MicroRNA-16 inhibits the TLR4/NF-κB pathway and maintains tight junction integrity in irritable bowel syndrome with diarrhea

Abstract

Irritable bowel syndrome with diarrhea (IBS-D) is a chronic and relapsing inflammatory disorder in which pathogenesis has been shown to be in part the result of miRNA-mediated signaling. Here, we investigated the alleviatory role of miR-16 in IBS-D. First, we established an IBS-D mouse model using colonic instillation of acetic acid and developed an IBS-D cell model using lipopolysaccharide exposure. The experimental data demonstrated that miR-16 was underexpressed in the serum of IBS-D patients, as well as in the colorectal tissues of IBS-D mouse models and lipopolysaccharide-exposed intestinal epithelial cells. Next, miR-16 and TLR4 were overexpressed or inhibited to characterize their roles in the viability and apoptosis of intestinal epithelial cells, inflammation, and epithelial tight junction. We found that miR-16 overexpression increased the viability of intestinal epithelial cells, maintained tight junction integrity, and inhibited cell apoptosis and inflammation. We showed that miR-16 targeted TLR4 and inhibited the TLR4/NF-κB signaling pathway. Additionally, inhibition of NF-κB suppressed the long noncoding RNA XIST, thereby promoting enterocyte viability, inhibiting apoptosis and cytokine production, and maintaining tight junction integrity. In vivo experiments further verified the alleviatory effect of miR-16 on IBS-D symptoms in mice. Taken together, we conclude that miR-16 downregulates XIST through the TLR4/NF-κB pathway, thereby relieving IBS-D. This study suggests that miR-16 may represent a potential target for therapeutic intervention against IBS-D. Irritable bowel syndrome with diarrhea (IBS-D) is a chronic and relapsing inflammatory disorder in which pathogenesis has been shown to be in part the result of miRNA-mediated signaling. Here, we investigated the alleviatory role of miR-16 in IBS-D. First, we established an IBS-D mouse model using colonic instillation of acetic acid and developed an IBS-D cell model using lipopolysaccharide exposure. The experimental data demonstrated that miR-16 was underexpressed in the serum of IBS-D patients, as well as in the colorectal tissues of IBS-D mouse models and lipopolysaccharide-exposed intestinal epithelial cells. Next, miR-16 and TLR4 were overexpressed or inhibited to characterize their roles in the viability and apoptosis of intestinal epithelial cells, inflammation, and epithelial tight junction. We found that miR-16 overexpression increased the viability of intestinal epithelial cells, maintained tight junction integrity, and inhibited cell apoptosis and inflammation. We showed that miR-16 targeted TLR4 and inhibited the TLR4/NF-κB signaling pathway. Additionally, inhibition of NF-κB suppressed the long noncoding RNA XIST, thereby promoting enterocyte viability, inhibiting apoptosis and cytokine production, and maintaining tight junction integrity. In vivo experiments further verified the alleviatory effect of miR-16 on IBS-D symptoms in mice. Taken together, we conclude that miR-16 downregulates XIST through the TLR4/NF-κB pathway, thereby relieving IBS-D. This study suggests that miR-16 may represent a potential target for therapeutic intervention against IBS-D. Irritable bowel syndrome (IBS) is a chronic and relapsing bowel disorder affecting 11% of the global population with notable disease burdens, for example, decreased productivity and reduced life quality (1Lovell R.M. Ford A.C. Global prevalence of and risk factors for irritable bowel syndrome: a meta-analysis.Clin. Gastroenterol. Hepatol. 2012; 10: 712-721.e4Abstract Full Text Full Text PDF PubMed Scopus (1446) Google Scholar). IBS is characterized by chronic abdominal discomfort and alternations in frequency and appearance of stool (2Drossman D.A. Functional gastrointestinal disorders: history, pathophysiology, clinical features and Rome IV.Gastroenterology. 2016; https://doi.org/10.1053/j.gastro.2016.02.032Abstract Full Text Full Text PDF Scopus (1356) Google Scholar). Accordingly, IBS is further classified into three subtypes: IBS with diarrhea (IBS-D), IBS with constipation, and IBS with a mixed bowel pattern. Among these subtypes, IBS-D accounts for a quarter to a half of all IBS cases (1Lovell R.M. Ford A.C. Global prevalence of and risk factors for irritable bowel syndrome: a meta-analysis.Clin. Gastroenterol. Hepatol. 