The functional role of miRNAs in inflammatory pathways associated with intestinal epithelial tight junction barrier regulation in IBD

The intestinal epithelial barrier (IEB) consists of a single layer of epithelial cells connected by multiple intercellular junctions, including tight junction (TJ), adherent junction (AJ), and desmosome, which are the major protein complexes of the apical junctional complex (AJC) [6]. IEB acts as a physical barrier between luminal components and underlying tissue, limiting access of microbes, antigens, and toxins to the internal environment, while allowing ions, water, and nutrients to be absorbed [7]. Moreover, the epithelial cells play a significant role in bacteria phagocytosis, toxin neutralization, wound repair initiation, and innate and adaptive immunity activation [8]. Intercellular TJs are essential complexes that form a continuous sealed space of the IEB, guarding its proper functioning [7]. TJs are the apical complexes composed of transmembrane proteins: claudins (CLDN), occludin (OCLN), junctional adhesion molecules (JAMs); cytoplasmic scaffolding proteins: cingulin, zonula occludens (ZO) family, and regulatory proteins [9]. These dynamic structures are responsible for regulating epithelial permeability and integrity [9]. Loss of the intestinal epithelial barrier function, manifested by increased permeability or impaired integrity, leads to the disruption of intestinal homeostasis caused by exposure of luminal contents to the host's tissues. Permanent lasting intestinal barrier dysfunction combined with an aberrant immune response are both significantly involved in the development of the inflammatory process in IBD [7, 10]. The defective intestinal barrier is commonly observed in IBD patients’ colonic tissue, with the highest dysfunction for patients with active disease. It is directly correlated with persistence of intestinal inflammation [7, 11].

MiRNAs belong to noncoding and non-messenger RNAs, 18–25 nucleotide segments of single-stranded RNA, which are endogenously expressed and which play an important role in enzymatic, structural, regulatory functions by silence target mRNAs [12, 13, 14, 15]. The miRNA binds to complementary sequences of mRNA 3-untranslated regions (3-UTR). However, 100% complementarity to the 3-UTR of the mRNA is not required for the regulation of gene expression. Therefore 1 miRNA can target a lot of mRNAs [16, 17]. The human genome contains 1917 annotated hairpin precursors and 2654 mature sequences of miRNA [18]. They are transcribed from intergenic or intronic regions of DNA by RNA polymerase 2 [19, 20]. MiRNAs can be co-expressed with a gene, which they regulate, because often miRNA are clustered on chromosomes proximal to gene clusters [21, 22]. Essential miRNA's role in relevant biological processes such as cell differentiation, apoptosis, cell proliferation, and carcinogenesis has been proved in many diseases [23, 24, 25, 26]. In IBD miRNAs are involved in the Th1/Th17 immune response, inflammation processes, apoptosis, interleukin expression, intestinal barrier function, and many other processes. Therefore miRNAs are important molecules in the pathogenesis of IBD [27, 28]. Knowledge about the role of miRNAs in IBD is growing steadily, helping to understand the pathogenesis of IBD and creating an opportunity for new diagnostic and therapeutic possibilities.

In this review, we summarize and discuss the potential role of miRNAs in the regulation of inflammatory signaling pathways affecting the function of the intestinal epithelial barrier in IBD, with particular emphasis on the therapeutic potential. The aim of the review is also to determine the further development directions of the studies on miRNA potential in the modulation of the intestinal epithelial barrier in IBD.

