Neospora caninum infection specifically suppresses the expression of a host lncRNA XR_001919077.1 to facilitate parasite propagation by modulating host cell mitochondrial function and autophagy

ABSTRACT

Neospora caninum is one of the most common pathogens causing reproductive failure in ruminants (e.g., cattle and goats) worldwide. However, due to a poor understanding of the pathogenic mechanisms of N. caninum infection, no effective drugs and vaccines are currently available. Long non-coding RNAs (lncRNAs) have been reported to be important regulators involved in a great number of physiological and pathological processes. Our previous study found that N. caninum infection induced significantly aberrant expression of lncRNA profiles in caprine endometrial epithelial cells (EECs). In the present study, we found that N. caninum infection specifically suppressed the expression of a novel lncRNA, XR_001919077.1, and knockdown of XR_001919077.1 with small interfering RNA significantly promoted the propagation of N. caninum in caprine EECs. Rapid amplification of cDNA ends analysis generated six splice variants of XR_001919077.1, with lengths ranging from 592 to 694 nt. Transfection of the full length of each variant markedly inhibited the propagation of N. caninum in caprine EECs. Further study suggested that XR_001919077.1 acted as a sponge of Chi-miR-93-5p to promote the expression of sirt1, and the XR_001919077.1/Chi-miR-93-5p/sirt1 axis significantly delayed the in vitro growth of N. caninum in caprine EECs by regulating host cell mitochondrial function and autophagy. Our findings provide a novel insight to understand the interactions between N. caninum and host cells.

IMPORTANCE

The uterus is an indispensable reproductive organ for embryo implantation and fetal growth. The endometrium is more vulnerable to infection by pathogenic microorganisms resulting in an increased risk of miscarriage. Neospora caninum is one of the most common pathogens causing miscarriage in ruminants and is able to naturally inhabit the uterus, with N. caninum tissue cysts found in the endometrium. Recent advances in N. caninum research have revealed aberrant expression of long non-coding RNA (lncRNA) profiles in infected caprine endometrial epithelial cells. In the present study, N. caninum, but not Toxoplasma gondii, which has similar morphological and biological features to N. caninum, specifically suppresses the expression of a host lncRNA, XR_ 001919077.1, to impair host’s defense through the competitive endogenous RNA mechanism to modulate the host cell mitochondrial function and autophagy to facilitate parasite propagation. The findings suggest a novel immune evasion strategy of N. caninum to facilitate intracellular propagation and provide an alternative path to develop control strategies against neosporosis.

INTRODUCTION

Neosporosis, caused by Neospora caninum, induces abortions and high neonatal mortality in cattle and small ruminants (e.g., goats and sheep) (1, 2). In the cattle industries of 10 countries, the estimated median losses due to N. caninum abortions exceed US$1,298.3 million (3). Unfortunately, no effective drugs are available to prevent and control neosporosis, and the only commercial vaccine (Neoguard, MSD Animal Health, Millsboro, DE, USA) against N. caninum-induced abortions has been withdrawn from the market due to low protective efficacy in infected cattle (4, 5). The main reason for the scarcity of available drugs and vaccines is due to a poor understanding of the pathogenic mechanisms of N. caninum.

Long non-coding RNAs (lncRNAs) are a class of non-translated RNA molecules that are usually transcribed by RNA polymerase II (Pol II)-dependent processes and have a length of more than 200 nucleotides (6). Recently, this type of non-coding RNA (ncRNA) has been demonstrated to be an important regulator involved in a great number of physiological and pathological processes (7–9). Previous studies showed that host cells could use lncRNAs to enhance defense against pathogenic infection, while the pathogens also could hijack lncRNAs to benefit their survival (10–15). For example, Cryptosporidium parvum infection induces the upregulated expression of a panel of lncRNAs (e.g., NR_045064, U90926, XR_001779380, NR_033736, and Nostrill), and these lncRNAs promote the transcription of defense genes through epigenetic modifications to enhance intestinal epithelial defense against C. parvum infection (10–14). The lncRNA BTX is significantly upregulated in peritoneal macrophages infected with vesicular stomatitis virus, and this lncRNA promotes viral replication by regulating the translocation of DHX9 and ILF3 from the nucleus to the cytoplasm (15). Our previous study showed that a total of 181 lncRNAs were differentially expressed in caprine endometrial epithelial cells (EECs) infected with N. caninum by RNA sequencing (RNA-seq) (16). However, the roles of these differentially expressed lncRNAs in the pathogenesis of neosporosis are still unknown. In the present study, we characterized one of the downregulated lncRNAs, namely XR_001919077.1, and investigated the effects and corresponding mechanisms of this lncRNA on the in vitro propagation of N. caninum.

MATERIALS AND METHODS

Parasites, cell lines, and in vitro infection model

N. caninum Nc-1 wild-type strain and Toxoplasma gondii RH strain were kindly gifted by Prof. Qun Liu from China Agricultural University (Beijing, China) and Assoc. Prof. Ningbo Xia from South China Agricultural University (Guangdong, China). N. caninum and T. gondii tachyzoites were serially passaged in African green monkey kidney epithelial cells (Vero cells). Caprine EECs were kindly provided by Prof. Yaping Jin from Northwest A&F University (Shaanxi, China) and were used for establishing an in vitro model of N. caninum infection at a multiplicity of infection (MOI) of 3:1 (parasite:cell) according to our previous study (17).

