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Overexpression of miR-429 impairs intestinal barrier function in diabetic mice by down-regulating occludin expression

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Abstract

Diabetes mellitus (DM) is a group of metabolic diseases characterised by insulin deficiency/resistance and hyperglycaemia. We previously reported the presence of an impaired tight junction and decreased expression of occludin (Ocln) and zonula occludens-1 (ZO-1) in the intestinal epithelial cells (IECs) of type 1 DM mice, but the exact mechanism remains unclear. In this study, we investigated the role of microRNAs (miRNAs) in impairing the tight junction in IECs of DM mice. Using an integrated comparative miRNA microarray, miR-429 was found to be up-regulated in IECs of type 1 DM mice. Then, miR-429 was confirmed to directly target the 3’-UTR of Ocln, although it did not target ZO-1. Moreover, miR-429 down-regulated the Ocln expression in IEC-6 cells in vitro. Finally, exogenous agomiRNA-429 was shown to down-regulate Ocln and induce intestinal barrier dysfunction in normal mice, while exogenous antagomiRNA-429 up-regulated Ocln in vivo and improved intestinal barrier function in DM mice. In conclusion, increased miR-429 could down-regulate the expression of Ocln by targeting the Ocln 3′-UTR, which impaired intestinal barrier function in DM mice.

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Abbreviations

3’-UTR:

3’-untranslated region

4-kD FITC-dextran:

4-kD fluorescein isothiocyanate (FITC)-conjugated dextran

ATCC:

American Type Culture Collection

DE:

Diabetic enteropathy

DM:

Diabetes mellitus

DMEM:

Dulbecco’s modified Eagle’s medium

FBS:

Foetal bovine serum

FSS:

Fluorescein sodium salt

IEC:

Intestinal epithelial cell

LPS:

Lipopolysaccharide

miRNA/miR:

MicroRNAs

mRNA:

Messenger RNA

NC:

Negative control

Ocln:

Occludin

PBS:

Phosphate-buffered saline

qRT-PCR:

Quantitative real-time polymerase chain reaction

rRNA:

Ribosomal RNA

STZ:

Streptozocin

TEM:

Transmission electron microscopy

TJ:

Tight junction

ZO-1:

Zonula occludens-1

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Acknowledgment

This study was supported by the National Natural Science Foundation of China (No. 81270442 and No. 81370475).

Contribution statement

Tao Yu and Xi-Ji Lu carried out the molecular biology studies, participated in the sequence alignment and drafted the manuscript. Ti-Dong Shan and Can-Ze Huang carried out the immunofluorescence assays. Hui Ouyang and Ji-Hao Xu participated in the induction of the diabetes model. Jie-Yao Li and Hong-Sheng Yang conducted the statistical analysis. Wa Zhong and Zhong-Sheng Xia participated in the design of the study and participated in the sequence alignment. Qi-Kui Chen conceived the study, participated in its design and coordination, and helped to draft the manuscript. All authors read and approved the final manuscript.

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    Authors and Affiliations

    1. Department of Gastroenterology, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, 107 Yan Jiang Xi Road, Guangzhou, Guangdong, 510120, China

      Tao Yu, Xi-Ji Lu, Jie-Yao Li, Ti-Dong Shan, Can-Ze Huang, Hui Ouyang, Hong-Sheng Yang, Ji-Hao Xu, Wa Zhong, Zhong-Sheng Xia & Qi-Kui Chen

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    1. Tao Yu
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    Tao Yu and Xi-Ji Lu contributed equally to this work.

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    Figure 6

    MiR-429 did not affect the expressions of ZO-1, Claudin-2, and Claudin-3 in IEC-6. (a-c) The mRNA and protein expressions of ZO-1, Claudin-2, and Claudin-3 in IEC-6 cells (48 hours after transfection) were assessed by qRTPCR (a) and Western blot (b) in the three groups. The integrated intensities of bands (c) were calculated using β-actin as an endogenous control. (JPG 112 kb)

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    Yu, T., Lu, XJ., Li, JY. et al. Overexpression of miR-429 impairs intestinal barrier function in diabetic mice by down-regulating occludin expression. Cell Tissue Res 366, 341–352 (2016). https://doi.org/10.1007/s00441-016-2435-5

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