MiR-34c and PlncRNA1 mediated the function of intestinal epithelial barrier by regulating tight junction proteins in inflammatory bowel disease

doi:10.1016/j.bbrc.2017.01.115

Biochemical and Biophysical Research Communications

Volume 486, Issue 1, 22 April 2017, Pages 6-13

https://doi.org/10.1016/j.bbrc.2017.01.115 Get rights and content

Highlights

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Overexpression of PlncRNA1 protect intestinal epithelial injury that induced by DSS.
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MiR-34c targeted MAZ, while tight junction proteins ZO-1 and occluding were regulated by MAZ.
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PlncRNA1 and miR-34c bound together to regulate the expressions of MAZ and tight junction proteins.
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The protect effects of PlncRNA1 overexpression on intestinal epithelial barrier was reversed by overexpression of miR-34c.

Abstract

Background

Inflammatory bowel disease (IBD) is originated from uncontrolled inflammation, and desired methods for IBD therapy remains the main difficult. The network comprised with miRNA and lncRNA has been verified to play an important role on diverse human diseases. In this study, we demonstrated the role of miR-34c and lncRNA PlncRNA1 on the function of intestinal barrier.

Methods

Intestinal epithelial barrier model was constructed based on normal intestinal epithelial cell line Caco-2. 2% DSS was supplemented in the Apical side of the model cells to induce the injury of intestinal epithelial barrier. Real-time PCR or western blot was used to determine mRNA or protein expression of miR-34c, PlncRNA1, Myc-associated zinc finger protein (MAZ), zonula occludens 1 (ZO-1) and occludin.

Results

DSS induced injury of intestinal epithelial barrier, while overexpression of PlncRNA1 seemed to protect intestinal epithelial barrier from injury. Tight junction (TJ) proteins ZO-1 and occludin were regulated by MAZ, while, miR-34c targeted MAZ to regulate its expression, in addition, PlncRNA1 and miR-34c bound together to regulate the expressions of MAZ, ZO-1 and occludin. The protect effects of PlncRNA1 overexpression on intestinal epithelial barrier function was reversed by overexpression of miR-34c.

Conclusion

MAZ and TJ proteins were involved in the function of intestinal epithelial barrier, while miR-34c and PlncRNA1 regulated the intestinal dysfunction cooperatively.

Introduction

Inflammatory bowel disease (IBD) is a class of intestinal diseases with the character of loss intestinal barrier function [1]. However, intestinal mucosal barrier has been verified to play an important role in gastrointestinal microflora. Previous study have suggested that the main role of intestinal mucosal barrier was to prevent the invasion of pathogens [2], while, the intact of intestinal mucosal barrier would bring much invasive bacteria, and break the balance of gastrointestinal microflora. Among this, tight junctions (TJ) is an essential permeable intercellular barrier and regulates the function of intestinal barrier [3]. Several TJ proteins like zonula occludens 1 (ZO-1) and occludin often considered to regulate intestinal permeability. However, the deregulation TJ proteins might also cause unbalance of gastrointestinal microflora. IBD has been identified to originate from this unbalance, and the disease often affects people during their toddler period, and contributes to a reduction of life quality and irreversible negative outcomes such as death [4]. Patients suffering from this disease often develop disabling complications within 5–10 years' diagnosis because of their uncontrolled inflammation [5]. A current evidence suggested that lack of desired methods for IBD therapy was the main difficult for the patients. Therefore, found a promising treatment is necessary.

Recently, increasingly evidences supported that noncoding RNA (ncRNA) potentially regulating most coding genes in human genome, and usually involved in diverse biological functions as well as human diseases. MicroRNAs (miRNAs) are a class of RNA with the length of ∼22 nt. Presently, miRNAs have been verified to bind to 3'UTR of it's target genes to regulate variety biological processes, such as immune process, cell apoptosis, inflammation reaction, oxidative stress. Unlike miRNA, long noncoding RNAs (lncRNAs) are a set of ncRNA with the length of more than 200 nt, and with less sequence conservation [6]. Now, large amounts of lncRNAs have been well identified and used to regulate gene expression, such as HOTAIR [7], [8], MALAT1 [9], [10]. Study has supported that both miRNAs and lncRNAs are belong to the ncRNAs, and in most situation, they regulate genes expression and several biological processes cooperatively [11].