RNA extraction and reverse transcriptase-quantitative polymerase chain reaction

Caprine EECs infected with N. caninum tachyzoites or T. gondii tachyzoites were collected, and the total RNA was isolated from collected cell samples using AG RNAex Pro Reagent (Accurate Biology, Hunan, China) following the manufacturer’s instructions. The concentration and quality of each RNA sample were assessed using a Nano-100 spectrophotometer (Allsheng Instruments Co., Hangzhou, China). RNA (1,000 ng) of each sample was reverse transcribed to cDNA using Hifair V Reverse Transcriptase (Yeasen Biotechnology Co., Ltd., Shanghai, China) for reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR) analysis of mRNAs and lncRNAs, while 0.8 µg RNA of each sample was reverse transcribed using a Mir-X miRNA First-Strand Synthesis Kit (Takara Biomedical Technology, Dalian, China) for RT-qPCR analysis of microRNAs (miRNAs). The expression of mRNAs and lncRNAs was determined using 2× Universal SYBR Green Fast RT-qPCR Mix (ABclonal, Wuhan, China), with the glyceraldehyde-3-phosphate dehydrogenase (gapdh) gene as the internal reaction control. The expression of miRNAs was investigated using TB Green Fast qPCR Mix (Takara Biomedical Technology, Dalian, China), with the u6 small nuclear RNA gene as the internal reaction control. Each reaction was performed in triplicate, and three replicates were done for each experiment. The relative abundance of each gene was calculated using the 2 ^−ΔΔCt method (18). The information on the primers used in this study is listed in Table S1.

Rapid amplification of cDNA ends analysis and full-length amplification of XR_001919077.1

To obtain the 3′ and 5′ ends of unknown sequences of XR_001919077.1, 5′ and 3′ rapid amplification of cDNA ends (RACE) assays were performed using a SMARTer RACE 5′/3′ Kit (Clontech Laboratories, Inc., CA, USA) following the manufacturer’s protocol. The gene-specific primers used in RACE assays are listed in Table S1.

PCR primers (Table S1) were then designed based on 3′ and 5′ end sequences obtained from RACE assays to amplify the full length of XR_001919077.1. The PCR product was recovered, purified, ligated into a pMD19-T vector (Takara Biomedical Technology, Dalian, China), and transformed into Escherichia coli JM109 cells (Takara Biomedical Technology, Dalian, China). The positive colonies were sent to Tsingke Biotech (Beijing, China) for sequencing to determine the splice variants of XR_001919077.1.

Construction of overexpression plasmids

The overexpression plasmid of each splice variant of XR_001919077.1 was constructed into pcDNA 3.1 (+) vector (Invitrogen, Gaithersburg, MD, USA) using restriction endonucleases Hind III and Xho I (Takara Biomedical Technology, Dalian, China). The overexpression plasmid of sirt1 (pcDNA3.1 (+)-sirt1) was constructed into pcDNA 3.1 (+) vector by Tsingke Biotech (Beijing, China).

Cell transfection

Caprine EECs were seeded into 12-well plates (Invitrogen, Waltham, MA, USA) for 24 h. Then, small interfering RNAs (siRNAs) (100 pmol/L, RiboBio Co., Ltd., Guangzhou, China) against XR_001919077.1 (si-XR_001919077.1–1 and si-XR_001919077.1–2) or sirt1 (si-sirt1), overexpression plasmid of each splice variant or sirt1 (1.6 µg/well), or Chi-miR-93-5p mimics and inhibitor (60 pmol/L, GenePharma, Shanghai, China) were transfected into caprine EECs using Lipofectamine 2000 reagent (Invitrogen, Gaithersburg, MD, USA) according to the manufacturer’s instructions.

Fluorescence in situ hybridization analysis

The subcellular location of XR_001919077.1 was detected using Ribo Fluorescence in Situ Hybridization Kit (RiboBio Co., Ltd., Guangzhou, China) according to the manufacturer’s instructions. Briefly, caprine EECs were cultured onto glass coverslips in 24-well plates (Invitrogen, Waltham, MA, USA) for 24 h, and the cells were washed three times with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized with PBS containing 0.5% Triton-X 100 at 4°C for 5 min, and blocked with prehybridization solution at 37°C for 30 min. The treated cells were incubated overnight at 4°C with rabbit anti-N. caninum MIC13 antibody (1:80) made in the parasitology laboratory of Northwest A&F University, Shaanxi, China. Secondary antibodies were FITC-conjugated donkey anti-rabbit IgG (1:200, Shanghai Sangon Biotech, Shanghai, China). The cells were then incubated with a hybridization solution containing a cy3-conjugated against XR_001919077.1 probe (RiboBio Co., Ltd., Guangzhou, China) overnight at 37°C in the dark, with the u6 probe (RiboBio Co., Ltd., Guangzhou, China) as a control. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI) for 10 min in the dark. The images were photographed under confocal immunofluorescence microscopy (Leica Microsystems, Wetzlar, Germany).

Western blot analysis

Cell samples were lysed on ice in RIPA lysis buffer (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China) supplemented with 1 mM phenylmethylsulfonyl fluoride (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China), and the proteins were separated using sodium dodecyl sulfate-polyacrylamide gel and transferred to polyvinylidene difluoride membranes (Millipore, Billerica, USA). The membranes were blocked with 5% non-fat milk at room temperature for 2 h and then incubated with primary antibodies against SIRT1 (1:2,000, Abways, Shanghai, China), LC-3B (1:5,000, Abways, Shanghai, China), p62 (1:5,000, Abways, Shanghai, China), or β-actin (1:5,000, ABclonal, Wuhan, China) at 4°C overnight. Horseradish peroxidase-conjugated donkey anti-rabbit antibody (1:5,000, ABclonal, Wuhan, China) was used to incubate the membranes at room temperature for 1 h. Each reaction was performed in triplicate, and three replicates were done for each experiment. The protein blots were visualized using an enhanced chemiluminescence system, and gray values of the blots were calculated using ImageJ software (19).