Tumor necrosis factor alpha inhibitor protein 3 (TNFAIP3)/A20 is a negative regulator of NfkB [29, 30, 31]. TNFAIP3/A20 plays an important role in the maintenance of epithelial barrier integrity by OCLN stabilization at the tight junction [32]. R. K. Felwick et al. examined miRNA-23a expression in CD and its role in the pathophysiology of CD. They demonstrated that miRNA-23a, by targeting TNFAIP3 mRNA 3’UTR, caused altered IEB function. miRNA-23a promotes an increase in the transcriptional activity of NFkB, but only in the presence of TNFa. Overexpression of miRNA-23a with TNFa leads to significant impairment of the intestinal epithelial barrier function. Moreover, upregulated expression of miRNA-23a throughout increased NFkB activity contributes to the significant pro-inflammatory effect. In active and inactive Crohn's disease, patient's biopsy miRNA-23a expression was significantly up-regulated. miRNA -23a expression positively correlated with loose stool frequency. There were no significant differences between TNFAIP3 mRNA expression in a Crohn's disease donors’ group and a healthy cohort, but in encoded protein TNFAIP/A20 changes were observed. Using laser confocal microscopy (LCM) assayed for RNA strong immunostaining at the apical tight junction and subapical membrane regions of epithelial cells in healthy tissue was observed, while in active and inactive Crohn's disease tissue there was full or partial loss of staining in these cellular regions. In active and inactive Crohn's disease group, there was a significant decrease of TNFAIP3/A20 protein level at apical and subapical membrane regions [33].

Activin receptor-like type 1 (ALK1 or ACVRL1) is a cell-surface serine/threonine receptor for the TGF-beta superfamily of ligands [34]. Altered intestinal immunity associated with dysregulated TGF-beta signaling is known in the pathophysiology of IBD [35]. ALK1 is expressed in many tissues and cells, including human colonic intestinal epithelial cells (IECs) [36, 37, 38]. Toyonaga et al. examined the clinical impact of colonic ALK1 signaling dysregulation on intestinal barrier integrity and clinical outcomes in patients with CD. ALK1 signaling plays an important role in regulating NOTCH target gene expression, the stemness, and the differentiation of human colonic IECs. Decreased ALK1 expression observed in CD patients colonic tissue contributes to lower NOTCH activity, higher colonic IECs stemness, and inhibited colonic IECs differentiation. Moreover, reduced ALK1 signaling impairs IEC barrier integrity by down-regulating tight junction protein expression – exactly tight junction protein 1 (TJP1), CLDN8, OCLN. ALK1 protein expression is directly regulated by miRNA-31-5p and significantly negatively correlated with miRNA-31-5p expression, increased in CD patient's colonic mucosa. Thus, downregulated ALK1 expression is caused by miRNA-31-5p overexpression. Toyonaga et al. also revealed that the subset of CD patients with markedly lower ALK1 expression (low-ALK1 CD subset) than the other CD patients (hi-ALK1 CD subset) have an increased risk of surgical resection and endoscopic relapse [39].

Data shows lncRNA gene H19 can play an important role in the development of inflammatory diseases and cancer [40, 41]. Vitamin D receptor (VDR) is a nuclear receptor whose signaling pathways play a significant role in the regulation of inflammation and carcinogenesis in different tissues through the mediation of the functions of 1,25(OH)2D3 [42, 43, 44, 45]. Investigations showed that 1,25(OH)2D3 protects the intestinal barrier, with injuries induced by destructive reagents and decreased VDR signaling perhaps playing an essential role in pathogenesis mechanisms of UC [46, 47, 48, 49, 50, 51]. Chen et al. investigated the expression and correlation of H19, VDR, and miRNA-675-5p in UC and miRNA-675-5p-induced effects on epithelial barrier [52]. In Caco-2 cells, miRNA-675-5p mimics transfection-caused significantly increased paracellular permeability, decreased TEER, and expression level of ZO-1, OCLN, and VDR. In contrast, miRNA-675-5p inhibitor co-transfection importantly impaired monolayer barrier disruption in Caco-2 cells with H19 upregulated expression, validating the important role of miRNA-675-5p in the effects of H19. Moreover, overexpression of H19 importantly correlated with decreased expression of VDR, which was also significantly impaired by miRNA-675-5p inhibitors. H19 overexpression caused decreased mRNA levels of tight junction proteins and miRNA-675-5p inhibitors impaired lower expression level of ZO-1. In UC tissues, related to healthy tissues, notably decreased VDR expression levels were observed, as well as significantly increased expression levels of H19 and miRNA-675-5p. Besides, miRNA-675-5p expression negatively correlated with the expression level of VDR. Chen et al. also proved that the direct target of miRNA-675-5p is VDR mRNA [52]. MiRNA-675-5p is transcribed from the first exon of H19 and the function of H19 can be modulated by that miRNA in many diseases [41, 53].

