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. 2024 May 8;11(5):206.
doi: 10.3390/vetsci11050206.

miR-329b-5p Affects Sheep Intestinal Epithelial Cells against Escherichia coli F17 Infection

Yeling Xu  1 , Weihao Chen  1 , Huiguo Yang  2 , Zhenghai Song  3 , Yeqing Wang  4 , Rui Su  5 , Joram M Mwacharo  6 , Xiaoyang Lv  7   8 , Wei Sun  1   7   8
Affiliations

miR-329b-5p Affects Sheep Intestinal Epithelial Cells against Escherichia coli F17 Infection

Yeling Xu et al. Vet Sci. .

Abstract

Diarrhea is the most common issue in sheep farms, typically due to pathogenic Escherichia coli (E. coli) infections, such as E. coli F17. microRNA, a primary type of non-coding RNA, has been shown to be involved in diarrhea caused by pathogenic E. coli. To elucidate the profound mechanisms of miRNA in E. coli F17 infections, methods such as E. coli F17 adhesion assay, colony counting assay, relative quantification of bacterial E. coli fimbriae gene expression, indirect immune fluorescence (IF), Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), Western blotting (WB), and scratch assay were conducted to investigate the effect of miR-329b-5p overexpression/knock-down on E. coli F17 susceptibility of sheep intestinal epithelial cells (IECs). The findings indicated that miR-329b-5p enhances the E. coli F17 resistance of sheep IECs to E.coli F17 by promoting adhesion between E. coli F17 and IEC, as well as IEC proliferation and migration. In summary, miR-329b-5p plays a crucial role in the defense of sheep IECs against E. coli F17 infection, providing valuable insights into its mechanism of action.

Keywords: Escherichia coli F17; intestinal epithelial cells; micro RNA; sheep diarrhea.

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Conflict of interest statement

Yeqing Wang and Rui Su are employed in Suzhou Stud Farm Co., Ltd. The remaining authors have no conflicts of interest to declare.

Figures

Figure 1
Figure 1
Efficiency verification of miR-329b-5p mimics and miR-329p-5p inhibitors. (A) Transfection of miR-329b-5p mimics and NC into sheep IECs. Expression of miR-329b-5p was detected by RT-qPCR 48 h after transfection. (B) Transfection of miR-329b-5p inhibitor and inhibitor NC into sheep IECs. Expression of miR-329b-5p was detected by RT-qPCR 48 h after transfection. * p < 0.05.
Figure 2
Figure 2
miR-329b-5p affects susceptibility of sheep IEC to E. coli F17. (A) Colony count of E. coli F17 after up-regulation of miR-329b-5p. (B) Colony count of E. coli F17 after down-regulation of miR-329b-5p. (C) RT-qPCR of E. coli F17 fimbriae gene after up-regulation of miR-329b-5p. (D) RT-qPCR of E. coli F17 fimbriae gene after down-regulation of miR-329b-5p. (E) In the immunofluorescence assay, cells were observed under the fluorescence microscope (100×). * p < 0.05 and ** p < 0.01.
Figure 3
Figure 3
Effect of miR-329b-5p on vimentin expression. (A) RT-qPCR assay of vimentin expression after up-regulation of miR-329b-5p. (B) RT-qPCR assay of vimentin expression after down-regulation of miR-329b-5p. (C,D) WB of vimentin expression after up-regulation of miR-329b-5p. (E,F) WB of vimentin expression after down-regulation of miR-329b-5p. * p < 0.05 and ** p < 0.01.
Figure 4
Figure 4
Effect of miR-329b-5p on proliferation of IECs. (A) CCK-8 assay after up-regulation of miR-329b-5p. (B) CCK-8 assay after down-regulation of miR-329b-5p. (C,D) EDU assay after up-regulation of miR-329b-5p (100×). (E,F) EDU assay after -regulation of miR-329b-5p (100×). * p < 0.05 and ** p < 0.01.
Figure 5
Figure 5
Effect of miR-329b-5p on proliferation of IECs detected by proliferation markers. (A) mRNA relative expression of PCNA and CCND1 after up-regulation of miR-329b-5p. (B) mRNA relative expression of PCNA and CCND1 after down-regulation of miR-329b-5p. (C,D) Protein expression of PCNA after up-regulation of miR-329b-5p. (E,F) Protein expression of PCNA after down-regulation of miR-329b-5p. * p < 0.05 and ** p < 0.01.
Figure 6
Figure 6
Effect of miR-329b-5p on migration of IECs. (A,B) Scratch assay after up-regulation of miR-329b-5p. (C,D) Scratch assay after down-regulation of miR-329b-5p. ** p < 0.01.
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