MiRNA-29c-3p Promotes Intestinal Inflammation via Targeting Leukemia Inhibitory Factor in Ulcerative Colitis

Abstract

Background: Dysregulation of micro-RNAs (miRNAs) is profoundly linked to inflammatory bowel diseases (IBD), but little is known about the specific biological functions of miRNAs in IBD. This study sought to elucidate the effect and the underlying target of miR-29c-3p in ulcerative colitis (UC).

Methods: The levels of miR-29c-3p and leukemia inhibitory factor (LIF) were measured in inflamed lesions of UC patients and dextran sulfate sodium (DSS)-induced colitis mice by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting. MiR-29c-3p was predicted to target LIF by bioinformatics software, which was verified via luciferase reporter assay and transfection of miR-29c-3p mimics or inhibitor. The role of miR-29c-3p/LIF axis in intestinal inflammation was explored in experimental colitis mice and Caco-2 cells.

Results: MiR-29c-3p was markedly downregulated while LIF was upregulated in colon tissues of UC patients and DSS-challenged colitis mice as well as in primary intestinal epithelial cells (IECs) and LPS-treated Caco-2 cells. MiR-29c-3p inhibited LIF expression at the transcriptional level via binding to LIF 3'-untranslated region (UTR) in Caco-2 cells. Targeting miR-29c-3p/LIF axis regulated inflammatory cytokines production, cell proliferation and apoptosis. Overexpression of miR-29c-3p aggravated mice experimental colitis via suppressing LIF.

Conclusion: Our findings demonstrate that the upregulation of miR-29c-3p promotes gut inflammation and the expression of pro-inflammatory mediators via suppressing LIF, thereby modulating the pathogenesis of UC.

Keywords: apoptosis; inflammation; leukemia inhibitory factor; microRNA-29c-3p; ulcerative colitis.

Figure 1

The expression of miR-29c-3p and LIF in UC tissues and IECs. (A) The mRNA levels of LIF in UC and normal tissues. (B) Representative Western blots and densitometric quantitative analysis of LIF proteins levels in UC and normal tissues. (C) The mRNA levels of LIF in primary IECs of UC and normal colon. (D) Representative Western blots and densitometric quantitative analysis of LIF protein levels in IECs of UC and normal colon. (E) The expression of miR-29c-3p in UC and normal tissues. (F) The expression of miR-29c-3p in primary IECs of UC and normal colon. (G) Pearson’s correlation analysis of the expression levels of miR-29c-3p and LIF protein. (H) Pearson’s correlation analysis of the expression levels of miR-29c-3p and LIF mRNA. **P<0.01 versus normal group.

Figure 2

Identification of LIF as a direct target of miR-29c-3p. (A) Schematic description of a putative binding site of miR-29c-3p in the 3ʹ-UTR of human LIF. (B) Luciferase reporter activity in Caco-2 cells. (C) QRT-PCR analysis of miR-29c-3p expression in Caco-2 cells pre-transfected with miR-29c-3p mimics, miR-29c-3p inhibitor or corresponding scramble controls. (D) qRT-PCR analysis of LIF mRNA levels in pre-transfected Caco-2 cells. (E) Representative Western blots and densitometric quantitative analysis of LIF protein levels in pre-transfected Caco-2 cells. ** and *** indicates P<0.01 and 0.001, respectively.

Figure 3

The role of miR-29c-3p and LIF in LPS-mediated cell inflammation. (A) The expression of miR-29c-3p in LPS-treated Caco-2 cells pre-transfected with miR-29c-3p mimics or scrambled control. (B) Representative Western blots and densitometric quantitative analysis of LIF protein levels in LPS-treated Caco-2 cells pre-transfected with miR-29c-3p mimics or scrambled control. (C) qRT-PCR analysis of LIF expression in LPS-treated Caco-2 cells pre-transfected with miR-29c-3p mimics or scrambled control. (D) The protein levels of IL-1β, IL-6, IL-8 and TNF-α in the culture supernatants of LPS-treated Caco-2 cells pre-transfected with miR-29c-3p mimics or together with LIF mimics. (E) Validation of si-LIF transfection efficiency in Caco-2 cells. (F) The protein levels of IL-1β, IL-6, IL-8 and TNF-α in the culture supernatants of LPS-treated Caco-2 cells pre-transfected with si-LIF or scrambled control. *P<0.05, **P< 0.01, ***P<0.001.

Figure 4

The role of miR-29c-3p and LIF on cell proliferation and apoptosis. (A) CCK-8 assays of Caco-2 cells after transfection with miR-29c-3p mimics and LIF mimics. (B) CCK-8 assays of Caco-2 cells after transfection with miR-29c-3p inhibitor and si-LIF. (C) CCK-8 assays of Caco-2 cells after transfection with si-LIF or scrambled control. (D)Flow cytometry analysis of cell apoptosis after transfection with miR-29c-3p mimics and LIF mimics (E) Flow cytometry analysis of cell apoptosis after transfection with miR-29c-3p inhibitor and si-LIF. (F) Flow cytometry analysis of cell apoptosis after transfection with si-LIF or scrambled control. *P<0.05, **P< 0.01, ***P<0.001.

Figure 5

The expression of miR-29c-3p and LIF in DSS-induced mice colitis. Mice were given 2.5% DSS in the drinking water for consecutive 7 days to induce experimental colitis. (A) The expression of miR-29c-3p in colon tissues and primary IECs of mice. (B) Representative Western blots and densitometric quantitative analysis of LIF protein levels in colon tissues and primary IECs. (C) qRT-PCR analysis of LIF expression in colon tissues and primary IECs. ** and *** indicates P<0.01 and 0.001, respectively.

Figure 6

MiR-29c-3p mimics treatment aggravates DSS-induced mice colitis. DSS-treated mice were intracolonically introduced with miR-29c-3p mimics. Mice were euthanatized 7 days after DSS treatment and the severity of colitis was evaluated. (A) Changes of body weight of mice of different groups are shown as the percentage of initial weight. ***Indicates P<0.001, DSS group versus control group. ^###Indicates P<0.001, DSS+PEI/miR-29c-3p mimics group versus DSS+PEI/miR-NC group. (B) Colon gross morphology of different groups. (C) Colon length in the indicated groups. (D) The disease activity index (DAI). (E) Macroscopic evaluation of colon inflammation. X200 Magnification. (F) The histological scores. (G) The protein levels of IL-1β, IL-6, IL-8 and TNF-α in the colon tissues of mice in different groups. ** and *** indicates P<0.01 and 0.001, respectively.