Coding and Noncoding RNA Expression Profiles of Spleen CD4+ T Lymphocytes in Mice With Echinococcosis

doi:10.21203/rs.3.rs-960369/v1

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Coding and Noncoding RNA Expression Profiles of Spleen CD4⁺ T Lymphocytes in Mice With Echinococcosis

https://doi.org/10.21203/rs.3.rs-960369/v1

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Background: Cystic echinococcosis (CE) is a severe and neglected zoonotic disease, which is caused by Echinococcus granulosus sensu lato and poses health and socioeconomic hazards. RNA molecules play important roles in genetic coding, translation, regulation, and gene expression and are classified into noncoding RNAs, such as long noncoding RNAs (lncRNAs), miRNAs, and circular RNAs (circRNAs), and coding RNAs (mRNAs) based on whether they encode proteins.

Methods: Peripheral blood serum from E. granulosus-infected and uninfected female BALB/c mice was used to measure IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17A, and TNF-α levels using the cytometric bead array mouse Th1/Th2/Th17 cytokine kit. mRNA, lncRNA, miRNA, and circRNA profiles were analyzed in spleen CD4⁺ T cells from the two groups of mice using high-throughput sequencing.

Results: The results showed that the levels of serum IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17A, and TNF-α were significantly higher in the CE mice than in the control group (P < 0. 01). A total of 1,758 known mRNAs, 37 miRNAs, 175 lncRNAs, and 22 circRNAs were differentially expressed between infected and uninfected mice (|fold change| ≥ 0.585, P < 0.05). These differentially expressed molecules were closely related to the JAK/STAT, mitogen-activated protein kinase, p53, and PI3K/Akt signaling pathways, cell cycle, and metabolic pathways.

Conclusions: E. granulosus infection caused significant increases in the IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17A, and TNF-α levels in the peripheral blood of mice, as well as significant changes in the expression levels of a variety of coding and noncoding RNAs. A further study of these trends and pathways may help clarify the pathogenesis of CE and provide new insights into the prevention and treatment of this infection.

Parasitology

Echinococcosis

High-throughput sequencing

mRNA

miRNA

lncRNA

circRNA

Cystic echinococcosis (CE) refers to a serious zoonotic parasitic disease caused by Echinococcus granulosus sensu lato ^{[1, 2]}. The high incidence areas of CE are mainly concentrated in western China (Tibet, Qinghai, Gansu, and Ningxia), southern Meizhou, and East Africa ^[3]. The target organs of CE are mainly the liver and lungs but also the brain, spleen, kidneys, heart, and spine. Notoriously, CE cysts develop very slowly, and the disease may be asymptomatic for 10–15 years ^[4–6]. As a result, patients with CE often present to the hospital late, which prevents early disease management. There have been significant advances in the study of CE in recent years; however, imaging is still the main diagnostic tool, and it is difficult to identify cysts of less than 2 cm in diameter ^[7–9]. Clinically, serum testing can support the diagnosis of CE, but unfortunately, the sensitivity and specificity of the tests are low, and there are major limitations in terms of the prognosis ^{[10, 11]}. Data on the underlying mechanisms involved in the development and progression of CE in the host are scarce. Thus, given the limitations of the current diagnostic techniques, the ineffectiveness of drugs, and the inadequacy of surgery, there is an urgent need to explore molecular mechanisms and related pathways involved in the progression of CE to identify new drug and vaccine targets.

RNA is a macromolecule that plays an important biological role in the coding, decoding, regulation, and expression of genes ^[12]. RNA can be divided into coding and noncoding RNA (ncRNA), based on various functions. In particular, mRNA is a transmitter of genetic information that directs protein synthesis. RNAs that do not have the ability to encode proteins (ncRNAs) can be classified according to their length into miRNAs (18–24 nucleotides) and long ncRNAs (lncRNAs), which, similar to circular RNAs (circRNAs), are > 200 nucleotides in length ^{[13, 14]}. Recent studies have reported an increasing number of coding and ncRNAs that are widely expressed during E. granulosus infection in hosts ^[15–18]. Therefore, these molecules may be key regulators of worm–host interactions. A search for potentially effective molecules and related pathways is expected to provide novel perspectives and a meaningful clinical value for the diagnosis and treatment of the helminthiasis.