2012; 10: 712-721.e4Abstract Full Text Full Text PDF PubMed Scopus (1446) Google Scholar, 3Xu X.J. Liu L. Yao S.K. Nerve growth factor and diarrhea-predominant irritable bowel syndrome (IBS-D): a potential therapeutic target?.J. Zhejiang Univ. Sci. B. 2016; 17: 1-9Crossref PubMed Scopus (27) Google Scholar). The pathogenesis of IBS-D remains to be fully understood. Multiple etiological factors and triggers have been identified, including genetic susceptibility, visceral hypersensitivity, increased mucosal permeability, and altered gut microbiology (4Camilleri M. Peripheral mechanisms in irritable bowel syndrome.N. Engl. J. Med. 2012; 367: 1626-1635Crossref PubMed Scopus (253) Google Scholar). Emerging evidence has highlighted the significance of miRNAs as potential biomarkers against IBS (5Moein S. Vaghari-Tabari M. Qujeq D. Majidinia M. Nabavi S.M. Yousefi B. MiRNAs and inflammatory bowel disease: an interesting new story.J. Cell. Physiol. 2019; 234: 3277-3293Crossref PubMed Scopus (50) Google Scholar). Martinez et al. (6Martinez C. Rodino-Janeiro B.K. Lobo B. Stanifer M.L. Klaus B. Granzow M. et al.miR-16 and miR-125b are involved in barrier function dysregulation through the modulation of claudin-2 and cingulin expression in the jejunum in IBS with diarrhoea.Gut. 2017; 66: 1537-1538Crossref PubMed Scopus (98) Google Scholar) analyzed the differentially expressed miRNAs in IBS-D patients by RNA sequencing and identified miR-125b-5p and miR-16-5p as the most downregulated miRNAs in the context of IBS-D. The aforementioned findings encouraged us to undertake miRNA-involving IBS-D studies. miRNAs refer to short strands of RNA with the length of about 22 nucleotides that function as guide molecules to knock down the target mRNA and downregulate the corresponding proteins (7Ha M. Kim V.N. Regulation of microRNA biogenesis.Nat. Rev. Mol. Cell Biol. 2014; 15: 509-524Crossref PubMed Scopus (3953) Google Scholar). Moreover, miRNAs were matured in the cytoplasm and can be transported by extracellular vehicles to the neighboring cells or into the circulatory system (8Vishnoi A. Rani S. MiRNA biogenesis and regulation of diseases: an overview.Methods Mol. Biol. 2017; 1509: 1-10Crossref PubMed Scopus (521) Google Scholar). Of note, miRNAs in the serum are recognized as potential targets for therapeutic agents for a variety of diseases. In this sense, we started an investigation from microRNA-16 (miR-16) in the serum of IBS-D patients and explored its downstream effectors. In silico prediction of this study identified toll-like receptor 4 (TLR4) as a putative target of miR-16. Interestingly, TLR4 is a pathogen-recognition receptor of inflammation, which is contributory to IBS (9Brint E.K. MacSharry J. Fanning A. Shanahan F. Quigley E.M. Differential expression of toll-like receptors in patients with irritable bowel syndrome.Am. J. Gastroenterol. 2011; 106: 329-336Crossref PubMed Scopus (173) Google Scholar). In addition, intestinal barrier function could be recovered by wogonin through inactivating TLR4-dependent NF-κB pathway (10Wang W. Xia T. Yu X. Wogonin suppresses inflammatory response and maintains intestinal barrier function via TLR4-MyD88-TAK1-mediated NF-kappaB pathway in vitro.Inflamm. Res. 2015; 64: 423-431Crossref PubMed Scopus (87) Google Scholar). Furthermore, NF-κB signaling pathway has been involved in as and chronic inflammation, and has a role to in IBS B. et expression through NF-kappaB in a model of irritable bowel syndrome with chronic visceral J. Gastroenterol. 2015; PubMed Scopus Google Scholar, X. L. B. T. et of against irritable bowel syndrome via NF-kappaB and signaling 2015; PubMed Scopus Google Scholar). Moreover, has been that NF-κB signaling the expression of M. X. J. XIST epithelial cell inflammatory response via 2019; Scopus (98) Google and XIST was to the IBS L. W. to and chronic in the Sci. S. A. PubMed Scopus Google Scholar). we in the study that miR-16 may in of which may the In to the role of miR-16 in we the miR-16 expression in the serum from IBS-D shown in miR-16 was underexpressed in the IBS-D We developed an IBS-D mouse IBS-D were with decreased and reduced stool and abdominal of the were IBS-D with increased in and of the colorectal that the of IBS-D was decreased in to the mice. in the IBS-D was the mice. by and the cytokine was in the intestinal of IBS-D and of and were to be in IBS-D to the mice. was to study the of the or were that the IBS-D mouse model was The of miR-16 in the mouse colorectal was by the and miR-16 was to be expressed in the IBS-D mice. Moreover, the serum of miR-16 in IBS-D was to be downregulated as with that in Taken together, data demonstrated the of miR-16 in IBS-D patients and mice. the effect of miR-16 on the intestinal epithelial cells, we established the