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. 2020 Aug 31;13(1):435.
doi: 10.1186/s13071-020-04312-x.

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

Guiling Zhang  1 , Yajun Zhang  1 , Ziwen Niu  1 , Chenrong Wang  1 , Fujie Xie  1 , Juanfeng Li  1 , Sumei Zhang  1 , Meng Qi  2 , Fuchun Jian  1 , Changshen Ning  1 , Longxian Zhang  3 , Rongjun Wang  4
Affiliations

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

Guiling Zhang et al. Parasit Vectors. .

Abstract

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.

Keywords: Cryptosporidium parvum; HCT-8; NF-κB; TLRs; miR-942-5p.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
Promotion of TLR and NF-κB signaling molecules in HCT-8 cells by C. parvum. a Gel electrophoresis of qPCR products. b Expression of TLR2 and TLR4 at 4 and 12 hpi. Non-infected cells represent the control group (*P < 0.05 and ***P < 0.001 by t-test: test versus non-infected cells). c Western blots of TLR4, NF-κB, and IκBα protein at 4, 8 and 12 hpi; 0 hpi represents the control group
Fig. 2
Fig. 2
miR-942-5p expression in HCT-8 cells. a miR-942-5p expression in HCT-8 cells infected by C. parvum (0 hpi represents the control group). b miR-942-5p expression in C. parvum-infected HCT-8 cells after transformation with TLR2-specific siRNA. c miR-942-5p expression in C. 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)
Fig. 3
Fig. 3
miR-942-5p expression in C. parvum-infected HCT-8 cells pretreated with NF-κB inhibitors. a PDTC inhibition of NF-κB. b p65-dependent inhibition of NF-κB p65 by SC-514 (*P < 0.05 and **P < 0.01 by t-test: test versus inhibitor-negative group)
See this image and copyright information in PMC

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