The objectives of this study were (1) to determine coding and ncRNA expression profiles in mice infected with echinococcosis and (2) to identify the altered signaling pathways.

Parasite infection

Protoscoleces of E. granulosus were obtained by surgical removal of cysts from patients with CE at the General Hospital of Ningxia Medical University, Hepatobiliary Surgery. Four 4–6-week-old female BALB/c mice were purchased from the Ningxia Medical University Laboratory Animal Centre. The mice were randomly divided into two groups; mice in the infected group (n = 2) were subcutaneously injected into three spots with 2,000 E. granulosus protoscoleces in phosphate-buffered saline (PBS), and mice in the uninfected group (n = 2) were subcutaneously injected into three spots with an equal volume of PBS.

Sample isolation

Spleen CD4⁺ T cells from the infected and uninfected mice were isolated 6 months after infection. Total RNA was extracted from CD4⁺ T cells using the TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. RNA quality and purity were evaluated using a NanoDrop 2000c instrument (Thermo Scientific). The RevertAid first-strand cDNA synthesis kit (Thermo Fisher Scientific) was used for cDNA synthesis following the manufacturer’s directions.

Coding and ncRNA expression profiles and pathway analysis

High-throughput RNA sequencing was performed by Shanghai Novelbio (China). The original data were manipulated to obtain high-quality reads. The RNA tags were exactly matched to the mouse genome to identify the known RNAs. The relative RNA expression levels in the two groups of mice were determined using the DESeq R package. The fold change cutoff of ≥ 2 and P < 0.05 were used to identify differentially expressed RNAs. Volcano plots and heatmaps were used to visualize the differential RNA expression profiles between the two groups.

Cytokine measurement in the serum

Peripheral blood serum from two groups of mice (n = 10) was used to measure the levels of IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17A, and TNF-α using the cytometric bead array mouse Th1/Th2/Th17 cytokine kit (Becton, Dickinson and Company). The capture microspheres were mixed and centrifuged at 200 × g at room temperature for 5 min. The supernatant was aspirated, and an equal volume of a serum enhancement solution was added, followed by vortexing and incubation for 30 min at room temperature, protected from light. Subsequently, the mixture was added to an equal volume of serum, and all the tubes were incubated for 3 h at room temperature, protected from light, with equal amounts of a phycoerythrin-labeled antibody for cytokine detection.

Statistical analysis

All data were processed using GraphPad Prism 5 (GraphPad Software, La Jolla, CA, USA), and the Student’s t-test was used for comparison between the two groups of mice. Fisher's test was used to calculate the significance level of each Gene Ontology (GO) term to determine differentially significant GO terms and pathways. Statistical significance was set at P < 0.05.

Serum cytokine levels in CE mice

The levels of IFN-γ, IL-2, IL-4, IL-6, IL-10, IL-17A, and TNF-α were measured in the peripheral blood serum of 10 CE and 10 control mice (Fig. 1) and were found to be significantly higher in the CE mice than in the control group (P < 0. 01). These findings suggest a strong immune response in mice to reject the parasitism of E. granulosus, even at 6 months after the infection. This shows that the mouse model of E. granulosus infection is successful, which can continue the follow-up experiment.

Identification of differentially expressed coding and ncRNAs in association with E. granulosus infection

To identify coding and ncRNAs associated with CE, spleen CD4⁺ T cells were isolated from two infected and two control mice 6 months postinfection. There were a total of 1,758 known differentially expressed mRNAs, 37 differentially expressed miRNAs, 175 differentially expressed lncRNAs, and 22 differentially expressed circRNAs between the two groups (|fold change| ≥ 0.585, P < 0.05), which are shown in the volcano and cluster plots (Fig. 2). The detailed information for the top 20 differentially expressed molecules of each type is presented in Tables 1–4. The expression of these molecules was significantly altered in the CE mice compared with that in the control mice, which suggested that these RNAs might be involved in the development of this parasitic disease.