Aryl hydrocarbon receptor (AhR) belongs to the basic helix-loop-helix (bHLH) superfamily of ligand-inducible transcription factors, which are associated with cellular responses to environmental stimuli. AhR signaling modulates the development, function, and production of different cell types in the gastrointestinal tract and has an important role in the regulation of the immune response in IBD. AhR was implicated in several intestinal pathologies, such as intestinal inflammation, infection, and cancer [54]. The effect of AhR on the intestinal epithelial barrier may be directly or indirectly mediated through signaling with different cell types, including immune cells. AhR activation prevents intestinal barrier dysfunction by restoring the expression and distribution of the ZO-1, OCLN, and CLDN1 [55, 56]. AhR gene and protein expressions were downregulated in CD patients’ inflamed colonic tissue, but not in UC patients, compared with tissue from uninflamed areas and healthy group [57]. Zhao et al. revealed AhR is a target gene of miRNA-124a. In murine colitis models and Caco-2 cells overexpression of miRNA-124a led to decreased AhR expression and impaired intestinal barrier function. Meanwhile, miRNA-124a inhibitors restored TJ protein expression, such as ZO-1, OCLN, JAM-A, Claudin-1 enhancing intestinal barrier integrity. Expression of miRNA-124a in colon biopsy tissues from CD patients and in colon epithelial cells was markedly upregulated [58].

Myosin light chain kinase (MLCK) activated phosphorylation of the myosin II regulatory light chain (p-MLC) which is correlated with intestinal barrier tight junctions’ permeability [59, 60]. Colon cancer-associated-transcript-1 (CCAT1), an lncRNA oncogene promoting cancer cell proliferation, migration, and its over-expression, was observed in inflammatory colonic tissues [61, 62]. Ma et al. examined the interaction between miRNA-185-3p, CCAT1, MLCK, and IEB integrity. Increased MLCK activity and p-MLC expression in Caco-2 cells may disrupt intestinal barrier function through changes to normally smooth arc-like shapes ZO1 and OCLN to irregular undulations of ZO1 and OCLN. Higher activity of MLCK and inflammatory pathways is associated with overexpression of CCAT1, which was significantly increased in IBD tissues compared with normal colorectal tissues. The study indicated that CCAT1 may play an important role in the modulation of miRNA185-3p stability by acting as a miRNA sponge. MLCK and CCAT1 have the same miRNA-185-3p response elements in their RNA regions. The analysis revealed that the expression of miR-185-3p was negatively correlated with the expression of CCAT1 in IBD patients. Up-regulated expression of CCAT1 is disturbing the interaction between miRNA-185-3p and MLCK leading to disruption of IEB function caused by increased activity of MLCK. The investigation demonstrated that irregular undulations of ZO1 and OCLN, which were induced by CCAT1 overexpression or miRNA-185-3p inhibitors could be gradually decreased by miRNA-185-3p. The expression of miRNA-185-3p was significantly decreased in IBD biopsy tissue compared with tissues obtained from healthy patients [63].

Shen et al. proved MLCK and c-JUN are direct targets of miRNA-200b. In Caco-2 cells stimulated by TNF-alfa miRNA-200b treatment led to a significant a decrease in IL-8 expression compared with untreated cells. Moreover, overexpression of miRNA-200b induced decrease of phospho-JNK (p-JNK), phospho-c-Jun (p-c-Jun), and total-c-Jun expression levels likewise AP-1 activity. MiRNA-200b could inhibit IL-8 synthesis caused by TNF-a. During TNFa-induced epithelial barrier disruption, negative changes in TJ proteins distributions CLDN1 and ZO-1 exhibiting irregularly undulating shapes were observed. Moreover, CLDN1 partial removal into cytoplasmic vesicles was observed. The investigation showed miR-200b overexpression maintains TJ morphology and attenuates rearrangements of CLDN1 and ZO-1 induced by TNF-a. MiRNA-200b overexpression treatment significantly increased TEER and dramatically decreased paracellular permeability after TNF-a stimulation. MiRNA-200b overexpression caused decreased expression levels of MLCK and p-MLC in trials with or without TNF-alfa stimulation, so miR-200b can successfully inhibit the MLCK/p-MLC pathway and preserve TJ epithelial barrier function. Thus, upregulated expression of miRNA-200b suppresses the intestinal barrier disruption induced by TNF-alfa upregulated JNK/c-Jun/AP-1 signaling pathway [64].