Dual-luciferase reporter assay

To determine the sponging relationship between XR_001919077.1 and Chi-miR-93–5p, the sequence of XR_001919077.1, containing Chi-miR-93-5p binding site (WT) or mutated binding site (MUT), was cloned into the firefly luciferase pmirGLO reporter vector (Promega, Madison, WI, USA) to construct the recombinant plasmid pmirGLO-XR_001919077.1-WT or pmirGLO-XR_001919077.1-MUT. Caprine EECs were seeded into 96-well plates for 24 h, and the plasmid pmirGLO-XR_001919077.1-WT or pmirGLO-XR_001919077.1-MUT was transfected into caprine EECs in the presence of Chi-miR-93-5p mimics or control mimics using Lipofectamine 2000 reagent. Similarly, the 3′-untranslated region (UTR) sequences of sirt1 containing Chi-miR-93-5p binding site (WT) or mutated binding site (MUT) were cloned into the firefly luciferase pmirGLO reporter vector to construct the recombinant plasmid pmirGLO-sirt1-WT or pmirGLO-sirt1-MUT and transfected into caprine EECs to verify the target relationship between Chi-miR-93-5p and sirt1. At 48 h post-transfection, the firefly luciferase activity was measured using a Luc-Pair Duo-Luciferase HS Assay Kit (GeneCopoeia Inc., MD, USA) on a multifunctional fluorimeter microplate reader (Tecan, Männedorf, Switzerland), with Renilla luciferase activity used as an internal reference. Each reaction was performed in triplicate, and three replicates were done for each experiment.

Determination of reactive oxygen species, mitochondrial membrane potential, and adenosine triphosphate levels

The intracellular reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), and adenosine triphosphate (ATP) levels were, respectively, monitored using the 2′,7′-dichloro-fluorescin diacetate fluorescent probe (Beyotime Biotechnology, Shanghai, China), Mitochondrial Membrane Potential Assay Kit with JC-1 (Beyotime Biotechnology, Shanghai, China), and ATP Assay Kit (Beyotime Biotechnology, Shanghai, China) according to their manufacturer’s instructions. The fluorescence intensities of ROS and MMP were observed under an inverted fluorescence microscopy (Leica Microsystems, Wetzlar, Germany), and ATP contents were determined using a multifunctional fluorimeter microplate reader (Tecan, Männedorf, Switzerland) as previously described (20). Each reaction was performed in triplicate, and three replicates were done for each experiment.

Determination of mitochondrial DNA copy numbers

The mitochondrial DNA (mtDNA) copy numbers were measured by quantifying the expression of the nicotinamide adenine dinucleotide dehydrogenase subunit-1 (nd1) gene in genomic DNA (gDNA) (21). The gDNA samples were extracted from cell samples using a Blood/Cell/Tissue DNA Extraction Kit (Tiangen, Beijing, China) following the manufacturer’s protocol. A total of 200 ng gDNA was used as a template to perform RT-qPCR using 2× Universal SYBR Green Fast RT-qPCR Mix, with the 18s rRNA gene as an internal reaction control. The primer sequences of the nd1 gene and the 18s rRNA gene are listed in Table S1. Each reaction was performed in triplicate, and three replicates were done for each experiment.

Analysis of the propagation of N. caninum tachyzoites

Caprine EECs were infected with N. caninum tachyzoites at an MOI of 3:1 (parasite:cell) and cultured for 30 or 42 h post-infection (hpi). The number of N. caninum tachyzoites per parasitophorous vacuole was counted under inverted optical microscopy (Olympus Co., Tokyo, Japan).

The replication of N. caninum tachyzoites in the infected cells was also investigated based on the expression level of the Nc5 gene of N. caninum using qPCR as described previously (22, 23). Briefly, the gDNA samples were extracted from 1 × 10⁷ N. caninum tachyzoites or infected cells using a Blood/Cell/Tissue DNA Extraction Kit (Tiangen, Beijing, China) following the manufacturer’s protocol. A total of 200 ng of gDNA samples from infected cells were used as templates to perform qPCR using 2× Universal SYBR Green Fast RT-qPCR Mix. The number of N. caninum tachyzoites in 200 ng gDNA samples was calculated based on a standard curve with gDNA samples from serial dilutions of N. caninum tachyzoites (5–5 × 10⁵ tachyzoites) included in each run. Each reaction was performed in triplicate, and three replicates were done for each experiment.

Statistical analysis

Data obtained in each experiment were analyzed using the Student’s t test and one-way analysis of variance in GraphPad Prism 8.0.1 software (GraphPad Software Inc., San Diego, CA, USA). A P value < 0.05 was considered statistically significant.

RESULTS

Knockdown of XR_001919077.1 significantly promotes the propagation of N. caninum in caprine EECs

RT-qPCR analysis showed that the expression of XR_001919077.1 was significantly downregulated from 6 to 48 hpi in caprine EECs infected with N. caninum, with the lowest expression level found at 48 hpi (Fig. 1A), and N. caninum infection further decreased the expression of XR_001919077.1 in caprine EECs with the increase of MOIs at 48 hpi (Fig. 1B). However, T. gondii (MOI = 3:1), a pathogen with similar morphological and biological features to N. caninum, did not significantly alter the expression of XR_001919077.1 in caprine EECs at 48 hpi (Fig. S1A), and the expression of XR_001919077.1 was not affected in caprine EECs stimulated with lipopolysaccharide (200 nM; Abmole, Shanghai, China) (Fig. S1B) for 48 h. These findings suggest that N. caninum may specifically suppress the expression of XR_001919077.1 in caprine EECs.

Fig 1.

XR_001919077.1 is negatively correlated with the propagation of Neospora caninum in caprine endometrial epithelial cells. (A) The expression of XR_001919077.1 in caprine EECs during N. caninum infection for RT-qPCR. (B) The expression of XR_001919077.1 in caprine EECs infected with different MOIs (parasite:cell = 0:1, 1:1, 2:1, 3:1) of N. caninum tachyzoites at 48 h post infection (hpi) for RT-qPCR. (C) Interference efficiencies of two small interfering RNAs (si-XR_001919077.1–1 and si-XR_001919077.1–2) against XR_001919077.1 during N. caninum infection at 48 hpi for RT-qPCR. (D and E) Effect of knockdown of XR_001919077.1 on the propagation of N. caninum tachyzoites in caprine EECs at 30 hpi (D) and 42 hpi (E). (F) Effect of knockdown of XR_001919077.1 on the number of N. caninum in 200 ng DNA in infected caprine EECs at 48 hpi using qPCR. (G and H) Effect of overexpression of each XR_001919077.1 variant on the propagation of N. caninum tachyzoites in caprine EECs at 30 hpi (G) and 42 hpi (H). (I) Effect of overexpression of each XR_001919077.1 variant on the number of N. caninum in 200 ng DNA in infected caprine EECs at 48 hpi using qPCR. Data were obtained in triplicate and were analyzed using the Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001. TZ represents the tachyzoite.