GO and pathway analyses of differentially expressed genes between CE and normal mice

The results of the high-throughput sequencing of the differentially expressed genes were further analyzed based on GO annotations to associate the transcripts with the GO categories of biological process, molecular function, and cellular component. The differentially expressed mRNAs between the mice with active CE and the control mice were mainly involved in the regulation of DNA replication-independent nucleosome assembly, cell cycle, protein heterodimerization activity, and carbohydrate binding (Fig. 3a). Notably, these differentially expressed molecules were closely related to the cell cycle and the p53 signaling pathway (Fig. 3b).

The circRNAs identified were primarily associated with the control of mitotic nuclear division, deoxycytidyl transferase activity, the spindle pole, and kinetochore (Fig. 3c). Furthermore, the differentially expressed circRNAs were involved in the Hippo, JAK/STAT, oxytocin, and cGMP/PKG signaling pathways. These results suggested that these pathways might be promising targets for the treatment of CE.

Gene act network of differentially expressed mRNAs

Although we obtained important signaling pathways associated with CE, one gene could simultaneously be involved in multiple signaling pathways. Therefore, we constructed a gene act network based on the correlation between differentially expressed mRNAs, including their expression, binding, repression, activation, and complexes. This analytical approach can form corresponding regulatory affiliations, making it easier to identify important related genes. Analysis of the gene act network showed that all differentially expressed molecules between infected and uninfected mice were closely associated with the JAK/STAT, mitogen-activated protein kinase (MAPK), P53, and PI3K/Akt signaling pathways, cell cycle, and metabolic pathways (Fig. 4). In particular, three pathways, namely, the JAK/STAT, MAPK, and P53 signaling pathways, showed the largest numbers of arrows and may be the most likely new targets for the treatment of CE.

As a parasitic zoonosis, CE is prevalent in areas with well-developed livestock industries, especially sheep, and greatly hinders livestock productivity development and damages the regional economy ^[8]. Although albendazole has been used in the treatment of CE in recent decades, its efficacy has not been adequately demonstrated. Long-term use of benzimidazoles may cause a variety of adverse effects, especially in the liver ^[19–22], and the development of new alternative drugs would be of great importance for the treatment of CE. Following infection, echinococcosis activates a strong immune response in the host, eliminating most of the parasite within a few days ^[23]. Based on this, we constructed a mouse model of E. granulosus protoscoleces infection and detected differentially expressed mRNAs, lncRNAs, miRNAs, and circRNAs.

The progress in the study of differential expression profiles of RNA in both CE itself and in the infected host has been very limited in terms of molecular mechanisms. In in vitro cultured E. granulosus protoscoleces, a total of 172 genes and 15 miRNAs, which are mainly involved in neurological development and carbohydrate metabolic processes, were shown to be significantly altered during development. In addition, miR-71- and miR-219-regulated genes may be involved in redox processes during adult development ^[24]. Whole-genome sequencing of E. granulosus identified 42 mature miRNAs in three different model stages ^[25]. It has been suggested that some molecules, such as miR-19b, miR-71, and miR-222-3p, could serve as possible biomarkers for E. granulosus ^{[26, 27]}. In hosts infected with E. granulosus, restimulation of the patient's peripheral blood mononuclear cells using crude E. granulosus antigens induced the expression of Th1/Th2 cytokine mRNAs ^[28]. Differentially expressed miRNAs (miR-181, miR-30, miR-365, miR-378, miR-449, miR-99, miR-130, and miR-16) that were identified in the sheep intestine, liver, and serum, as well as in mouse macrophages, following infection with E. granulosus may be critically involved in the body's immune response ^[29–39]. Microarray analysis of circRNA expression profiles in adjacent tissues of CE-infected patients, showed that hsa_circRNA_006773, hsa_circRNA_049637, hsa_circRNA_104349, and hsa_circRNA_406281 might serve as CE prognostic biomarkers and therapeutic targets ^[40]. Our high-throughput sequencing data are consistent with previous results showing that miR-29c-3p and miR-30b-5p are significantly downregulated in CE ^[41].

