2, as a member of Sox transcription factor family, is actively expressed in germinating cells of E.multilocularis proliferation, and is a potential regulator of E.multilocularis germinating cells (Cheng et al., 2017a).

miRNA play an important role in the regulation of parasite infection. Surprisingly, there is a significant correlation between the expression level of miRNA and their corresponding target genes, mainly involved in E.multilocularis infection and regulation of cytokine activity and immune response (Guo & Zheng, 2017). Zheng (2018) confirmed through animal experiments that downregulated miR-222-3p is capable of modulating macrophage immune functions, possibly contributing to the pathogenesis during E.multilocularis infection. Similarly, specific amplification of two kinds of miRNAs (emu-miR-10 and emu-miR-227) derived from parasites has been found in previous reports that they contribute to the role of these two miRNAs as diagnostic targets for hydatidosis (Guo & Zheng, 2017). In this study, we found that miR-184 plays a regulatory role in both human and canine modules. Several targets for miR-184 have been described, including that of mediators of neurological development, apoptosis and it has been suggested that miR-184 plays an essential role in development (Li et al., 2011). Liu et al., (2010) showed that Methyl-CpG binding protein 1 (MBD1) regulates the expression miR-184. High levels of miR-184 promotes cell proliferation, whereas inhibition of miR-184 rescued phenotypes associated with MBD1 deficiency. We speculate that miR-184 played an important role in hydatidosis.

Some signaling pathways and key pathway factors are also involved in the infection of Echinococcus. EGFR/ERK signaling pathway can stimulate the growth and development of larvae by promoting the proliferation of germinating cells in host (Cheng et al., 2017b). Interestingly, the granular layer of E.granulosus has anti-inflammatory effects by inhibiting PI3K/Akt activation and growth factor-driven Akt phosphorylation and macrophage proliferation (Seoane et al., 2016). In addition, host insulin can stimulate the development of parasites in the liver (Hemer et al., 2014). Up to now, only benzimidazole, albendazole and tolbendazole have been allowed to be used in the treatment of human hydatidosis (Siles-Lucas et al., 2018). Previous clinical experience has shown that E.multilocularis can cause repeated infection of the host, and the clinical and pathological symptoms are gradually aggravated. Therefore, it is imperative to comprehensively explore the basic detailed mechanism and key regulatory molecules of E.multilocularis infection.

Here, we explore the potential pathogenic mechanism of E.multilocularis through in-depth analysis of the sequencing data of human and canine E.multilocularis infection. The comprehensive strategy based on functional modules provides rich resources and theoretical guidance for biologists to further explore its therapeutic mechanism.

Material and Methods

Result

Dysfunction of gene expression in hydatidosis

Gene expression profiles of humans and canine infected with E.multilocularis were obtained. Compared with the control group, we identified a total of 1,937 differentially expressed genes in the first-infection group canine, of which 1,400 were up-regulated and 537 were down-regulated. Compared with the control group, there are 1,698 differentially expressed genes in the repeated-infection group canine, of which 1,274 are up-regulated and 424 are down-regulated (Table S1). A total of 267 differentially expressed proteins, 101 up-regulated and 166 down-regulated were identified in human hepatic lesions and margins samples compared to control samples (Table S2). We suggest that these differentially expressed genes may be associated with hydatidosis. Then, for different host, we constructed protein-protein interaction (PPI) networks and identified hydatidosis-related modules in PPI network, respectively. Among them, in humans infected with E.multilocularis, a total of 16 modules were identified, namely m1 to m16 (Fig. 1A). In canine infected with E.multilocularis, a total of 5 modules were identified, namely M1 to M5 (Fig. 1B). These modules may participate in different functions and pathways, representing different post-infection regulatory mechanisms of echinococcosis.

Identification of dysfunction modules through function and pathway

In order to further understand the biological characteristics of the two module sets, we performed GO function and KEGG pathway enrichment analysis, respectively. We have collected a wealth of GO terms (Table S3). In GO terms, the human module was significantly enriched in 2,510 cell compositions, 3,071 molecular functions, and 23,185 biological processes. Canine modules were involved in 504 cell components, 866 molecular functional and 5,393 biological processes. Based on functional analysis, we observed that these modules tend to enrich a variety of disease-related functions, such as response to “endoplasmic reticulum stress” and response to “transforming growth factor beta”. KEGG enrichment analysis shows that human modular genes participate in 264 functional pathways and canine modular genes participate in 240 functional pathways (Table S4). Human modular genes are significantly enriched in PI3K-Akt signaling pathway and TNF signaling pathway, which are significantly related to the inflammatory reaction in the occurrence and development of hydatidosis in human. Canine modular genes were mainly involved in signal pathways such as MAPK signaling pathway and p53 signaling pathway. These pathways play important role in the apoptosis process of canine E.multilocularis. In view of the close relationship between the function and pathway of module genes and the progression of hydatidosis, we identified these two modules as dysfunction modules.