IL-9 can modulate intestinal inflammation in animal models by regulating intestinal barrier function [65]. In DSS-induced colitis murine models, inhibitors of the IL9 impair inflammation [66]. Overexpression of IL9 was noted in UC tissue and a positive correlation was found of IL9 expression level with the activity of the disease [67]. Previous data demonstrated substantial down-regulation of CLDN8 in active IBD patients’ tissues; additionally, CLDN8 seems to be one of the most important claudins in the pathology of IBD and its expression was the most reduced compared to other claudins genes in IBD [68, 69, 70]. Li et al. proved upregulation of IL-9 in colonic tissues of CD patients compared to the expression level in the no-inflammatory colonic tissue. In Caco-2 cells treatment with IL-9 reduced TEER, and co-treatment with a neutralizing antibody against IL-9 (anti-IL-9) significantly increased the tight junction protein expression. In treated cells a reduction of mRNA expression of CLDN8, CLDN1, ZO-1, and OCLN were detected. In murine models and cell cultures, IL-9 stimulation demonstrated significant overexpression of miRNA-21 and down-regulated expression of CLDN8. The investigation by using a miRNA-21 mimic and miRNA-21 inhibitor gave expected results in cell models that confirmed miRNA-21 directly targets CLDN8. In biopsy tissues of CD patients plentiful expression of miRNA-21, compared to the control group, was marked. Moreover, the expression of CLDN8 was significantly reduced in CD tissues, while up-regulated in the control group. Treatment with miRNA-21 inhibitor in TNBS-induced colitis in mice attenuated inflammation and revised permeability of the intestine barrier and other cardinal signs of colitis and restored CLDN8 level [71].

Previous studies showed the IL23/Th17 axis engagement in the regulation of IBD [72, 73]. Wang et al. proved that the IL23 pathway is associated with dysfunction of the intestinal epithelial barrier by interaction with miRNA-223. In the murine colitis model, IL23 treatment resulted in a decreased expression level of CLDN8 compared to untreated control. The same results were observed in cell models; IL-23 stimulation caused CLDN8 down-regulation and miRNA-223 upregulation. Inversely, co-treatment with anti-IL23P19 significantly increased CLDN8 expression level and attenuated up-regulated expression of miRNA-223. Epithelial barrier functions featured by TEER were attenuated after IL-23 stimulation and enhanced in trials using co-treatment with anti-IL23P19, which suggests an important role of the IL-23 pathway in cell barrier integrity. The study revealed that endogenous CLDN8 is inhibited by miRNA-223 in colonic epithelial cells; miRNA-223 mimics decreased the expression of CLDN8 and miRNA-223 inhibitor increased CLDN8 expression. In UC and CD biopsy samples, significantly higher expression of miRNA-223 compared to expression in healthy colonic mucosa and down-regulated expression of CLDN8 were noted. Treatment with miRNA-223 in a murine model of TNBS-induced colitis caused reactivation of CLDN8 in colitis tissues, reduction of important colitis signs, and enhancement of the intestinal epithelial barrier integrity. Thus, inhibitors of miRNA-223 could play a therapeutic role in IBD [68].