These differentially expressed molecules in infected mice are closely related to JAK-STAT signaling pathway, MAPK signaling pathway, p53 signaling pathway, PI3K-Akt signaling pathway, cell cycle and metabolic pathways. Further study of these direction and pathways may conduce to clarify the pathogenesis and provide a new perspective for the prevention and treatment of CE.

In this study, the use of high-throughput sequencing for RNA expression profiling of splenic CD4⁺ T cells after infection of mice with E. granulosus enriched our understanding of the molecular mechanisms underlying the development of CE. Although potentially important RNA molecules and associated signaling pathways were identified, further experiments and clinical trials are needed to determine their potential to serve as new targets for the treatment of CE.

CE: Cystic echinococcosis; circRNA: Circular RNA; GO: Gene Ontology; lncRNA: Long noncoding RNA; MAPK: Mitogen-activated protein kinase; ncRNA: Noncoding RNA; PBS: Phosphate-buffered saline.

Acknowledgements

We appreciate the support provided by the Ningxia Medical University Science and Technology Center.

We would like to thank Editage (www.editage.cn) for English language editing.

Funding

This work was supported by the Key Research and Development Project of the Ningxia Hui Autonomous Region (No. 2018BEG02003) and by the Key Natural Science Foundation of the Ningxia Hui Autonomous Region (2020AAC02039).

Availability of data and materials

All the data generated during this study are included in this article.

Authors’ contributions

WZ and MXZ designed and conducted the experiments. YZZ, TRZ, and YXL constructed a mouse model of E. granulosus infection. SMX and YQZ collected the data. XCD and SHY wrote and revised the manuscript. SHY and CW performed bioinformatics and statistical analyses, interpreted the data, and obtained financial support for this research. All authors have read and agreed to the final version of the manuscript.

Ethics approval and consent to participate

The animal experiments were authorized by the Ethics Committee of Ningxia Medical University and were in accordance with all institutional and national guidelines for laboratory animals. The data obtained complied with relevant ethical requirements.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no relevant conflicts of interest.

