Female BALB/c mice (age, 8 weeks; mean weight, 20.25±0.93 g) were obtained from the Shandong University Laboratory Animal Center. A total of 48 mice were randomly divided into two groups (control group, n=24; infected group, n=24). They were reared in groups of six mice per cage under specific pathogen-free conditions under a 12-h light/dark cycle at 25°C and had ad libitum access to tap water and self-produced mouse feed (8). All animal experiments were approved by the Ethics Committee on Animal Experiments of the Medical School of Shandong University (Jinan, China). Each BALB/c mouse (infected group) was challenged with 10 T. gondii cysts by gavage and the spleens of experimental mice were collected for RNA extraction one month after the challenge. All efforts were made to minimize suffering and humane endpoints were used. Mice with unkempt fur and diarrhea were euthanized. For euthanasia, mice were placed in a chamber and CO₂ was administered at a concentration of 60–70% over a 5-min exposure time, after which the cervical dislocation method was at times used to ensure that effective euthanasia had occurred.

T. gondii (low virulent Prugniaud strain, obtained from the Department of Pathogen Biology, Anhui Medical University, Hefei, China) was maintained in our laboratory using the passage of cysts in eight-week-old Kunming mice obtained from Shandong University Laboratory Animal Center (mean weight, 42.31±5.31 g).

RNA was obtained from different spleen samples (one mouse from the control group and one mouse from the infected group) using TRIzol reagent (Takara Bio, Inc.) according to the manufacturer's instructions. RNA was isolated using the improved cetyltrimethylammonium bromide (CTAB) method, applying isopropanol instead of lithium chloride for RNA precipitation. In brief, one gram of spleen sample was added to liquid nitrogen and ground into a fine powder, which was evenly mixed in 5 ml preheated (65°C) extraction buffer (2% CTAB, 2% polyvinylpyrrolidone, 0.1 M Tris-HCl, 2.0 M NaCl, 25 mM EDTA, 2% beta-mercaptoethanol; pH 8.0). The mixture was incubated for 5 min at 65°C and shaken three times during the incubation period. After a short cooling period, isopropanol (2.5 ml) was added to the mixture, after which the mixture was vortexed for 1 min and centrifuged at 10,900 × g for 15 min at 4°C. DNase was then used to treat the extract and RNA was precipitated at 25°C for 10 min using the same volume of isopropanol. The extracted RNA was first resuspended in an equal volume of phenol/chloroform/isopropanol mixture (25:24:1) and then in an equal volume of chloroform/isopropanol (24:1). Both a 0.1 volume of 3M NaOAC (pH 5.2) and 2.5 volumes of cold ethanol were added to the mixture to precipitate the RNA overnight at −20°C. The quality of the prepared RNA from each sample was analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc.). The fragmentation buffer was added to cut the mRNA into short fragments (200–700 nucleotides). First-strand cDNA was synthesized using the templates of the short fragments. DNA polymerase I (New England Biolabs, Inc.), dNTPs, RNase H (Invitrogen; Thermo Fisher Scientific, Inc.) and buffer were used to synthesize the second-strand cDNA. The fragments were purified using the QiaQuick PCR kit and washed with EB buffer (consists of sodium chloride, magnesium chloride, HEPES and sucrose) for end repair prior to adding polyA tails and adaptors. Fragments of suitable size were detected by agarose gel electrophoresis and amplified using PCR (8), after which the products were sequenced using Illumina HiSeq™ 2000 (Illumina, Inc.).

A total of two rat miRNA transcriptome libraries were sequenced using the Illumina HiSeq 2000 platform. Details of the raw data are listed in Table I. High-quality 20±21 nucleotide-long reads were processed with the CleaveL pipeline for small RNA target identification, as previously described (15). Sequences of ribosomal RNAs, transfer RNAs, small nucleolar RNAs and small nuclear RNAs were retrieved from the RNA families database (xfam.org). The control (S01) and infected (S02) sample data were analyzed separately. P≤0.01 and fold change ≥2 were used to identify significant differentially expressed genes. The miRDB software (16) was used to predict the target genes of differentially expressed miRNAs.

The selected sequence was constructed using BLASTX and the National Center for Biotechnology Information (NCBI) database (https://www.ncbi.nlm.nih.gov/guide/sequence-analysis/) to better observe the function of miRNA targets and the metabolic regulatory networks related to mouse miRNAs. BLASTX searches using the InterPro and KEGG databases were used to collect the predicted target proteins with an E-value of 1×10³⁰. Target gene function and metabolic pathways of miRNAs were verified using the best hits. Finally, the terms in the categories molecular function, biological process and cellular component of target genes were obtained using the GO and InterPro databases (https://ngdc.cncb.ac.cn/databasecommons/). The GO terms and KEGG pathways with P≤0.01 and fold change ≥2 were considered significant.