IL-21 plays an essential role in many processes involved in the progression of IBD, including interferon (IFN)-y production, Th1 cell differentiation [74, 75] promotion, and activation of transcription factors such as STAT1 and STAT3 regulation [76]. Wang et al. revealed IL-21 signaling pathway involvement in IEB disruption in IBD. They confirmed that IL-21 controls the level of CLDN5 by miRNA-423-5p, and 3’UTR of CLDN5 is a direct target of miRNA-423-5p. Up-regulated expression of miRNA-423-5p could attenuate CLDN5 level and miRNA-423-5p inhibitors can restore the level of CLDN5, both in Caco-2 cells and in a mouse model of acute colitis. CLDN5 transfection into Caco-2 cells restored the intestinal barrier function after forced miRNA-423-5p mimic stimulation by increasing epithelial barrier integrity and decreasing paracellular permeability. Therefore miRNA-423-5p could control the intestinal barrier function by targeting CLDN5. Moreover, miRNA-423-5p inhibitor treatment in a mouse model of acute colitis reduced inflammation by down-regulating the expression of IL1B, IL6, TNF-a and inhibiting phosphorylation of the components of the NF-kappaB/MAPKs/JNK signaling pathway. MiRNA-423-5p is regulated by IL-21; IL-21 induces expression of miRNA-423-5p, and IL-21 neutralizing antibody blocked the expression of miR-423-5p. In colonic tissue from UC patients, miRNA-423-5p and IL-21 expression level were abnormally increased compared to the healthy group [77].

Interleukin-1B is a proinflammatory cytokine increased in the course of IBD. IL-1B contributes to disruption of intestinal epithelial barrier function, playing an essential role in the development of IBD. The decrease of intestinal barrier tight junction integrity during IL-1B stimulation is partially caused by the up-regulated activity of MLCK induced by IL-1B [78, 79, 80]. Rawat et al. investigated the influence of IL-1B on IEB TJ permeability and TJ protein expression, and examined predicted miRNA to IL-1B-induced effects. Physiological concentration of IL-1B in Caco-2 cells leads to down-regulation of OCLN expression and increases TJ permeability. There was a strong correlation between the decreased OCLN expression level induced by IL-1B and the increased TJ permeability. IL-1B caused a significant increase of miRNA-200c-3p expression, while inhibitors of miRNA-200c-3p prevented OCLN down-regulated expression caused by IL-1B and increased TJ permeability. In the murine colitis model, IL-1B stimulation also caused a significant increase of miRNA-200c-3p expression, a decrease in OCLN mRNA level, and increased intestinal permeability. Inhibitors of miRNA-200c-3p transfected in vivo inhibited the up-regulated expression of miRNA-200c-3p induced by IL-1B, decreased OCLN expression, and inhibited impaired intestinal barrier integrity [81]. The pre-miRNA-200c-3p transfection caused miRNA-200c-3p overexpression [82], decreased OCLN mRNA level and degradation of intestinal tissue OCLN protein thereby enhanced intestinal barrier disruption in murine intestine. In a murine colitis model up-regulated expression of IL-1B, miRNA-200c-3p, and decreased OCLN expression was noted. Treatment with miRNA-200c-3p inhibitor resulted in inhibition of miRNA-200c-3p expression, a decrease of OCLN mRNA, and an increase of colonic permeability. In colonic tissue from active UC patients, a significant up-regulated miRNA-200c-3p and IL-1B expression was marked compared to the healthy colonic tissue from patients after colonic resection for noninflammatory conditions [81].