Crosstalk between modules and key genes in modules

The relationship and regulation between genes have always been complex and changeable. Exploring the complex interaction between modules will help us to understand the molecular mechanism during the progression of hydatidosis. Therefore, we performed crosstalk analysis between modules based on the interaction relationships between module genes. We identified 60 significant crosstalk relationships among human dysfunction modules (Fig. 2A), and five relationships among canine dysfunction modules (Fig. 2B). The crosstalks may represent the relationship between modules, which together regulate the progression of hydatidosis. In addition, the role of important genes in modules was more crucial. Therefore, based on the degree distribution of genes in modules, the genes with the highest connectivity in each module are considered as key genes. Then, we analyzed 16 human modules and found that key genes in 12 modules played a regulatory role in the occurrence and development of hydatidosis. In addition, in 5 canine-related modules, key genes related to hydatidosis were identified (Table S5). It is worth noting that among all the key genes of patients with hydatidosis, the connectivity of RPS5 is 148, which is the most connected endogenous gene. In canine, EHMT1 with a connectivity of 73 is considered the most critical endogenous gene.

The regulation of miR-184 mediated by ncRNAs/TFs in the progression of hydatidosis

To explore the role of ncRNA during the progression of hydatidosis, pivot analysis was performed to identify ncRNAs, which significantly regulated modules. In total, we identified 158 ncRNAs that significantly regulate pathogenic module in canine E.multilocularis infection and 1,129 ncRNAs that significantly regulate pathogenic module in human E.multilocularis infection (Table S6, Figs. 3A) and 3B. Especially, miR-184 has homologues in E.multilocularis. SLC38A3, a target of miR-184, is homologous in human and canine. Therefore, we infer that miR-184 aggravates the pathological changes of human infected by regulating SLC38A3. It can be concluded that miR-184 plays an important role in the pathogenesis of hydatidosis. The pathogenic process of hydatidosis is also closely related to the disorder of TFs. Similar to ncRNAs, we performed pivot analysis to predict the regulation relationships between TFs and modules (Table S7). The results showed that 110 TFs had significant transcriptional regulation effects on the dysfunction module of E.multilocularis infection in humans. Statistical analysis showed that NFKB1 significantly regulated 6 dysfunction modules, which may participate in the inflammatory process and promote physiological disorders in the process of E.multilocularis infection. RELA and SP1 also play an indispensable role in the process. We also found that 9 TFs regulate the dysfunction of canine E.multilocularis infection. TP53 regulated one dysfunction module and plays a central role in the response to oxidative stress and DNA damage. In the regulatory network, miR-184 regulates target genes through 9 TFs and participates in 13 KEGG pathways (Fig. 4A). Among the TFs, FOSL2 may up-regulates CLU of module 11 and regulates inflammatory response. CDX2 could down-regulates UGT2B15 of module 12, and participates in cellular response to xenobiotic stimulus and other functions. In the canine regulatory network, miR-184 regulates 4 target genes through 2 TFs and participates in 6 KEGG pathways (Fig. 4B). Similarly, miR-184 may participate in BMP signaling pathway by down-regulating GDF3 of module 5 mediated by TFs NANOG. MiR-184 also will participate in signaling pathways such as Endocrine resistance by up-regulating ESR1 of module 1 mediated by STAT5B. In addition, the expression levels of key genes CLU, ESR1, GDF3, and UGT2B15 have been verified by qPCR, consistent with the results predicted previously. In addition, miR-184 expression levels have been verified in human and canine (Fig. 4C).