miRNAs were extracted from spleen samples using the miRNeasy Serum/Plasma Kit (Qiagen GmbH) according to the manufacturer's protocol. Subsequently, miRNA was reverse transcribed into cDNA using the One Step PrimeScript & Reg miRNA cDNA Synthesis Kit (Takara Bio, Inc.). PCR was performed with the Prime-Script™ RT reagent kit (Takara Bio, Inc.) according to the manufacturer's protocol and in an ABI 7000 real-time PCR system (Applied Biosystems; Thermo Fisher Scientific, Inc.) using the following thermocycling conditions: 95°C for 5 sec and 58°C for 20 sec for 35 cycles, and 72°C for 30 sec. The experiments were performed once and set up in triplicate. The mRNA levels of large tumor suppressor kinase (Lats)2, Lats1 and TNF receptor superfamily member 11b (Tnfrsf11b) were detected by qPCR with the 2^−∆∆Cq quantification method (17) and the primers are listed in Table II.

Selected sequences from wild-type (WT) 3′-UTRs were cloned into the pMir-reporter vector (Ambion; Thermo Fisher Scientific, Inc.) and the mutant 3′-UTRs were generated by altering the predicted miR-31-5p, miR-135a-5p, miR-145a-5p, miR-204-5p, miR-211-5p and miR-429-3p 3′-UTR binding sites via a two-step PCR approach (the template was obtained from mouse miRNAs) (18). The miRNA mimics are listed in Table III. 293-T cells were purchased from FuXiang Biotechnology Co. 293-T cells were cultivated in Dulbecco's modified Eagles medium (DMEM) with antibiotics and Fetal Bovine Serum at 37°C in a humidified 5% CO₂ atmosphere. Cells were co-transfected with either a WT or mutant 3′-UTR reporter vector and miR-31-5p, miR-135a-5p, miR-145a-5p, miR-204-5p, miR-211-5p and miR-429-3p mimics or negative control constructs and were cultured for 24 h. The cells were then assessed to ascertain their luciferase activity using a dual-luciferase reporter assay system (Promega Corporation). The experiments were performed once and set up in triplicate.

In brief, the samples were treated with RIPA lysis buffer (50 mM Tris, pH 7.4, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing 1 mM protease inhibitor phenylmethanesulfonyl fluoride and centrifuged at 12,100 × g at 4°C for 10 min. The supernatant was then separated and mixed with 50 µl of SDS-PAGE sample buffer and boiled for 5 min, after which 5 µg (the protein concentration was determined by bicinchoninic acid protein assay kit) per lane was loaded onto the polyacrylamide gel and SDS-PAGE was performed with a 10% gel. Proteins were transferred onto polyvinylidene fluoride membranes (Beyotime Institute of Biotechnology) via electrophoresis, performed at 80 V for 3 h, using a Bio-Rad transfer system (Bio-Rad Laboratories, Inc.). The membranes were blocked for 2 h with skimmed milk (Beyotime Institute of Biotechnology) at room temperature and probed with the corresponding antibody (rabbit) diluted in blocking buffer at 1:10,000 (anti-LATS1, cat. no. ab70561; anti-LATS2, cat. no. ab111054; Anti-Tnfrsf11b, cat. no. ab183910; all from Abcam) at 4°C for 12 h. The membrane was then incubated for 2 h at room temperature with horseradish peroxidase-labeled goat anti-rabbit IgG antibody (cat. no. 18772; MilliporeSigma), diluted in blocking buffer at 1:20,000, and signals were detected with a super-sensitive signal enhanced chemiluminescence system (Beyotime Institute of Biotechnology). The levels of Lats2, Lats1 and Tnfrsf11b proteins were measured in infected and control mice in this experiment. The experiments were performed once and set up in triplicate.

Values are expressed as the mean ± standard deviation. Statistically significant differences between two groups were determined using Student's t-test. ANOVA and least-significant difference test were used for comparison among multiple groups. The results of the western blot were analyzed using ImageJ software (v1.51; National Institutes of Health). P<0.05 was considered to indicate a statistically significant difference.

A total of 1,899 miRNAs were identified after a series of filtering. Of these, 327 miRNAs were identified as known miRNAs, whereas 477 were identified as homologs in miRbase. Specifically, the expression of 1,000 miRNAs was detected in the control group, among which 320 miRNAs were known miRNAs and 362 were novel miRNAs. Furthermore, the expression of 996 miRNAs was detected in the infected group, of which 304 were known miRNAs and 202 were novel miRNAs. Of the 1,899 miRNAs identified, 114 were specifically expressed in the control group, while 110 were specifically expressed in the infected group. Furthermore, it was observed that most of the known miRNAs, such as miR-99a-5p, miR-143-5p and miR-27b-5p, were highly expressed in the control and infected groups, which was not unexpected. However, various novel miRNAs exhibited low expression or were expressed in only one group (Table SI).