The B-cell translocation gene (BTG)/TOB family of antiproliferation proteins are responsible for the regulation of genotoxic and cellular stress signaling pathways, cell differentiation, apoptosis, cell-cycle progression, gene expression, and also they are associated with protection mechanisms in oncogenic transformation [83]. He et al. demonstrated that miRNA-301A expression is significantly up-regulated in the peripheral blood mono-nuclear cells and colorectal tissues of IBD patients [84]. Early data showed that miRNA-301A could act as an NFkB activator in pancreatic cancer cells [85]. He et al. also examined the regulation of intestinal inflammation and epithelial barrier integrity by miRNA-301A [86]. BTG1 is a direct target of miRNA-301A; up-regulated miRNA-301A expression caused significantly down-regulated BTG1 mRNA expression in human cells. Overexpression of BTG1 caused inhibition of IL-1B-induced c-Jun phosphorylation and NFkB activation. Therefore miRNA-301A probably promotes NFkB activation by targeting BTG1. In IBD patients the levels of IEC-derived BTG1 are negatively correlated with miRNA-301A expression. In murine colitis model with MiRNA-301A gene knockout observed a smaller increase of epithelial permeability, IL-1B, IL-6, IL-8, TNF IEC production compared with the control colitis group and increased E-cadherin expression level in epithelia. In cell models, up-regulated miRNA-301A expression led to an increase of epithelial permeability, enhancement of paracellular permeability, transepithelial electrical resistance (TEER) reduction, and down-regulation of E-cadherin expression level. A positive correlation was found between increased miR-301A expression in IEC of IBD patients and mRNA level of NFkB p65. Moreover, miRNA-301A influences the activation of NFkB signaling in IEC by promoting that signaling and increasing the level of NFkB-associated proteins like IL-1B, IL-6, IL-8, and TNF. MiRNA-301A up-regulated expression is significantly induced by IL-1B and gently induced by IL-6. In intestinal epithelial cells (IEC) isolated from IBD patients compared with healthy control group cells, miRNA-301A expression level was importantly increased and BTG1 expression was decreased, both in CD and UC patients’ cells. Also, there were significant differences between IEC isolated from the inflamed area and IEC from the healthy tissue area. In the IEC from uninflamed mucosa, miRNA-301A expression was similar to IEC from the healthy patients’ control group and BTG1 expression was also down-regulated in inflamed mucosa compared to unaffected parts of the tissue. Apart from this, He et al. found a positive correlation between the IL-1B levels in IBD patient's serum and miRNA-301A expression in IEC from CD and UC patients as IL-1B mRNA and miRNA-301A expression in IEC. These results reveal the role of IL-1B in increasing miR-301A expression in IEC of IBD patients. MiRNA-301A up-regulated expression caused by IL-1B can occur through the JNK MAPK-dependent pathway. The promoter of miR-301A could be bound by c-Jun, and c-Jun phosphorylation is strongly induced by IL-1B. Summarizing, miRNA-301A could play a part in intestinal epithelial barrier disruption in IBD. In colonic tumor tissues from patients with CAC, miRNA-301A is increased compared with healthy controls. MiRNA-301A could putatively function as an oncogene [86].

Signal transducer and activator of transcription 3 (STAT3) is a transcription factor that belongs to a seven-member family of proteins responsible for peptide hormone signal transduction from the cell surface to the nucleus [87]. STAT3 expression in UC is localized in mucosal epithelial cells, lamina propria mononuclear cells, as well as in neutrophils granulocyte. STAT3 activity is regulated by phosphorylation through Janus kinase-signal transducer and activator of transcription (JAK) [88]. In UC STAT3 up-regulated expression could inhibit anti-inflammatory cytokine expression [89]. X.-Q. Chu et al. proved that STAT3 is a direct target of miRNA-495. Up-regulated miR-495 expression caused decreased STAT3 and JAK expression level, but increased expression of CLDN1. Therefore miR-495 inhibits the JAK/STAT3 signaling pathway by directed targeting STAT3 contributing to the maintenance of intestinal epithelial barrier functions. STAT3 positive cells in intestinal mucosa were statistically significantly more frequent in the UC group compared with the normal group. STAT3 and JAK expression levels in UC tissue of mice were increased, and miR-495 and CLDN1 expression were decreased [90].

The epidermal growth factor receptor (EGFR) is a cell surface glycoprotein that binds the epidermal growth factor family of extracellular protein ligands. EGFR plays an important role in the activation of signaling pathways associated with cellular differentiation, proliferation, and survival [91]. Zhang et al. examined the role of miRNA-122a in regulating intestinal barrier function and revealed miRNA-122a overexpression could up-regulate the expression level of zonulin by directly targeting EGFR, leading to enhancement of intestinal permeability and inflammation in the murine model and Caco-2 cells [92]. Increased zonulin level associated with increased expression of miRNA-122a could be an indicator of intestinal permeability [93]. TNF-a and LPS treatment increase the miRNA-122a expression in cultured cells and enterocytes [94], whereas LPS treatment also elicits decreased expression of EGFR, CLDN1, OCLN, and increased zonulin expression [92]. Moreover, IL-6 and TNF-a expression in mice-miRNA-122a treatment group serum were both significantly increased compared to the control group. In colonic tissues of IBD patients, miRNA-122a and zonulin expression were significantly up-regulated relative to colonic tissues from the healthy group [92].