To determine the role of XR_001919077.1 during N. caninum infection, two siRNAs against XR_001919077.1 (si-XR_001919077.1-1 and si-XR_001919077.1-2) were designed, and the expression of XR_001919077.1 was significantly decreased by both si-XR_001919077.1-1 and si-XR_001919077.1-2, but the interference effect of si-XR_001919077.1-2 (∼70%) was more profound (P < 0.01) than si-XR_001919077.1-1 (∼30%) (Fig. 1C). Therefore, si-XR_001919077.1-2 was used for further study. Interestingly, transfection with si-XR_001919077.1-2 significantly increased the number of N. caninum tachyzoites per vacuole in caprine EECs at 30 hpi (Fig. 1D) and 42 hpi (Fig. 1E). qPCR of the Nc5 gene showed that the number of N. caninum was also dramatically increased in infected caprine EECs treated with si-XR_001919077.1-2 at 48 hpi (Fig. 1F). These findings suggest that the expression of XR_001919077.1 is negatively correlated with the growth and propagation of N. caninum tachyzoites in vitro.

XR_001919077.1 expresses six splice variants

The 5′ and 3′ RACE assays of XR_001919077.1 obtained two sequences and one sequence with a Poly-A tail, respectively (Fig. S2). To obtain the full length of XR_001919077.1, two pairs of primers (Table S1) were designed based on the sequences from the 5′ and 3′ RACE assays and used for PCR amplification and subsequent sequencing. Sequence analysis showed that six splice variants were generated by XR_001919077.1, named lnc592, lnc616, lnc632, lnc633, lnc638, and lnc694 according to their lengths (Fig. S3). The coding potential of each variant was investigated using CPC 2.0, but none of them had coding potential (data not shown). Notably, si-XR_001919077.1-2 could partially inhibit the expression of each XR_001919077.1 variant in caprine EECs infected with N. caninum (Fig. S4A).

Overexpression of XR_001919077.1 inhibits the propagation of N. caninum in vitro

To further examine the role of XR_001919077.1 on the propagation of N. caninum, the recombinant plasmids of six XR_001919077.1 variants, namely pcDNA3.1 (+)-lnc592, pcDNA3.1 (+)-lnc616, pcDNA3.1 (+)-lnc632, pcDNA3.1 (+)-lnc633, pcDNA3.1 (+)-lnc638, and pcDNA3.1 (+)-lnc694, were transfected into caprine EECs at 24 h before infection of N. caninum, respectively. RT-qPCR showed that the expression of each XR_001919077.1 variant was significantly upregulated after transfection (Fig. S4B). Notably, overexpression of each of the six variants significantly decreased the number of N. caninum tachyzoites per vacuole at 30 hpi (Fig. 1G) and 42 hpi (Fig. 1H). qPCR of the Nc5 gene showed that the number of N. caninum was also dramatically decreased in infected caprine EECs transfected with each variant of XR_001919077.1 at 48 hpi (Fig. 1I). These results suggest that XR_001919077.1 significantly inhibits intracellular propagation of N. caninum tachyzoites in caprine EECs.

XR_001919077.1 promotes the expression of sirt1 by sponging Chi-miR-93-5p in caprine EECs infected with N. caninum

To investigate the mechanisms of XR_001919077.1 that affect the intracellular propagation of N. caninum tachyzoites in vitro, the location of XR_001919077.1 in caprine EECs was investigated using a cy3-conjugated probe against XR_001919077.1, with u6 localized in the nucleus as a control. Fluorescence in situ hybridization (FISH) analysis showed that XR_001919077.1 was found in both cytoplasm and nucleus in caprine EECs with or without infection of N. caninum at 48 hpi (Fig. 2; Fig. S5).

Fig 2.

XR_001919077.1 localizes in both cytoplasm and nucleus in caprine endometrial epithelial cells. The nucleus was stained with DAPI. Scale bar: 10 µm.

The most common function of lncRNAs is to act as competitive endogenous RNAs (ceRNAs) to regulate the expression of targeted genes through a competitive combination of miRNAs (24, 25). Bioinformatics analysis using the miRanda database identified 18 potential sponging miRNAs for XR_001919077.1. RT-qPCR showed that six (Chi-miR-30e-5p, Chi-miR-495-3p, Chi-miR-93-5p, Chi-miR-211, Chi-miR-134, and Chi-miR-128–5p) of them were significantly upregulated in caprine EECs infected with N. caninum at 48 hpi (Fig. S6A). However, the expression of only one miRNA, named Chi-miR-93-5p, was significantly inhibited by overexpression of each XR_001919077.1 variant (Fig. 3A; Fig. S6B), while knockdown of XR_001919077.1 using si-XR_001919077.1-2 promoted the expression of Chi-miR-93-5p during N. caninum infection (Fig. 3B). The dual-luciferase reporter assay, based on predicting potential binding sites between XR_001919077.1 and Chi-miR-93-5p (Fig. 3C), showed that Chi-miR-93-5p mimics markedly reduced the luciferase activity of the pmirGLO-XR_001919077.1-WT vector but did not affect that of the pmirGLO-XR_001919077.1-MUT vector in caprine EECs (Fig. 3D). These results suggest the sponging relationship between XR_001919077.1 and Chi-miR-93-5p in caprine EECs during N. caninum infection.

Fig 3.