Discussion

Hydatidosis is a zoonotic parasitic disease caused by the larva of Echinococcus. It not only seriously affects human health, but also causes economic losses in many countries (Longman & Martin, 1987). In addition, alveolar Echinococcosis (AE) is the main disease caused by the larval E.multilocularis (Gottstein et al., 2017). The infection often involves the liver and may transfer to the lungs and brains (Detry et al., 2018). In recent years, the exploration of echinococcosis has focused on some genes or proteins, as well as related signaling pathways, and some achievements have been made. However, the global regulation of these genes, proteins and signaling pathways in hydatidosis remains unclear. We have synthesized a series of analytical methods to explore the potential molecular mechanism of the occurrence and development of alveolar echinococcosis. Firstly, two groups of dysfunction modules were identified based on protein interaction network and functional enrichment analysis. One is about 16 modules of human-related diseases and the other is about 5 modules of dog-related diseases. Furthermore, we found that TFs as pivot regulators can connect a series of functions and pathways, thus exacerbating the pathological changes of echinococcosis. Based on the results of the enrichment analysis, we found that both human and canine module genes are mainly involved in the “TGF- β signaling pathway” and the “BMP signaling pathway”. According to the enrichment analysis, we observed that the physiological process of apoptosis was significantly enriched in seven dysfunction modules in humans and in four dysfunction modules in canine. It happens to confirm that apoptosis is involved in the pathogenesis of E.multilocularis (Zhang et al., 2012). Human have 5 dysfunction modules that significantly enrich the regulation of interleukin, and canine have 4 dysfunction modules that significantly enrich the biological process. Samten B et al. also pointed out that the expression levels of interleukin-21 and interleukin-4 in CE patients were significantly higher than those in normal people, which indicated that interleukin was also closely related to the pathogenesis of E.multilocularis (Zhang et al., 2015). These functions and pathways involved by module genes have a comprehensive network effect, which comprehensively regulates the physiological disorders caused by E.multilocularis.

Secondly, we identified the key genes in these dysfunction modules (including RPS6, RPS3 and RPL5 in humans, and EHMT1 and ACTL9 in canine). Among them, RPS6 phosphorylation has a certain regulatory role in the process of cell cycle. Casein kinase 1 (CK1) dependent phosphorylation of RPS6 promotes the binding of RPS6 to the cap-binding complex. In addition, protein phosphatase 1 (PP1) can antagonize the phosphorylation of RPS6 C terminal and cap binding in intact cells. These findings further deepen our understanding of the regulation of phosphorylation of RPS 6 (Hutchinson et al., 2011; Rosner, Schipany & Hengstschlager, 2013). EHMT1 plays a negative regulatory role in the gene induction pathway mediated by NF-kappa B and interferon I (Ea et al., 2012).

Finally, we identified ncRNA (including CRNDE both in human and canine) and TFs (including NFKB1, TP53, BRCA1 and KLC2) that significantly regulated these dysfunction modules. Statistical analysis showed that long non-coding RNA (lncRNA), mainly CRNDE, played an important regulatory role in the functional dysfunction modules of human and canine. NFKB1 significantly regulates 4 dysfunction modules of human hydatidosis. Related studies have reported that NFKB1 is the core TF regulating hydatidosis, and it may participate in the inflammatory process of hydatidosis (Jiang et al., 2016). Therefore, NFKB1 may play an important regulatory role in the pathogenesis of E.multilocularis. In addition, TP53 plays a key role in the regulation of oxidative stress and apoptotic network, so it is considered that TP53 is also involved in the infection of hydatid (Ichimura et al., 2002). The interaction between KLC2 and 14 − 3 − 3 protein depends on the phosphorylation of KLC2, and then mediates the signal transduction pathway (Cheng et al., 2015). These studies indicate that KLC2 plays an important role in the functional pathway of hydatidosis. It is worth noting that the miR-184 of hydatid worms can target multiple module genes through TF and ncRNA, and play multiple regulatory roles. Therefore, we boldly speculate that it is involved in mediating oxidative stress, and that cytotoxic cytokine-induced beta cell dysfunction aggravates the pathological changes after human infection with E.multilocularis (Barrett et al., 2013). In addition, miR-184 may regulate the function of pancreatic beta-cells through glucose metabolism, alter insulin secretion, and thus down-regulate the GDF3 gene, leading to the final infection of hydatidosis in canine (Dalgaard & Eliasson, 2017).

Conclusions

Based on the results of this study, we have obtained a more comprehensive pathogenic module of E.multilocularis. These modules involve many hydatidosis-related genes which been confirmed and candidate factors to be tested, which provide a theoretical basis for further research on hydatidosis. After systematic research, we believe that miR-184 is a key regulatory gene for E.multilocularis to promote host disease progression.

Supplemental Information