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Abstract
BackgroundMicro (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.MethodsIn 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.ResultsHCT-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.ConclusionsmiR-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.
Highlights
MicroRNAs are small noncoding RNA molecules that function in RNA silencing and post-transcriptional regulation of gene expression
This study investigated the upregulation of miR-942-5p in cultured HCT-8 human ileocecal adenocarcinoma cells following activation of the TLR2/TLR4-nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway by C. parvum
Cryptosporidium parvum activation of TLR2 and TLR4 in HCT‐8 TLR1 to TLR10 expression was assayed by quantitative real-time polymerase chain reaction (qPCR) at 8 and 12 hpi
Summary
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. 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. Micro (mi)RNAs are small noncoding RNA molecules found in plants, animals, viruses, and some parasite species They function in RNA silencing and post-transcriptional regulation of gene expression [4]. In biliary epithelial cells for example, C. parvum reduces the expression of let-7 family miRNAs, which target the synaptosome associated protein 23 (SNAP23) gene, and loss of SNAP23 expression interferes with the release of exosomes carrying antimicrobial-peptides [7]
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