XR_001919077.1 functions as a sponge for Chi-miR-93-5p in caprine endometrial epithelial cells infected with N. caninum. (A) The expression of Chi-miR-93-5p in caprine EECs infected with N. caninum by overexpression of each XR_001919077.1 variant at 48 h post-infection (hpi). (B) The expression of Chi-miR-93-5p in caprine EECs infected with N. caninum by interference with si-XR_001919077.1-2 at 48 hpi. (C) Potential binding sites between XR_001919077.1 and Chi-miR-93-5p predicted by miRanda database. (D) Sponging relationship between XR_001919077.1 and Chi-miR-93-5p identified using the dual-luciferase reporter assay. Data were obtained in triplicate and were analyzed using the Student’s t test. *P < 0.05 and **P < 0.01.

SIRT1, a conserved protein NAD (+)-dependent deacetylases, is a key modulator involved in metabolic and various physiological processes, including senescence, inflammatory response, and mitochondrial biogenesis, and has been identified as a target for Chi-miR-93-5p in rats with type 2 diabetic retinopathy, old-age rats, and human dermal fibroblasts (26–28). Coincidentally, our previous study showed that N. caninum infection suppressed the expression of SIRT1 in caprine EECs (20). In the present study, the mRNA and protein levels of sirt1 were also significantly downregulated from 12 to 48 hpi during N. caninum infection (Fig. 4A and B). To verify the targeting relationship between Chi-miR-93-5p and sirt1 in caprine EECs, the dual-luciferase reporter assay was performed based on predicting potential binding sites between Chi-miR-93-5p and sirt1 3′-untranslated region (Fig. 4C). Chi-miR-93-5p mimics significantly inhibited the luciferase activity of the pmirGLO-sirt1-WT but did not affect that of the pmirGLO-sirt1-MUT in caprine EECs (Fig. 4D). To further determine the effects of Chi-miR-93-5p on the expression of sirt1 during N. caninum infection, Chi-miR-93-5p mimics or Chi-miR-93-5p inhibitor was transfected into caprine EECs. In caprine EECs infected with N. caninum, the expression of Chi-miR-93-5p was significantly increased by Chi-miR-93-5p mimics but was significantly decreased by Chi-miR-93-5p inhibitor (Fig. S7). Meantime, Chi-miR-93-5p mimics significantly decreased both the mRNA (Fig. S8A) and protein (Fig. 5A) levels of sirt1, while the reverse effects were detected for Chi-miR-93-5p inhibitor (Fig. 5A; Fig. S8A). These data suggest that Chi-miR-93-5p could directly target sirt1 in caprine EECs during N. caninum infection.

Fig 4.

Chi-miR-93-5p targets sirt1 in caprine endometrial epithelial cells infected with N. caninum. (A) mRNA level of sirt1 analyzed in caprine EECs infected with N. caninum tachyzoites from 6 to 48 h post infection (hpi) by RT-qPCR. (B) Protein level of sirt1 analyzed in caprine EECs infected with N. caninum tachyzoites from 6 to 48 hpi by western blotting. Relative protein levels of SIRT1 compared to β-actin were determined by densitometry. (C) Potential binding sites between Chi-miR-93-5p and 3′-UTR of sirt1. (D) Target relationship between sirt1 and Chi-miR-93-5p identified using the dual-luciferase reporter assay. Data were obtained in triplicate and were analyzed using the Student’s t test. *P < 0.05 and **P < 0.01. TZ represents the tachyzoite.

Fig 5.

XR_001919077.1 promotes the expression of sirt1 by sponging Chi-miR-93-5p in caprine endometrial epithelial cells following N. caninum infection. (A) The expression of the SIRT1 protein analyzed in caprine EECs transfected with Chi-miR-93-5p mimics or inhibitor during N. caninum infection at 48 h post-infection (hpi) using western blotting. (B) The expression of the SIRT1 protein analyzed in caprine EECs transfected with recombinant plasmids of six XR_001919077.1 variants during N. caninum infection at 48 hpi using western blotting. (C) The expression of the SIRT1 protein analyzed in caprine EECs transfected with si-XR_001919077.1–2 during N. caninum infection at 48 hpi using western blotting. (D) The expression of the SIRT1 protein analyzed in caprine EECs co-transfected with recombinant plasmid of each of six XR_001919077.1 variants and Chi-miR-93-5p mimics during N. caninum infection at 48 hpi using western blotting. Relative protein level of SIRT1 compared to β-actin was determined by densitometry. Data were obtained in triplicate and were analyzed using the Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.

Interestingly, overexpression of each XR_001919077.1 variant significantly increased the mRNA (Fig. S8B) and protein levels (Fig. 5B) of sirt1, but knockdown of XR_001919077.1 using si-XR_001919077.1-2 significantly decreased the mRNA (Fig. S8C) and protein levels (Fig. 5C) of sirt1. Furthermore, compared with control mimics, co-transfection of Chi-miR-93-5p mimics with each of six XR_001919077.1 variants significantly decreased the protein level of SIRT1 (Fig. 5D). These results suggest that XR_001919077.1 promotes the expression of sirt1 by sponging Chi-miR-93-5p in caprine EECs during N. caninum infection.

XR_001919077.1 delays in vitro propagation of N. caninum tachyzoites by regulating the Chi-miR-93-5p/sirt1 axis