There have been reports previously that the well-orchestrated regulatory interaction networks in our body were always involved in diverse ncRNA regulations, for example, miRNA-miRNA interaction, miRNA-mRNA interaction, lncRNA-miRNA interaction [12], [13], [14]. Among those, the widespread network lncRNA-miRNA interaction was largely explored, where ncRNAs regulate the expression of RNA by binding the protein coding messengers. For example, lncRNA HULC regulated the onset and progression of hepatocellular carcinoma by binding to miR-372, both of them formed the regulatory network cooperatively [15]. LncRNA MD1 combined with miR-133 and miR-135 to regulate the muscle differentiation [16].

This study we performed lncRNA PlncRNA1 and miR-34c to study whether they regulate intestinal barrier function of IBD cooperatively, and our aims were to explore the potential mechanism underlying the onset and progression of IBD.

Section snippets

Cell line and culture

The normal intestinal epithelial cell line Caco-2 was purchased from iCell Biotechnology Co., Ltd (China). The cells were cultured in RPBM 1640 medium supplemented with 10% fetal calf serum and maintained at 37 °C with 5% CO₂ in a humidified atmosphere.

Intestinal epithelial barrier model construction and Trans-epithelial electrical resistance (TER) determination

Caco-2 cells were planted in a Transwell system for monolayers, an epithelial voltohmmeter ERS-2 (Merck Millipore) was used for measurement of TER values. TER was measured until the similar values were occurred on three consecutive measurements.

Overexpression of PlncRNA alleviated the injury of intestinal mucosal barrier

To construct the intestinal mucosal barrier model, the Caco-2 cells with TER value more than 500 Ω cm² was recognized as successfully establishment. Then the model cells were divided into control group and LV-PlncRNA1 transfected group (LV-PlncRNA1 group). According to the result, overexpressed PlncRNA1 significantly decreased TER value compared with the control (Fig. 1A). While DSS supplementation significantly decreased TER values in both control and PlncRNA1 group. Moreover, the TER value in

Discussion

Intestinal barrier has the ability to delete and prevent exogenous substrates, while the intact barrier often brings much invasive pathogens. Studies have reported that the enterocyte of TJ plays an important role in intestinal barrier. From this study, we explored how the TJ proteins affected intestinal barrier function, and explored the mechanism between them.

Recently, studies have reported that various of miRNAs were involved in the IBD to regulate its occurrence and development. For

Conflict of interest

All authors declare that there is no conflict of interest.

Acknowledgements

This study was supported by Wenzhou Science&Technology Bureau (No. Y20160026) and Zhejiang Provincial Natural Science Foundation (No. LY17H030010).