Agudelo Higuita NI. Brunetti E, McCloskey C. Cystic echinococcosis. J Clin Microbiol. 2016;54:518–23.
Wang J. Gao C, Steverding D. Wang X, Shi F. Yang Y. Differential diagnosis of cystic and alveolar echinococcosis using an immunochromatographic test based on the detection of specific antibodies. Parasitol Res. 2013;112:3627–33.
Wen H. Vuitton L, Tuxun T. Li J, Vuitton DA. Zhang W, et al. Echinococcosis: advances in the 21st century. Clin Microbiol Rev. 2019;32:e00075-18.
McManus DP. Gray DJ, Zhang W. Diagnosis, treatment, and management of echinococcosis. BMJ 2012;344:e3866.
Kern P. Menezes da Silva A, Akhan O. Müllhaupt B, Vizcaychipi KA. Budke C, et al. The echinococcoses: diagnosis, clinical management and burden of disease. Adv Parasitol. 2017;96:259–369.
Sioutis S. Reppas L, Bekos A. Soulioti E, Saranteas T. Koulalis D, et al. Echinococcosis of the spine. EFORT Open Rev. 2021;6:288–96.
Craig PS. Giraudoux P, Wang ZH. Wang Q. Echinococcosis transmission on the Tibetan Plateau. Adv Parasitol. 2019;104:165–246.
Larrieu E. Gavidia CM, Lightowlers MW. Control of cystic echinococcosis: background and prospects. Zoonoses Public Health 2019;66:889–99.
Stojković M. Weber TF, Junghanss T. Clinical management of cystic echinococcosis: state of the art and perspectives. Curr Opin Infect Dis. 2018;31:383–92.
Ray R. De PK, Karak K. Combined role of Casoni test and indirect haemagglutination test in the diagnosis of hydatid disease. Indian J Med Microbiol. 2002;20:79–82.
Manzano-Román R. Sánchez-Ovejero C, Hernández-González A. Casulli A, Siles-Lucas M. Serological diagnosis and follow-up of human cystic echinococcosis: a new hope for the future? Biomed Res Int. 2015;2015:428205.
Duffy K. Arangundy-Franklin S, Holliger P. Modified nucleic acids: replication, evolution, and next-generation therapeutics. BMC Biol. 2020;18:112.
Lekka E. Hall J. Noncoding RNAs in disease. FEBS Lett. 2018;592:2884–900.
Bartel DP. Metazoan microRNAs Cell. 2018;173:20–51.
Ancarola ME. Marcilla A, Herz M. Macchiaroli N, Pérez M. Asurmendi S, et al. Cestode parasites release extracellular vesicles with microRNAs and immunodiagnostic protein cargo. Int J Parasitol. 2017;47:675–86.
Yu A, Wang Y, Yin J, Zhang J, Cao S, Cao J, et al. Microarray analysis of long non-coding RNA expression profiles in monocytic myeloid-derived suppressor cells in Echinococcus granulosus-infected mice. Parasites Vectors 2018;11:327.
Macchiaroli N, Maldonado LL, Zarowiecki M, Cucher M, Gismondi MI, Kamenetzky L, et al. Genome-wide identification of microRNA targets in the neglected disease pathogens of the genus Echinococcus. Mol Biochem Parasitol. 2017;214:91–100.
Cai P. Gobert GN, McManus DP. MicroRNAs in parasitic helminthiases: current status and future perspectives. Trends Parasitol 2016;32:71–86.
Dehkordi AB. Sanei B, Yousefi M. Sharafi SM, Safarnezhad F. Jafari R, et al. Albendazole and treatment of hydatid cyst: review of the literature. Infect Disord Drug Targets 2019;19:101–4.
Li S. Han XM, Guo YM. [Progress of researches on the development of non-benzimidazoles for the treatment of echinococcosis]. Zhongguo Xue Xi Chong Bing Fang Zhi Za Zhi 2020;33:213–7. [in Chinese].
Hong ST. Albendazole and praziquantel: review and safety monitoring in Korea. Infect Chemother. 2018;50:1–10.
Aasen TD. Nasrollah L, Seetharam A. Drug-induced liver failure requiring liver transplant: report and review of the role. Exp Clin Transplant. 2018;16:344–7.
Tamarozzi F. Mariconti M, Neumayr A. Brunetti E. The intermediate host immune response in cystic echinococcosis. Parasite Immunol. 2016;38:170–81.
Bai Y, Zhang Z, Jin L, Zhu Y, Zhao L, Shi B, et al. Dynamic changes in the global transcriptome and microRNAome reveal complex miRNA-mRNA regulation in early stages of the bi-directional development of Echinococcus granulosus protoscoleces. Front Microbiol. 2020;11:654.
Bai Y, Zhang Z, Jin L, Kang H, Zhu Y, Zhang L, et al. Genome-wide sequencing of small RNAs reveals a tissue-specific loss of conserved microRNA families in Echinococcus granulosus. BMC Genomics 2014;15:736.