In caprine EECs, Chi-miR-93-5p mimics significantly increased the number of N. caninum tachyzoites per vacuole at 30 hpi (Fig. 6A) and 42 hpi (Fig. 6B), while the opposite effect on the infection burden of this parasite was detected for Chi-miR-93-5p inhibitor at 30 hpi (Fig. 6A) and 42 hpi (Fig. 6B). qPCR of the Nc5 gene showed that although the number of N. caninum was not significantly increased in infected caprine EECs transfected with Chi-miR-93-5p mimics at 48 hpi, the number of N. caninum was significantly decreased for transfection of Chi-miR-93-5p inhibitor (Fig. 6C). Our previous study found that EX527, an inhibitor of SIRT1, promoted the propagation of N. caninum tachyzoites in caprine EECs, while resveratrol (an activator of SIRT1) inhibited the in vitro propagation of N. caninum tachyzoites (20). To exclude extensive pharmacological effects, an overexpression plasmid, pcDNA3.1 (+)-sirt1, was constructed, and a siRNA, si-sirt1, was synthesized in the present study. In caprine EECs infected with N. caninum, the mRNA and protein levels of sirt1 were significantly upregulated by transfection with pcDNA3.1 (+)-sirt1 (Fig. S9) but were significantly downregulated by transfection with si-sirt1 (Fig. S9). Meantime, overexpression of sirt1 significantly decreased the number of N. caninum tachyzoites per vacuole at 30 hpi (Fig. 6D) and 42 hpi (Fig. 6E), while the opposite effect on the infection burden of this parasite was detected for si-sirt1 at 30 hpi (Fig. 6D) and 42 hpi (Fig. 6E). qPCR of the Nc5 gene showed that the number of N. caninum was dramatically decreased in infected caprine EECs treated with overexpression plasmids of pcDNA3.1 (+)-sirt1 at 48 hpi, while the opposite effect was detected for si-sirt1 (Fig. 6F). Furthermore, compared with the control mimics, co-transfection of Chi-miR-93-5p mimics and pcDNA3.1(+)-sirt1 inhibited the propagation of N. caninum tachyzoites in caprine EECs, but co-transfection of Chi-miR-93-5p mimics with each of the six XR_001919077.1 variants significantly promoted the propagation of this parasite in caprine EECs (Fig. 6G and H). These results suggest that the expression of XR_001919077.1 is negatively correlated with the propagation of N. caninum by promoting the expression of sirt1 by sponging Chi-miR-93-5p in caprine EECs.

Fig 6.

The XR_001919077.1/Chi-miR-93-5p/sirt1 axis delays the propagation of N. caninum tachyzoites in caprine endometrial epithelial cells. (A and B) Effect of Chi-miR-93-5p mimics or inhibitor on the propagation of N. caninum tachyzoites in caprine EECs at 30 h post infection (hpi) (A) and 42 hpi (B). (C) The number of N. caninum in 200 ng DNA in infected caprine EECs transfected with Chi-miR-93-5p mimics or inhibitor at 48 hpi using qPCR. (D and E) Effect of pcDNA3.1 (+)-sirt1 or si-sirt1 on the propagation of N. caninum tachyzoites in caprine EECs at 30 hpi (D) and 42 hpi (E). (F) The number of N. caninum in 200 ng DNA in infected caprine EECs transfected with pcDNA3.1 (+)-sirt1 or si-sirt1 at 48 hpi using qPCR. (G and H) Effect of co-transfection of Chi-miR-93-5p mimics and each of the six XR_001919077.1 variants, or pcDNA3.1(+)-sirt1 on the propagation of N. caninum tachyzoites in caprine EECs at 30 hpi (G) and 42 hpi (H). Data were obtained in triplicate and were analyzed using the Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.

XR_001919077.1/Chi-miR-93-5p/sirt1 axis regulates mitochondrial function in caprine EECs during N. caninum infection

Our previous study found that N. caninum infection downregulated the expression of SIRT1 to facilitate the propagation by damaging the mitochondrial function (20). In the present study, we asked whether the XR_001919077.1/Chi-miR-93–5p/sirt1 axis delayed the in vitro propagation of N. caninum by regulating mitochondrial function. To address this issue, caprine EECs were respectively transfected with each of the six XR_001919077.1 variants, si-XR_001919077.1-2, Chi-miR-93-5p mimics or inhibitor, pcDNA3.1 (+)-sirt1, or si-sirt1, and then infected with N. caninum at 24 h post-transfection. Overexpression of each variant of XR_001919077.1 significantly decreased ROS production (Fig. 7A) but increased MMP levels (Fig. 7B), ATP contents (Fig. 8A), and mtDNA copy numbers (Fig. 8B) in infected caprine EECs, while opposite effects were detected for si-XR_001919077.1-2 (Fig. 7C, D, 8C, and D). Transfection of Chi-miR-93-5p mimics induced the accumulation of intracellular ROS (Fig. 7E) and a marked reduction of MMP levels (Fig. 7F), ATP contents (Fig. 8E), and mtDNA copy numbers (Fig. 8F) in infected caprine EECs. However, contrary results were detected for the transfection of Chi-miR-93–5p inhibitor (Fig. 7E, F, 8E, and F). The effects for intracellular ROS, MMP levels, ATP contents, and mtDNA copy numbers induced by overexpression of sirt1 were opposite to transfection of Chi-miR-93-5p mimics but were consistent with transfection of each of the six XR_001919077.1 variants (Fig. 7G, H, 8G, and H), while the opposite effects were observed for si-sirt1 (Fig. 7G, H, 8G, and H). These results suggest that N. caninum infection induces mitochondrial dysfunction to facilitate intracellular propagation through the XR_001919077.1/Chi-miR-93–5p/sirt1 axis.

Fig 7.

The XR_001919077.1/Chi-miR-93-5p/sirt1 axis affects the ROS levels and MMP levels in caprine EECs following N. caninum infection. (A and B) ROS levels (A) and MMP levels (B) determined in caprine EECs transfected with recombinant plasmids of six XR_001919077.1 variants during N. caninum infection at 48 h post-infection (hpi). (C and D) ROS levels (C) and MMP levels (D) determined in caprine EECs transfected with si-XR_001919077.1-2 during N. caninum infection at 48 hpi. (E and F) ROS levels (E) and MMP levels (F) determined in caprine EECs transfected with Chi-miR-93-5p mimics or inhibitor during N. caninum infection at 48 hpi. (G and H) ROS levels (G) and MMP levels (H) determined in caprine EECs transfected with pcDNA3.1 (+)-sirt1 or si-sirt1 during N. caninum infection at 48 hpi. Data were obtained in triplicate and were analyzed using the Student’s t test. Scale bar: 200 µm. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig 8.