lipopolysaccharide IBS-D model in the colonic epithelial cell X. S. C. et the inflammatory response of intestinal epithelial cells by the Sci. PubMed Scopus Google miR-16 was to the miR-16 expression in the intestinal epithelial cells was to the miR-16 the miR-16 expression and and that cell viability in the intestinal epithelial cells was and the apoptosis was the cells, which could be in response to miR-16 by and expression of and was in the intestinal epithelial cells. miR-16 suppressed and tight junction proteins and were downregulated in the IBS-D cell and their were the cells were with miR-16 as verified by the tight junction was by as by reduced and miR-16 to tight junction data the that overexpression of miR-16 enterocyte viability, inhibited apoptosis and cytokine production, and maintained tight junction integrity. The downstream modulation factors of miR-16 were by The of the downstream was analyzed by and by as in were in the of the in of the and the and TLR4 further the modulation pathway, we analyzed the pathway the using The TLR4 and IBS was by (10Wang W. Xia T. Yu X. Wogonin suppresses inflammatory response and maintains intestinal barrier function via TLR4-MyD88-TAK1-mediated NF-kappaB pathway in vitro.Inflamm. Res. 2015; 64: 423-431Crossref PubMed Scopus (87) Google Scholar). The of miR-16 and TLR4 was from the and by and miR-16 reduced the was in TLR4 and miR-16 and the miR-16 to TLR4 mRNA on the The expression of NF-κB in the colorectal of the IBS-D model was by which were to be expressed in the IBS-D the expression of NF-κB and was by and in response to or of miR-16 in the colonic epithelial cell and miR-16 was NF-κB and were downregulated in the of miR-16 In miR-16 to the we that miR-16 inhibited the TLR4/NF-κB signaling pathway. of of miR-16 and and overexpression of miR-16 with TLR4 were on the cells. The of and and that miR-16 was in the cells with miR-16 expression of NF-κB and was of TLR4 the of NF-κB and in cells with miR-16 The of and miR-16 of NF-κB and in cells, as with miR-16 and and that of miR-16 the cell viability and the further overexpression of TLR4 the to miR-16 of and miR-16 the cell viability and the were by and and miR-16 the and and the of and which could be by of in the cells miR-16 further the and expression and the of and that the to tight junction was in response to miR-16 and the effect of miR-16 was by TLR4 overexpression miR-16 was with miR-16 inhibited the TLR4/NF-κB signaling pathway to enterocyte viability, to apoptosis and cytokine production, and to tight junction integrity. We the XIST expression in the colorectal of the IBS-D model using which that XIST was expressed in the IBS-D. with NF-κB for decreased the XIST the the role of NF-κB and XIST in we the cells with with or XIST overexpression data that the of NF-κB and in cells. The showed XIST was downregulated in response to in cells. and were to the cell viability and apoptosis in and cells in response to to the to viability and apoptosis in the cells, XIST overexpression the of and tight junction proteins were by and was found that the cell viability, the downregulated the and and the expression of and in XIST overexpression the aforementioned of of that the tight junction that been by XIST overexpression the tight junction effect of In inhibition of NF-κB suppresses XIST, thereby promoting enterocyte viability, apoptosis and cytokine production, and maintains tight junction integrity. the of on the cells, we overexpressed or with XIST, in the cells. The and showed that miR-16 expression of NF-κB and XIST in cells. XIST in in the of and NF-κB and were to the cell viability and apoptosis and and tight junction proteins were by and and miR-16 in increased cell viability, the suppressed the and and the expression of and and overexpression of XIST the of miR-16 overexpression the tight junction effect of miR-16 as by increased was miR-16 was with XIST The aforementioned data demonstrated that miR-16 inhibited the TLR4/NF-κB pathway to XIST, enterocyte viability, apoptosis and cytokine production, and maintained tight junction integrity. that miR-16 IBS through the XIST we overexpressed or with XIST, in the colorectal of the IBS-D mice. The of and and showed that miR-16 suppressed the NF-κB and XIST in IBS-D mice. overexpression of XIST in notable in NF-κB and XIST expression in the of miR-16 Next, we IBS-D with NF-κB and to the and expression of XIST and NF-κB was in the NF-κB and XIST was reduced in The of showed that NF-κB inhibited the serum of and in IBS-D In addition, found that with the IBS-D the inflammatory cell was reduced in the mucosal