Lakner AM. Steuerwald NM, Walling TL. Ghosh S, Li T. McKillop IH, et al. Inhibitory effects of microRNA 19b in hepatic stellate cell-mediated fibrogenesis. Hepatology 2012;56:300–10.
Song J. Ouyang Y, Che J. Li X, Zhao Y. Yang K, et al. Potential value of miR-221/222 as diagnostic, prognostic, and therapeutic biomarkers for diseases. Front Immunol. 2017;8:56.
Fauser S. Kern P. T-lymphocyte cytokine mRNA expression in cystic echinococcosis. Acta Trop. 1997;64:35–51.
Guo X, Zheng Y. Expression profiling of circulating miRNAs in mouse serum in response to Echinococcus multilocularis infection. Parasitology 2017;144:1079–87.
Jiang S, Li X, Wang X, Ban Q, Hui W, Jia B. MicroRNA profiling of the intestinal tissue of Kazakh sheep after experimental Echinococcus granulosus infection, using a high-throughput approach. Parasite 2016;23:23.
Jin X, Guo X, Zhu D, Ayaz M, Zheng Y. miRNA profiling in the mice in response to Echinococcus multilocularis infection. Acta Trop. 2017;166:39–44.
Jiang MM. Mai ZT, Wan SZ. Chi YM, Zhang X. Sun BH, et al. Microarray profiles reveal that circular RNA hsa_circ_0007385 functions as an oncogene in non-small cell lung cancer tumorigenesis. J Cancer Res Clin Oncol. 2018;144:667–74.
Wang X. Yong C, Yu K. Yu R, Zhang R. Yu L, et al. Long noncoding RNA (lncRNA) n379519 promotes cardiac fibrosis in post-infarct myocardium by targeting miR-30. Med Sci Monit. 2018;24:3958–65.
Gao M. Li C, Xu M. Liu Y, Cong M. Liu S. LncRNA MT1DP aggravates cadmium-induced oxidative stress by repressing the function of Nrf2 and is dependent on interaction with miR-365. Adv Sci (Weinh), 2018;5:1800087.
Diao L. Wang S, Sun Z. Long noncoding RNA GAPLINC promotes gastric cancer cell proliferation by acting as a molecular sponge of miR-378 to modulate MAPK1 expression. OncoTargets Ther. 2018;11:2797–804.
Qu L. Ding J, Chen C. Wu ZJ, Liu B. Gao Y, et al. Exosome-transmitted lncARSR promotes sunitinib resistance in renal cancer by acting as a competing endogenous RNA. Cancer Cell 2016;29:653–68.
Yan T. Qiu C, Sun J. Li WH. MiR-877-5p suppresses cell growth, migration and invasion by targeting cyclin dependent kinase 14 and predicts prognosis in hepatocellular carcinoma. Eur Rev Med Pharmacol Sci. 2018;22:3038–46.
Yu MJ. Zhao N, Shen H. Wang H. Long noncoding RNA MRPL39 inhibits gastric cancer proliferation and progression by directly targeting miR-130. Genet Test Mol Biomarkers 2018;22:656–63.
Xie ZZ. Xiao ZC, Song YX. Li W, Tan GL. Long non-coding RNA Dleu2 affects proliferation, migration and invasion ability of laryngeal carcinoma cells through triggering miR-16-1 pathway. Eur Rev Med Pharmacol Sci. 2018;22:1963–70.
Kalifu B. Maitiseyiti A, Ge X. Chen X, Meng Y. Expression profile of circular RNAs in cystic echinococcosis pericystic tissue. J Clin Lab Anal. 2021;35:e23687.
Eroglu F. Dokur M, Ulu Y. MicroRNA profile in immune response of alveolar and cystic echinococcosis patients. Parasite Immunol. 2021;43:e12817.

Due to technical limitations, table 1 to 4 is only available as a download in the Supplemental Files section.

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Journal Publication

published 08 Apr, 2022

Read the published version in Contrast Media & Molecular Imaging →

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You are reading this latest preprint version

Coding and Noncoding RNA Expression Profiles of Spleen CD4⁺ T Lymphocytes in Mice With Echinococcosis

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Methods

Parasite infection

Sample isolation

Coding and ncRNA expression profiles and pathway analysis

Cytokine measurement in the serum

Statistical analysis

Results

Serum cytokine levels in CE mice

GO and pathway analyses of differentially expressed genes between CE and normal mice

Gene act network of differentially expressed mRNAs

Discussion

Conclusions

Abbreviations

Declarations

References

Tables

Supplementary Files

Status:

Journal Publication

Version 1