The XR_001919077.1/Chi-miR-93-5p/sirt1 axis affects the adenosine triphosphate (ATP) contents and mtDNA copy numbers in caprine endometrial epithelial cells following N. caninum infection. (A and B) ATP contents (A) and mtDNA copy numbers (B) determined in caprine EECs transfected with recombinant plasmids of six XR_001919077.1 variants during N. caninum infection at 48 h post-infection (hpi). (C and D) ATP contents (C) and mtDNA copy numbers (D) determined in caprine EECs transfected with si-XR_001919077.1-2 during N. caninum infection at 48 hpi. (E and F) ATP contents (E) and mtDNA copy numbers (F) determined in caprine EECs transfected with Chi-miR-93-5p mimics or inhibitor during N. caninum infection at 48 hpi. (G and H) ATP contents (G) and mtDNA copy numbers (H) determined in caprine EECs transfected with pcDNA3.1 (+)-sirt1 or si-sirt1 during N. caninum infection at 48 hpi. Data were obtained in triplicate and were analyzed using the Student’s t test. Scale bar: 200 µm. *P < 0.05, **P < 0.01, and ***P < 0.001.

XR_001919077.1/Chi-miR-93-5p/sirt1 axis regulates cell autophagy in caprine EECs during N. caninum infection

Our previous studies showed that N. caninum infection inhibited the expression of sirt1 to promote autophagy in caprine EECs (20). We then asked whether cell autophagy induced by N. caninum infection was regulated by the XR_001919077.1/Chi-miR-93-5p/sirt1 axis. In the present study, caprine EECs were transfected with each of the six XR_001919077.1 variants, si-XR_001919077.1-2, Chi-miR-93-5p mimics or inhibitor, pcDNA3.1 (+)-sirt1, or si-sirt1, and then infected with N. caninum at 24 h post-transfection. The protein levels of LC3-II (a marker of autophagosome formation) and p62 (an autophagy receptor protein involved in the degradation of ubiquitinated cargo and a common marker for monitoring the occurrence of autophagy flow) were examined at 48 hpi. Transfection with each XR_001919077.1 variant significantly decreased the expression of LC3-II protein (Fig. 9A), while the opposite effect was detected for transfection with si-XR_001919077.1-2 (Fig. 9B). Increased expression of LC3-II protein was found following transfection with Chi-miR-93-5p mimics, but the contrary result was detected following transfection with Chi-miR-93-5p inhibitor (Fig. 9C). Additionally, transfection with pcDNA3.1 (+)-sirt1 significantly decreased the protein level of LC3-II, but the contrary result was detected for transfection with si-sirt1 (Fig. 9D). However, the protein levels of p62 decreased upon transfection of each XR_001919077.1 variant (Fig. 9E), si-XR_001919077.1–2 (Fig. 9F), Chi-miR-93–5p mimics and inhibitor (Fig. 9G), pcDNA3.1 (+)-sirt1, and si-sirt1 (Fig. 9H) in caprine EECs during N. caninum infection. These results suggest that the XR_001919077.1/Chi-miR-93–5p/sirt1 axis inhibits autophagy in caprine EECs induced by N. caninum infection.

Fig 9.

The XR_001919077.1/Chi-miR-93–5p/sirt1 axis affects the expression of the LC3B protein and p62 protein in caprine endometrial epithelial cells following N. caninum infection. (A–D) The protein levels of LC3B determined in caprine EECs transfected with recombinant plasmids of the six XR_001919077.1 variants (A), si-XR_001919077.1-2 (B), Chi-miR-93-5p mimics or inhibitor (C), pcDNA3.1 (+)-sirt1, or si-sirt1 (D) during N. caninum infection at 48 h post-infection (hpi) by western blotting. (E–H) The protein levels of p62 determined in caprine EECs transfected with recombinant plasmids of the six XR_001919077.1 variants (E), si-XR_001919077.1–2 (F), Chi-miR-93-5p mimics or inhibitor (G), pcDNA3.1 (+)-sirt1, or si-sirt1 (H) during N. caninum infection at 48 hpi by western blotting. The relative protein levels of LC3-II (A–D) or p62 (E–H) compared to β-actin were determined by densitometry. Data were obtained in triplicate and were analyzed using the Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001.

DISCUSSION

Reproductive failure in pregnant animals is the main consequence induced by N. caninum infection (1). Therefore, uncovering the intracellular survival and pathogenicity of N. caninum in reproductive organs is essential to exploring novel effective targets for controlling neosporosis. The uterus, an indispensable reproductive organ that forms the mother-fetal interface, embryo implantation, and maintenance of gestation, has been reported naturally inhabited by N. caninum (29–31). In the present study, our data indicate that a host lncRNA, namely XR_001919077.1, is specifically significantly downregulated during N. caninum infection, and the XR_001919077.1/Chi-miR-93-5p/sirt1 axis significantly delayed the propagation of N. caninum in caprine EECs by regulating host cell mitochondrial function and autophagy.

LncRNAs are usually transcribed by RNA Pol II and undergo extensive alternative splicing to greatly increase the reservoir of transcriptional diversity (32, 33). Emerging evidence suggests that splice variants of lncRNAs are involved in various pathological processes (34–39). For example, lncRNA SOX2OT is significantly upregulated in esophageal squamous cell carcinoma and has two splice variants (namely SOX2OT-S1 and SOX2OT-S2). Each variant is markedly increased in sub-G1 phase of cell cycle and is significantly downregulated in the induction of neural differentiation (34). In tongue squamous carcinoma, a hypoxia-associated lncRNA, LINC00887, generates two variants (namely 887S and 887L) that share the same downstream target, Carbonic Anhydrase IX (CA9), a well-known hypoxia-induced gene during tumor progression. Interestingly, 887S and 887L interact under normoxia, while upon hypoxia, 887S and 887L have opposite effects on tumor progression (35). The lncRNA RP11-369C8.1 produces six splice variants, two of which (TRMP and TRMP-S) have been characterized to promote the growth of cancer cells by modulating cell cycle progression through distinct mechanisms of action (38, 39). In the present study, we found that XR_001919077.1 expresses six splice variants, but all these variants significantly inhibit intracellular propagation of N. caninum tachyzoites in caprine EECs.

