Cryptosporidium parvum upregulates miR-942-5p expression in HCT-8 cells via TLR2/TLR4-NF-κB signaling

Background Micro (mi)RNAs are small noncoding RNA molecules that function in RNA silencing and post-transcriptional regulation of gene expression. This study investigated host miRNA activity in the innate immune response to Cryptosporidium parvum infection. Methods In vitro infection model adopts HCT-8 human ileocecal adenocarcinoma cells infected with C. parvum. The expression of miR-942-5p was estimated using quantitative real-time polymerase chain reaction (qPCR). The TLRs-NF-κB signaling was confirmed by qPCR, western blotting, TLR4- and TLR2-specific short-interfering (si)RNA, and NF-κB inhibition. Results HCT-8 cells express all known toll-like receptors (TLRs). Cryptosporidium parvum infection of cultured HCT-8 cells upregulated TLR2 and TLR4, and downstream TLR effectors, including NF-κB and suppressed IκBα (nuclear factor of kappa light polypeptide gene enhancer in B cells inhibitor, alpha). The expression of miR-942-5p was significantly upregulated at 4, 8, 12 and 24 h post-infection, and especially at 8 hpi. The results of TLR4- and TLR2-specific siRNA and NF-κB inhibition showed that upregulation of miR-942-5p was promoted by p65 subunit-dependent TLR2/TLR4-NF-κB pathway signaling. Conclusions miR-942-5p of HCT-8 cells was significantly upregulated after C. parvum infection, especially at 8 hpi, in response to a p65-dependent TLR2/TLR4-NF-κB signaling. TLR4 appeared to play a dominant role.


Open Access
Parasites & Vectors *Correspondence: zhanglx8999@henau.edu.cn; wrj-1978@henau.edu.cn 1 College of Veterinary Medicine, Henan Agricultural University, Zhengzhou 450046, P. R. China Full list of author information is available at the end of the article Background Cryptosporidium is an emerging zoonotic pathogen that causes diarrhea in both immunocompetent and immunosuppressed hosts, and is second only to rotavirus as a cause of moderate-to-severe diarrhea in children under two years of age [1]. In immunocompromised patients, it can cause severe, life-threatening prolonged disease. In 2016, the disease burden of cryptosporidiosis in children younger than five years was more than 12 million disability-adjusted life-years (DALYs) [2]. Thirtyeight Cryptosporidium valid species and approximate 60 Cryptosporidium genotypes have been identified or described in animals, humans, and environmental samples [3] but C. parvum and C. hominis are responsible for more than 90% of infections in humans. Despite recent efforts, effective prophylaxis and treatment are not available.
As with other intracellular pathogens, Cryptosporidium infection influences apoptosis. Microarray analysis of 51 apoptosis-associated genes indicated biphasic regulation by Cryptosporidium, with an anti-apoptotic state at 6 and 12 h post-infection (hpi) and a moderately proapoptotic state at 24, 48 and 72 hpi [14]. Inhibition of apoptosis in infected cells increases parasite survival and continuing apoptosis in uninfected bystander cells act to decrease the host immune response and may contribute to evasion of host defenses [15]. Previous studies have reported that Cryptosporidium inhibited of host-cell apoptosis by activating NF-κB [16,17]. Little is known about the regulation of host-cell apoptosis by miRNAs following Cryptosporidium infection. A previous study found that downregulation of miR-513 was followed by the upregulation of B7-H1 expression and decreased apoptosis [11].

Real-time quantitative PCR (qPCR)
HCT-8 cells were washed three times with phosphate buffered saline (PBS) before adding 1 ml TRIzol reagent (Invitrogen, Waltham, MA, USA) to each well. Total RNA was isolated following the kit manufacturer's instructions subsequent to treatment with Recombinant DNase I (Takara, Kyoto, Japan). RNA was reverse transcribed to cDNA with SuperScript IV Reverse Transcriptase (Invitrogen) by oligo (dT) and random primers. The cDNA was amplified using the TB Green Premix Ex Taq II (Takara, Kyoto, Japan) and the gene-specific primers shown in Table 1. GAPDH or β-actin genes were internal references for toll-like receptors (TLRs), the U6 gene was the internal reference for miR-942-5p. miR-942-5p was reverse transcribed to cDNA using the stemloop primer (5′-GTC GTA TCC AGT GCA GGG TCC GAG GTA TTC GCA CTG GAT ACG ACC ACA TGG C-3′) and the primer (5′-CGC TTC ACG AAT TTG CGT GTC AT-3′) for U6. PCR included one 30 s cycle at 95 °C, 40 cycles of 5 s at 95 °C, 10 s at 55 °C, and 15 s at 72°C, and a final 15 s cycle at 95 °C, 1 min at 60 °C, and infinite at 25 °C. The Cq values were analyzed using the comparative Cq (ΔΔCq) method and the amount of target was obtained by normalizing to internal reference and comparing with the control group.

