MiR-942-5p targeting the IFI27 gene regulates HCT-8 cell apoptosis via a TRAIL-dependent pathway during the early phase of Cryptosporidium parvum infection

Abstract

Background: MicroRNAs (miRNAs) are involved in the regulation of both the innate and adaptive immune response to Cryptosporidium parvum infection. We previously reported that C. parvum upregulated miR‑942‑5p expression in HCT‑8 cells via TLR2/TLR4‑NF‑κB signaling. In the present study, the role of miRNA-942-5p in the regulation of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated HCT-8 cell apoptosis induced by C. parvum was investigated.

Methods: Quantitative real-time polymerase chain reaction, western blotting, flow cytometry, and immunofluorescence were used for analysis.

Results: Forced expression of miRNA-942-5p resulted in decreased apoptosis and an increased C. parvum burden in HCT-8 cells. The opposite results were observed using the suppressed expression of miRNA-942-5p. The miRNA-942-5p led to the translational suppression of IFI27 gene through targeting the 3'-untranslated region of the IFI27 gene. Moreover, overexpression of the IFI27 gene produced a high apoptotic ratio and low C. parvum burden. In contrast, a low apoptotic ratio and a high C. parvum burden were observed following downregulation of the IFI27 gene. Both miR-942-5p and the IFI27 gene influenced TRAIL and caspase-8 expression induced by C. parvum in HCT-8 cells. Moreover, TRAIL promoted HCT-8 cell apoptosis in a concentration-dependent manner.

Conclusions: These data suggested that C. parvum induced the downregulation of IFI27 via relief of miR-942-5p-mediated translational suppression. IFI27 downregulation was affected the burden of C. parvum by regulating HCT-8 cell apoptosis through TRAIL-dependent pathways. Future studies should determine the mechanisms by which C. parvum infection increases miR-942-5p expression and the role of miR-942-5p in hosts' anti-C. parvum immunity in vivo.

Keywords: Apoptosis; Cryptosporidium parvum; HCT-8 cell; Parasite burden; miR-942-5p.

Fig. 1

miR-942-5p is involved in cell apoptosis and parasite eradication during the early period following C. parvum infection. A The effect of miR-942-5p on cell apoptosis in HCT-8 cells. Cellular apoptosis was suppressed after transfection with miR-942-5p mimics, whereas apoptosis was enhanced after transfection with an miR-942-5p inhibitor. B and C The effect of miR-942-5p on the number of parasites in HCT-8 cells. The burden of C. parvum infection was significantly increased after transfection with an miR-942-5p mimic, whereas it was suppressed after in vitro transfection with an miR-942-5p inhibitor 24 h after initial parasite exposure. Cryptosporidium parvum parasites were stained in green, and the cell nuclei were stained blue. Bar = 20 µm. All data represent the combined mean ± SD of three independent experiments, with two to three technical replicates per experiment, and were analyzed with a t-test compared to cells transfected with a non-specific control. ^*P < 0.05; ^**P ≤ 0.01; ^***P ≤ 0.001; ^****P ≤ 0.0001

Fig. 2

The miR-942-5p targets the IFI27 3′-UTR, causing translational suppression. A The IFI27 mRNA schematic showed one potential binding site for miR-942-5p in its 3′-UTR. The IFI27 3′-UTR sequence covering the potential binding site was inserted into the pmirGLO-REPORT luciferase plasmid. Control plasmids with the mutant 3′-UTR sequence were also generated as a control. B HCT-8 cells were exposed to C. parvum sporozoites for as long as 24 h, followed by western blot to evaluate IFI27 protein expression and real-time PCR analysis for IFI27 mRNA expression. Representative western blots from three independent experiments are shown. The densitometric levels of protein signals and mRNA levels were quantified and expressed as the ratio to actin. C Targeting the IFI27 3′-UTR resulted in translational suppression. Cells were transfected with a pMIR-REPORT luciferase construct containing the miR-942-5p binding site in the IFI27 3′-UTR and treated with an miR-942-5p mimic for 24 h, followed by a luciferase analysis. D Manipulation of miR-942-5p function can result in reciprocal alterations in IFI27 protein expression in HCT-8 cells. Cells were treated with an miR-942-5p mimic or miR-942-5p inhibitor for 6 h and exposed to C. parvum sporozoites, followed by a western blot for IFI27 protein and real-time PCR for IFI27 mRNA 12 hpi. Representative western blot images and quantification of IFI27 mRNA levels from three independent experiments are shown. Densitometric levels of IFI27 signals were quantified and expressed as the ratio to β-actin. Densitometric levels of protein signals and mRNA levels were quantified and expressed as the ratio to β-actin. All data represent the combined mean ± SD of three independent experiments, with two to three technical replicates per experiment, and were analyzed with a t-test vs. the controls. ^*P < 0.05; ^**P ≤ 0.01