tissues of NF-κB IBS-D stool and of the were and in IBS-D with miR-16 showed decreased in and of XIST symptoms of colorectal and of and increased and suppressed and in the IBS-D with miR-16 the XIST the symptoms of mice. was to the of the intestinal inflammatory and in the was The of showed that the of cells was reduced in the of miR-16 the of which could be overexpression in we down with the that miR-16 inhibited the to IBS-D. IBS is a relapsing inflammatory disorder of the gastrointestinal that can to disease and of and and disease 2017; PubMed Scopus Google Scholar). that of miR-16 in and miR-16 was involved in barrier function dysregulation through the modulation of and cingulin expression in IBS (6Martinez C. Rodino-Janeiro B.K. Lobo B. Stanifer M.L. Klaus B. Granzow M. et al.miR-16 and miR-125b are involved in barrier function dysregulation through the modulation of claudin-2 and cingulin expression in the jejunum in IBS with diarrhoea.Gut. 2017; 66: 1537-1538Crossref PubMed Scopus (98) Google Scholar). of are on the mechanisms effect of miR-16 in the of IBS-D. findings that miR-16 inhibited the TLR4/NF-κB pathway to XIST, intestinal epithelial cell viability, the apoptosis and inflammatory and tight junction in IBS-D the regulation of miR-16 potential to and therapeutic for IBS-D. Emerging evidence has the of miRNAs in in which may be to intestinal and gastrointestinal symptoms R.M. S.K. of miRNAs in irritable bowel syndrome using a expression 2016; PubMed Scopus Google Scholar, for the irritable bowel Biol. 2011; PubMed Scopus Google Scholar). This study that miR-16 was underexpressed in the IBS-D patients and mouse has been identified in a study by et al. C. S. M. et al.miR-16 and receptor and with in irritable bowel 2017; PubMed Scopus Google which demonstrated that miR-16 was expressed in the jejunum of IBS-D that miR-16 overexpression enterocyte viability, apoptosis and cytokine production, and maintained tight junction in IBS-D. is that of miR-16 expression is with intestinal through its target (6Martinez C. Rodino-Janeiro B.K. Lobo B. Stanifer M.L. Klaus B. Granzow M. et al.miR-16 and miR-125b are involved in barrier function dysregulation through the modulation of claudin-2 and cingulin expression in the jejunum in IBS with diarrhoea.Gut. 2017; 66: 1537-1538Crossref PubMed Scopus (98) Google Scholar). a the targets were and have to be in silico we and verified TLR4 as a miR-16 study has the of miR-16 in through TLR4 F. Yu X. B. X. et al.miR-16 by TLR4 in 2019; PubMed Scopus Google Scholar). et al. S. B. M. The colonic of TLR4 and in patients with irritable bowel Med. 2016; PubMed Scopus Google Scholar) have found of TLR4 in IBS-D patients, which was to with disorder with We on to the downstream mechanisms and found that the effect of miR-16 was by inhibiting the TLR4/NF-κB signaling pathway. of miRNAs has been to intestinal inflammatory through the NF-κB signaling pathway. In X. S. J. W. S. et of may to pathogenesis of via NF-kappaB 2012; Scopus Google L. J. Liu et of in colonic epithelial experimental and 2011; PubMed Scopus Google and et from experimental by 2014; PubMed Scopus Google Scholar) and via the NF-κB signaling with is that miR-16 targets and TLR4 in inflammatory pathway the is by a W. C. J. X. the of on inflammatory 106: PubMed Scopus Google Scholar). et al. X. C. et of by toll-like receptor in diarrhea-predominant irritable bowel 2017; PubMed Scopus Google Scholar) that expression of TLR4 and NF-κB were in IBS-D with increased and factor the downstream of NF-κB was In is that TLR4 NF-κB to and visceral in an model of IBS B. et expression through NF-kappaB in a model of irritable bowel syndrome with chronic visceral J. Gastroenterol. 2015; PubMed Scopus Google Scholar). In to a of have the effect of miR-16 on through TLR4/NF-κB signaling F. Yu X. B. X. et al.miR-16 by TLR4 in 2019; PubMed Scopus Google Scholar, S. B. M. The colonic of TLR4 and in patients with irritable bowel Med. 2016; PubMed Scopus Google Scholar, X. X. J. Liu et mouse and Cell. Mol. Med. 2016; PubMed Scopus Google Scholar). miR-16 has been recognized as of inflammatory through the of TLR4 and X. X. Liu X. X. J. S. et are in the serum of patients and the inflammatory J. Med. 2015; Google Scholar). miR-16 can downregulate the expression of and inflammatory factors by thereby the in a model F. Yu X. B. X. et al.miR-16 by TLR4 in 2019; PubMed Scopus Google and with with decreased NF-κB as well as apoptosis T. F. T. et targeted to 2019; Full Text Full Text PDF PubMed Scopus Google Scholar). the study with in of miR-16 TLR4/NF-κB for the to the potential of