Western blotting
HCT-8 cells were grown to 80% confluence in 6-well culture plates and exposed to C. parvum sporozoites. The cells were lysed with a total protein extraction kit (Solarbio Life Sciences, Beijing, China), and the protein concentrations were determined with a Pierce Bicinchoninic Acid (BCA) Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer's instructions. The proteins in 30 µg samples of lysate were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto nitrocellulose membranes. Membranes were incubated with TLR4, NF-κB, IκBα, and β-actin primary monoclonal antibodies (Abcam, Cambridge, UK), and then with 0.2 µg/ml horseradish peroxidase (HRP)-conjugated secondary antibodies. The blots were read by an electrochemiluminescence (ECL) substrate (Thermo Fisher Scientific).

Short-interfering (si)RNA
SiRNAs targeting TLR-2 and TLR4 mRNAs were designed by the Sangon Biotech (Shanghai, China). HCT-8 cells were grown to 60-70% confluency in 12-well cell culture plates and transfected with siRNAs using Lipofectamine 3000 (Thermo Fisher Scientific). The extent of inhibition was determined by qPCR assays of TLR2 and TLR4 expression at 48 h post-transfection. The siRNAs that caused the greatest inhibition of TLR2, TLR4 expression were TLR2, GGA AGA UAA UGA ACA CCA ATT (sense) and UUG GUG UUC AUU AUC UUC CTT (antisense); TLR4, CCA GGU GCA UUU AAA GAA ATT (sense) and UUG GUG UUC AUU AUC UUC CTT (antisense). The siRNA oligonucleotides had no significant overlap with homologous gene sequences. Nonspecific siRNAs containing the same nucleotides in an irregular sequence were used as controls. The siRNAs were labeled with Cy3 using a silencer siRNA labeling kit (Thermo Fisher Scientific) for identification of transfected cells by confocal microscopy. HCT-8 cells were infected with C. parvum sporozoites 6 h after siRNA transfection. Total RNA was extracted at 0, 4, 8, 12, 24 and 48 hpi.

Data analysis
Data are represented as the mean ± standard deviation (SD) from three independent experiments. Each independent experiment was conducted by three replicates of qPCR and the mean value was used for data analysis. One-way ANOVA or t-test was carried out using the software of GraphPad Prism version 8.02 (https ://www.graph pad.com/).

Discussion
Cultured HCT-8 cells expressed all known TLRs (TLR1-TLR10) and C. parvum infection induced the upregulation of TLR2 and TLR4, but not other TLRs, as was previously found in H69 human choanocyte cells [23]. Upregulation of TLR4 was stronger than that of TLR2 (Fig. 1b), but activation of either receptor recruited downstream components, with increased NF-κB expression and decreased expression of IκBα, an NF-κB inhibitor. Nuclear translocation of NF-κB activated transcription. TLR2-and TLR4-induced activation of NF-κB has previously been reported in H69 cells infected by C. parvum [23]. The upregulation of miR-942-5p after C. parvum infection was dependent on TLR2/TLR4-NF-κB signaling. TLR4 may have had a stronger effect than TLR2, especially at 4 hpi, but both TLR2 and TLR4 contributed to  parvum-infected HCT-8 cells after transformation with TLR4-specific siRNA. Black control represents the group of non-infected cells (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001 by one-way ANOVA: test versus control group) the upregulation of miR-942-5p expression (Fig. 2b, c). There are few data on the difference in the contributions of TLR2 and TLR4 during C. parvum infection, but TLR4-NF-κB signaling has been reported more frequently. TLR2 may be involved in C. parvum-induced stabilization of iNOS mRNA expression in biliary epithelial cells [13]. Post-transcriptional suppression of TLR4 expression by let-7i has been shown to contribute to immune responses to C. parvum infection in cultured human cholangiocytes, and mu-miR-92a-2-5p, which targets TLR2, relieves Schistosoma japonicum-induced liver fibrosis [6,24].
A microarray analysis found that miR-942-5p was strongly upregulated during the early phase of C. parvum infection, and in this study qPCR confirmed that C. parvum infection was followed by significant upregulation of miR-942-5p at 4, 8, 12 and 24 hpi (Fig. 2a). Bioinformatics analysis indicated that miR-942-5p may be involved in the regulation of host-cell apoptosis. Previous studies have shown that miR-942 regulated cell apoptosis in response to microbial infection. For example, downregulation of miR-942 enhanced the apoptosis of HLCZ01 cells in response to hepatitis C virus infection [25]. Targeting of the IFI27 gene by miR-942-5p has been shown to inhibit apoptosis role in HCT-8 cells during the early phase of C. parvum infection (our unpublished data).

Conclusions
HCT-8 cells expressed all known TLRs, and TLR2 and TLR4 were upregulated following C. parvum infection with activation of downstream signaling. miR-942-5p was significantly upregulated after C. parvum infection, especially at 8 hpi, in response to a p65-dependent TLR2/ TLR4-NF-κB signaling. TLR4 appeared to play a dominant role.