The differentially expressed miRNAs identified are listed in Table SII. The expression levels of a total of 398 miRNAs were significantly changed (P<0.05; cut-off criterion: Fold change ≥2) after T. gondii challenge. Compared with the control group, 111 miRNAs were upregulated and 287 miRNAs were downregulated in the infected group. The heatmap indicated that 72 miRNAs were markedly differentially expressed (P<0.01; cut-off criterion: Fold change ≥3) between the infected and control groups. Among these differentially expressed miRNAs, 18 were upregulated and 54 were downregulated (Fig. 1).

As presented in Fig. 2, the top enriched GO terms for cancer-related genes were determined. Furthermore, KEGG enrichment analysis was performed using the target mRNAs of the differentially expressed miRNAs. The functional terms and numbers of genes in the categories cellular component, molecular function and biological process are listed in Fig. 2. In the category cellular component, genes were associated with cell part, envelope and organelle. In the category molecular function, terms associated with binding, catalytic activity, enzyme regulation and molecular transduction were significant. In the category biological process, significantly enriched terms were associated with biological regulation, cellular process, developmental process, death, metabolic process and pigmentation. In Fig. 3, KEGG pathways associated with cancer signaling are presented. Various miRNAs, including miR-145, miR-31, miR-21, miR-107, miR-125, miR-135 and miR-96, were analyzed. Compared with the control group, the genes PDCD4, PKC, ITGA5 and ATM were upregulated, while genes such as CYP24, Fzd3, p21 and RDX were downregulated in the infected group (Table SIII). Certain genes did not change (e.g. ERBB2, RAF1 and ERBB3) in the infected group.

Through the degradation of target gene mRNAs or the inhibition of the translation of target transcripts, miRNAs have important roles in cell proliferation and differentiation, apoptosis and a variety of diseases. A total of 8,116 target genes were analyzed for 45 differentially expressed cancer-related genes using miRDB (Table SIV). On the one hand, miRNAs are able to target a variety of cancer-related genes. For instance, miR-211-5p targets Tnfaip8, C1qtnf3, Lats2, Lats1 and St7; miR-145a-5p targets Tnfrsf11b, Etaa1 and C1qtnf9; and miR-429-3p targets Lats2, Lrp1b, Mtus1 and Tnfrsf11b. On the other hand, a single cancer-related gene may be targeted by multiple miRNAs. For instance, the genes Tnfaip8, C1qtnf3, Lats2, Lats1 and St7 may all be targeted by both miR-211-5p and miR-204-5p. Furthermore, miR-211-5p, miR-31-5p, miR-135a-5p, miR-204-5p and miR-429-3p are all able to target the Lats2 gene. The Lats1 gene is able to be targeted by four miRNAs (miR-201-5p, miR-211-5p, miR-204-5p and miR-107-3p), whereas Lzts3 is only targeted by miR-138-5p.

To further confirm the miRNA-seq results, RT-qPCR was performed in the present study. Candidate miRNAs were selected from cancer metabolic signaling pathways. Compared with the control group, miR-210-5p, miR-135b-5p, miR-21a-3p and miR-146a-3p were upregulated, while miR-135a-5p, miR-125a-5p, miR-145a-5p, miR-107-3p, miR-211-5p, miR-429-3p, miR-96-5p and miR-31-3p were downregulated in the infected group (Fig. 4). The RT-qPCR results suggested that the levels of miR-210-5p, miR-135b-5p and miR-21a-3p were significantly upregulated after T. gondii infection, whereas no difference in miR-146a-3p levels was observed in the infected group. The levels of miR-210-5p and miR-135b-5p in the infected group were five times higher than those in the control group. In addition, the levels of miR-135a-5p, miR-125a-5p, miR-145a-5p and miR-31-3p were markedly downregulated after infection with T. gondii, while no difference in the levels of miR-96-5p or miR-138-5p was observed between the infected and control groups. The levels of miR-135a-5p and miR-145a-5p in the infected group were only one-quarter of those in the control group. These results were in line with those of the RNA-seq.

Targets of miR-31-5p, miR-135a-5p, miR-145a-5p, miR-204-5p, miR-211-5p and miR-429-3p were verified by luciferase activity assays in 293T cells (Fig. 5). While miR-31-5p, miR-135a-5p, miR-211-5p and miR-429-3p were confirmed to be able to target the Lats2 gene, miR-204-5p and miR-211-5p target the Lats1 gene. In addition, miR-145a-5p and miR-429-3p were indicated to be targets of Tnfrsf11b. Furthermore, miR-211-5p was able to target the Lats1 and Lats2 genes, and miR-429-3p was confirmed to target the Tnfrsf11b and Lats2 genes.

As presented in Fig. 6, the protein levels of Lats1 and Lats2 in spleen samples of infected mice were higher than those in the control group, while the levels of Tnfrsf11b protein from the infected group were similar to those of the control mice.