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The rat weight, liver index, liver and colon phenotype, and serum biochemical indexes showed that QDT could significantly reduce liver and intestine injury and inhibit the progress of ACLF and endotoxemia. Toluidine blue staining and cytokine indexes showed that QDT could inhibit the activity of MCs and reduce the release of inflammatory factors. Mechanistically, QDT can inhibit the activity of MCs, activate miR-34c/MAZ/TJs pathway and miR-122a/Zonulin/EGFR pathway in colon, promote the recovery of intestinal barrier homeostasis, reduce and restore the damage of endotoxemia.
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Deficiency of microRNA-10b promotes DSS-induced inflammatory response via impairing intestinal barrier function
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Citation Excerpt :
To investigate the effect of miR-10b on systemic inflammation, we examined the serum levels of cytokines and showed that the serum levels of IL-1β (Fig. 4F), IL-6 (Fig. 4G), and TNF-α (Fig. 4H) pro-inflammatory factors were significantly upregulated, suggesting that miR-10b participated in DSS-induced systemic inflammation in mice. Dozens of miRNAs, such as miR-126 [19], miR-340 [20], and miR-34c [21], miR425 [22], have been reported to regulate the inflammatory response and the pathological process of IBD. However, the effect of miR-10b on IBD has never been studied.
Inflammatory bowel disease (IBD) is a non-specific inflammatory disease of the intestine with the pathogenesis to be largely unknown. We found that microRNA (miR)-10b knock-out mice displayed mild IBD symptoms, suggesting that miR-10b may be involved in the onset and development of IBD. This study focuses on elucidating the role of miR-10b in IBD.
The colitis model was induced by feeding the mice with 2.5% dextran sodium sulfate (DSS), and the expression levels of miR-10b in colon tissue and blood samples were examined. The severity of colitis was assessed by disease activity index, colon length, histopathological damage, intestinal permeability and ELISA. Then, after transfection of Caco-2 cells with miR-10b mimic and inhibitor, qRT-PCR was used to detect the expression levels of intestinal barrier related genes in colon tissues and cells.
miR-10b levels were significantly reduced in mice with DSS-induced acute colitis. Compared with wild-type (WT) mice, miR-10b knockout mice were more sensitive to DSS-induced colitis characterized by increased inflammatory cell infiltration and more severe disruption of colonic barrier function. In addition, by inhibiting miR-10b and thus increasing intestinal barrier gene expression in Caco-2 cells, we found that miR-10b suppressed inflammatory responses and enhanced intestinal barrier function both in vivo and in vitro.
miR-10b inhibits the inflammatory response in DSS-induced acute colitis mice in vivo and enhances intestinal barrier function in vitro, suggesting that miR-10b plays a key role in the developmental process of IBD. Thus, miR-10b may be expected to be a new target for the treatment of IBD.
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Increasing studies have confirmed that lncRNAs play critical role in the modulation of inflammation and intestinal epithelial cell function and diseased intestinal barrier.12–14 Lnc-PlncRNA1 (prostate cancer-up-regulated long noncoding RNA 1, also known as CBR3-AS1) was reported to protect intestinal epithelial barrier through modulating tight junction proteins.15 LncRNA-DANCR (differentiation antagonizing non-protein coding RNA) plays an important role in a variety of tumors, and the level of LncRNA-DANCR is increased in patients with colorectal cancer.16
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LncRNA-DANCR was up-regulated in DSS-induced groups of rats. TNF-α, IL-1β and IL-6 expression was significantly increased in DSS-induced groups of rats and cells. Zo-1 and MUC2 expression levels were decreased in DSS-induced groups of rats. Silencing LncRNA-DANCR reduced inflammation, cell apoptosis and up-regulated ZO-1, MUC2 and Claudin-1 in DSS-induced cells. MiR-125b-5p was the downstream LncRNA-DANCR target. All LncRNA-DANCR effects in the colitis model were reversed by the miR-125b-5p inhibitor.
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LncRNA-DANCR está involucrado en la inflamación y es uno de los mayores contribuyentes al cáncer de colon. Los efectos y el mecanismo de LncRNA-DANCR se investigaron por primera vez en el modelo de colitis inducido por DSS in vivo e in vitro.
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Atractylodis macrocephalae polysaccharides protect against DSS-induced intestinal injury through a novel lncRNA ITSN1-OT1
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Citation Excerpt :
Understanding of the probable association between them is still at an initial stage. Similar to RAMPtp induced ITSN1-OT1, overexpression of the lncRNA PlncRNA1 appeared to defend the intestinal epithelial barrier from damage stimulated by DSS [21]. On the contrary, LncRNAs also have the main part in controlling the intestinal epithelial barrier activity by varying TJ proteins expression, such as lncRNA SPRY4-IT1, which reduce the stability of mRNAs encoding TJ proteins and influence their translation [47].
Many dietary polysaccharides have been shown to protect the intestinal barrier integrity against several noxious stimuli. Previously, we have isolated a polysaccharide RAMPtp from Atractylodis macrocephalae Koidz, and analyzed its structure. However, the effects of RAMPtp on intestinal barrier function have not been investigated. Here, we evaluated the protective effects of RAMPtp on Dextran sulfate sodium (DSS)-induced intestinal epithelial cells (IECs) injury. The findings showed that RAMPtp boosted the proliferation and survival of IECs during DSS stimulation. Furthermore, we found that RAMPtp protected the IECs from injury induced by DSS through maintaining the barrier function and inflammation response. Mechanistically, we identified a novel lncRNA ITSN1-OT1, which was induced by RAMPtp during DSS stimulation. It blocked the nuclear import of phosphorylated STAT2 to prevent the DSS induced decreased expression and structural destroy of tight junction proteins. Hence, the study clarified the protective effects and mechanism of polysaccharides RAMPtp on DSS-induced intestinal barrier dysfunction.
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MiR-34c and PlncRNA1 mediated the function of intestinal epithelial barrier by regulating tight junction proteins in inflammatory bowel disease

Highlights

Abstract

Background

Methods

Results

Conclusion

Introduction

Section snippets

Cell line and culture

Intestinal epithelial barrier model construction and Trans-epithelial electrical resistance (TER) determination

Overexpression of PlncRNA alleviated the injury of intestinal mucosal barrier

Discussion

Conflict of interest

Acknowledgements

Am. J. Pathol.

Clin. Gastroenterol. Hepatol. Off. Clin. Pract. J. Am. Gastroenterol. Assoc.

Trends Mol. Med.

Biochem. Biophys. Res. Commun.

Cell

Cell. Signal.

Urol. Oncol.

Cancer Lett.

Nutr. Res.

Functional bowel symptoms in quiescent inflammatory bowel diseases: role of epithelial barrier disruption and low-grade inflammation