Toll-like receptor 4 (TLR4) is a transmembrane receptor responsible for inducing the secretion of inflammatory cytokines by NFkB activation [95]. In healthy tissue, TLR4 is uncommonly expressed, but in UC and CD patients colonic tissue shows marked TRL4 overexpression [96, 97]. Wu et al. examined TLR4 and miRNA-375 expression in colonic tissue from IBD patients and from the healthy control group. In the IBD patients, colonic tissue miRNA-375 expression was decreased and TLR4 expression was increased compared with the healthy control group. A significant negative correlation between miRNA-375 and TLR4 mRNA levels was revealed. TLR4 mRNA is a direct target of miRNA-375. miRNA-375 mimics transfection led to a decrease in TLR4 mRNA level, and miRNA-375 inhibitors gave the opposite effect in TLR4 expression. Moreover, miRNA-375 inhibition caused increased paracellular permeability, decreased TEER, but also apoptotic rate elevation in Caco-2 cells. MiRNA-375 could play a protective role in the intestinal epithelial barrier during inflammation throughout the mediation of the TLR4/NFkB signaling pathway. LPS stimulation in Caco-2 cells induced TLR4 overexpression and upregulated NFkB protein level, but in the presence of high-level miRNA-375, TLR4 expression was downregulated. Additionally in LPS-induced Caco-2 cells, miRNA-375 inhibition led to over-expression of TNF-a, IL-1B, IL-6, IL-8 and decrease in the relative expression level of ZO-1, OCLN, and IL-10 [98].

Protein phosphatase 2 (PP2A) is a major protein phosphatase holoenzyme that interacts with the TJ complex. The upregulated activity of PP2A causes dephosphorylation of the TJ proteins, ZO-1, OCLN, and claudin-1. In contrast, inhibition of PP2A induces the phosphorylation and recruitment of ZO-1, OCLN, and claudin-1. Therefore, the high activity of PP2A induces increased paracellular permeability and has an important role in the regulation of TJ function and assembly [99]. PP2A also can interact with JAM-A, and dephosphorylates JAM-A in the way of antagonization of aPKCζ-mediated phosphorylation of JAM-A at TJs [100]. Cell division cycle (Cdc)42 is a small GTPase of the Rho family that controls different cellular functions such as adhesion and transcription important to the formation of a functional intestinal epithelial barrier [101, 102, 103, 104]. Cordes et al. demonstrated probiotic E. Coli Nissle 1917 (EcN) treatment resulted in enhanced epithelial barrier integrity [105] and significantly upregulated expression of miRNA-320a, while enteropathogenic E. coli E2348/69 transfection caused the decrease of miRNA-320a level in T84 cells [5]. Moreover, both probiotic EcN and miRNA-320a significantly down-regulated PPA2 with its subunit R5B (PPP2R5B), Cdc42, and up-regulated the expression of JAM-A, whereas co-incubation with EPEC revealed inverse results. Trials with a high concentration of miRNA-320a co-incubated with EPEC revealed that miRNA-320a caused increased epithelial barrier integrity by attenuation of EPEC-induced PPP2R5B upregulation and JAM-A down-regulation. In murine colitis models, there was no correlation between disease activity and miRNA-320a tissue expression level; miRNA-320a was not significantly altered in DSS-induced colitis and not increased in T-cell transfer colitis, and colitic IL-10-/- mice. However, miRNA-320a is produced during proinflammatory stimulation, and as the elevated blood level of miRNA-320a positively correlates with the histological damage in murine colitis models, miRNA-320a may be used for monitoring of disease activity. In active CD patients, miRNA-320a blood concentration was significantly higher in relation to patients with CD in remission and the healthy group [106].