Fig. 3

IFI27 is involved in cell apoptosis and parasite eradication following early C. parvum infection. A Effect of IFI27 on cell apoptosis in HCT-8 cells. The cells were transfected with pcDNA3.1-IFI27-OE or siRNA-IFI27 for 24 h. Cells were exposed to an equal number of C. parvum sporozoites for 24 h after digestion and staining, and the cell apoptosis ratio was assessed by flow cytometry. Cellular apoptosis was enhanced after transfection with pcDNA3.1-IFI27-OE, but was restrained after transfection with siRNA-IFI27. B, C Effect of IFI27 on the number of parasites in HCT-8 cells. Cells were transfected with pcDNA3.1-IFI27-OE or siRNA-IFI27 for 24 h. Cells were exposed to an equal number of C. parvum sporozoites for 2 h, followed by extensive washing with culture medium. To determine the initial attachment and cellular invasion of C. parvum, the cells were immediately harvested after washing and C. parvum was quantified by real-time PCR. A similar number of parasites was detected in cells transfected with pcDNA3.1-IFI27-OE or siRNA-IFI27 following the initial exposure to C. parvum for 2 h. To determine the parasite burden after the initial cell attachment and invasion, infected HCT-8 cells were cultured for another 22 h after washing, followed by real-time PCR analysis. The C. parvum infection burden was obviously suppressed after transfection with pcDNA3.1-IFI27-OE but was increased after transfection with siRNA-IFI27 in vitro 24 h after the initial parasite exposure. All data represent the combined mean ± SD of three independent experiments with two to three technical replicates per experiment. The data were analyzed with a t-test and compared to cells transfected with an empty vector or nonspecific control siRNA. ^*P < 0.05; ^**P ≤ 0.01

Fig. 4

IFI27 is involved in cell apoptosis and parasite eradication following early C. parvum infection via a TRAIL-mediated pathway. A The effect of IFI27 on apoptosis-related molecules in HCT-8 cells. The cells were transfected with pcDNA3.1-IFI27-OE or siRNA-IFI27 for 24 h. Cells were exposed to an equal number of C. parvum sporozoites for up to 48 h, followed by a western blot for TRAIL protein expression and real-time PCR analysis for TRAIL and caspase-8 mRNA. Apoptosis-related molecules were enhanced after transfection with pcDNA3.1-IFI27-OE, but were restrained after transfection with siRNA-IFI27. B, C The effect of rTRAIL on cell apoptosis in HCT-8 cells. Cells were transfected with siRNA-IFI27 for 24 h. The cells were exposed to an equal number of C. parvum sporozoites for 2 h, followed by an incubation with varying concentrations of recombinant TRAIL (10, 25, and 50 ng/ml). After 6 h, the cells were digested and stained, and the apoptosis ratio was assessed by flow cytometry. With the increased concentration, the proportion of apoptosis was gradually increased. All data represent the combined mean ± SD of three independent experiments with two to three technical replicates per experiment. The data were analyzed with a one-way ANOVA followed by a Dunnett’s test for multiple comparisons. ^*P < 0.05; ^**P ≤ 0.01; ^***P ≤ 0.001; ^****P ≤ 0.0001

Fig. 5

The miR-942-5p regulates TRAIL expression after C. parvum infection in HCT-8 cells. Cells were transfected with an miR-942-5p mimic or miR-942-5p inhibitor for 24 h. Cells were exposed to an equal number of C. parvum sporozoites for up to 48 h, followed by a western blot for TRAIL protein and real-time PCR analysis for TRAIL and caspase-8 mRNA. All data represent the combined mean ± SD of three independent experiments with two to three technical replicates per experiment. The data were analyzed with a one-way ANOVA followed by a Dunnett’s test for multiple comparisons. ^*P < 0.05; ^**P ≤ 0.01; ^***P ≤ 0.001; ^****P ≤ 0.0001