The functions of lncRNAs are closely related to their subcellular locations. To some extent, lncRNAs located in the nucleus are mainly involved in histone modifications, chromatin remodeling, and scaffolding of nuclear complexes at the epigenetic and transcription levels, while lncRNAs in the cytoplasm participate in mRNA translation and stability and act as miRNA sponges at post-transcriptional and translational programs (6, 40). In the present study, XR_001919077.1 was found in both the cytoplasm and nucleus of caprine EECs using the FISH assay. Previous studies showed that the most well-known function of lncRNAs in the cytoplasm is to act as ceRNAs or microRNA decoys to modulate gene expression (41–43). For example, the lncRNA WAC-AS1 is significantly upregulated in serum samples of patients and HepG2 2.15 cells infected with hepatitis B virus (HBV), and this lncRNA induces host cell autophagy to facilitate HBV replication in HepG2.2.15 cells by targeting the miR-192-5p/ATG7 axis (41). LncRNA DANCR is significantly upregulated in peripheral blood samples of pulmonary tuberculosis patients and THP-1 cells infected with Mycobacterium tuberculosis H37Ra, but it inhibits intracellular survival of M. tuberculosis by sponging miR-1301-3p and miR-5194 to induce host cell autophagy (42). In the present study, we find that the expression of XR_001919077.1 negatively affects the propagation of N. caninum tachyzoites by regulating the Chi-miR-93-5p/sirt1 axis, suggesting the defense role of the XR_001919077.1/Chi-miR-93-5p/sirt1 axis during N. caninum infection.

SIRT1 is an important conserved mammalian NAD (+)-dependent protein deacetylase in mitochondrial biogenesis and autophagy (44, 45). The growth of Leishmania infantum is significantly reduced in bone marrow macrophages knocked out of SIRT1, which inhibits the metabolic switch from an early aerobic glycolytic environment toward mitochondrial oxidation (44). During infection of T. gondii, 4-hydroxybenzaldehyde, an aromatic compound, induces SIRT1-mediating autophagy by increasing the intracellular co-localization of autophagic vacuoles and T. gondii-containing parasitophorous vacuoles in bone marrow-derived macrophages, and the SIRT1-mediating autophagy restricts the intracellular propagation of T. gondii (45). Our previous study found that downregulation of sirt1 is beneficial for the propagation of N. caninum in caprine EECs by regulating mitochondrial function and autophagy (20). In the present study, we found that N. caninum infection induces mitochondrial dysfunction and autophagy to facilitate intracellular propagation through targeting the XR_001919077.1/Chi-miR-93-5p/sirt1 axis.

Conclusions

We characterized one caprine lncRNA, XR_001919077.1, which is specifically suppressed by N. caninum infection, impairing host defense to facilitate parasite propagation (immune evasion by this parasite), and clarified the function of XR_001919077.1 during N. caninum infection. Six splice variants of XR_001919077.1 are detected, and these variants promote the expression of sirt1 by sponging Chi-miR-93-5p. The XR_001919077.1/Chi-miR-93-5p/sirt1 axis negatively affects the propagation of N. caninum tachyzoites in vitro by regulating mitochondrial function and autophagy (Fig. 10). To the best of our knowledge, this is the first study on the significance of lncRNAs during N. caninum infection. The findings of this study provide a novel insight to unveil the mechanisms of intracellular survival and pathogenesis of N. caninum in reproductive organs/tissues.

Fig 10.

A hypothetical mechanism graph of XR_001919077.1/Chi-miR-93-5p/sirt1 axis regulates the propagation of N. caninum in vitro. N. caninum infection significantly downregulates the expression of XR_001919077.1 and sirt1 but upregulates the expression of Chi-miR-93-5p. XR_001919077.1 promotes the expression of sirt1 by sponging Chi-miR-93-5p in caprine EECs infected with N. caninum. The XR_001919077.1/Chi-miR-93-5p/sirt1 axis negatively affects the propagation of N. caninum tachyzoites in vitro by regulating mitochondrial function and autophagy.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/spectrum.01580-24.

Figure S1. spectrum.01580-24-s0001.tif.

The expression of XR_001919077.1 is not altered in caprine endometrial epithelial cells (EECs) infected with Toxoplasma gondii or stimulated with lipopolysaccharide (LPS).

Figure S2. spectrum.01580-24-s0002.tif.

3' and 5' end sequences of XR_001919077.1 are obtained by using rapid amplification of cDNA ends (RACE) assays.

Figure S3. spectrum.01580-24-s0003.tif.

Sequence alignment of six variants of XR_001919077.1.

Figure S4. spectrum.01580-24-s0004.tif.

Expression of each XR_001919077.1 variant is analyzed.

Figure S5. spectrum.01580-24-s0005.tif.

XR_001919077.1 localizes in both cytoplasm and nucleus in caprine endometrial epithelial cells (EECs) infected with Neospora caninum at 48 h post infection (hpi).

Figure S6. spectrum.01580-24-s0006.tif.

The expression of potential sponged miRNAs by XR_001919077.1 are analyzed in caprine endometrial epithelial cells (EECs) during N. caninum infection.

Figure S7. spectrum.01580-24-s0007.tif.

Transfection efficiencies of Chi-miR-93-5p mimics and Chi-miR-93-5p inhibitor are determined in infected caprine EECs.

Figure S8. spectrum.01580-24-s0008.tif.

The XR_001919077.1/Chi-miR-93-5p axis affects the mRNA levels of sirt1 in caprine endometrial epithelial cells (EECs) following N. caninum infection.

Figure S9. spectrum.01580-24-s0009.tif.

Transfection efficiency of overexpression plasmid or small interfering RNAs (siRNAs) of sirt1 is determined.

Table S1. spectrum.01580-24-s0010.xlsx.

Nucleotide sequences of primers are used in this study.

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