miR-16 in this new that inhibition of NF-κB suppressed XIST to enterocyte viability, apoptosis and cytokine production, and tight junction integrity. XIST is a in the and maintains A. A. and new from Biol. 2015; PubMed Scopus Google Scholar). evidence has that of XIST the NF-κB pathway could in the XIST cell apoptosis and via in 2019; PubMed Scopus Google Scholar). The XIST expression in IBS-D has been We that NF-κB to the XIST in IBS-D. junction is as an as a barrier the of the and the extracellular C. C. from to Rev. Mol. Cell Biol. 2016; 17: PubMed Scopus Google and was identified in epithelial cells as an the tight junction of with is a of IBS-D In IBS-D intestinal and epithelial barrier function by and S. X. et intestinal in IBS-D by and Physiol. 2017; PubMed Scopus Google Scholar). be that in this we to NF-κB signaling pathway. is as a NF-κB W. X. W. Yu L. et a effect on cells by PubMed Scopus Google Scholar, D. T. X. Liu D. and by Res. PubMed Scopus Google is a and has been to roles in a variety of cell Kim et effect of on cell viability is by the regulation of and and Res. PubMed Scopus Google Scholar, S. B. T. of in cells, and in with J. 2014; PubMed Scopus Google Scholar, J. S. et and by Res. 2015; PubMed Scopus Google Scholar). mechanisms to enterocyte viability may further Of note, data demonstrated that overexpression of miR-16 the of and a of tight junction in epithelial cells, the which has been well established may be from three First, has been to through thereby affecting the of intestinal cells D. S. regulation of intestinal epithelial tight junction 2011; Full Text Full Text PDF PubMed Scopus Google and miR-16 has been with X. S. M. miR-16-5p and apoptosis in epithelial cells by J. Google Scholar). miR-16 may and on its with the by in colorectal can be to cells to the expression of and by factors X. F. J. et by and PubMed Scopus Google and miR-16 as a of F. L. M. et of biomarkers with the and of via 2019; PubMed Scopus Google may be that miR-16 and in a Moreover, have been in the of tight for to and C. C. tight junction proteins and barrier 2014; PubMed Scopus Google and miR-16 has been to a S. A. C. of through the receptor J. 2017; PubMed Scopus Google Scholar). is that miR-16 and through Moreover, has been that could be by and be the cell in the of C. M. is by as PubMed Google Scholar). a in response to the of in was a regulation by and S. A. and regulation of 2019; 15: PubMed Scopus Google and has further been that could be by in cells to D. and mucosal barrier in cells by the of Biol. 2019; PubMed Scopus Google Scholar). The aforementioned evidence that can be of the cells as a in and in vivo experiments have that miR-16 inhibited the TLR4/NF-κB pathway, XIST to intestinal epithelial tight junction and to the evidence in the study showed that miR-16 targeted TLR4 to NF-κB and the downstream XIST, in enterocyte viability, tight junction and and We that miR-16 may be a target for or IBS-D as well as intestinal by the in we have the of the miR-16 may in the extracellular or extracellular Moreover, the that miR-16 is the involved in the NF-κB pathway against IBS-D. miRNAs may target XIST, or in the pathway, which be in using to inflammation, including and may further the model and the Moreover, and are to of the effect of miR-16 on of inflammatory cells in tissues in its this study the significance of miR-16 in IBS-D. the patients and The study was by the of to of and with the of experiments were with of the of to of and in with the for the and of by the of The downstream of miR-16 were by The were by the were analyzed by and using The was for further were in the using the bowel and with the target using further the modulation pathway, we for the pathway was for of the for miR-16 and target from to of from to were in this including IBS-D patients and The of IBS-D patients and are shown in IBS-D patients were to the abdominal or abdominal for for the by the bowel or discomfort in in abdominal or discomfort IBS-D to the stool and for of the and for of the were for the and patients were Peripheral of the patients were was for this of serum were mixed with of and to for The was with of for and to for Next, the was for and the was of which was into a new and mixed with into the for the was with and for which was The was into the and for with the The was into a to was and to for by for The was the RNA and the RNA was using a RNA was using and into using and a was to from was with and system and as The was to the expression of are in a with were from the of to of The were into experimental and with in The IBS-D mouse model was established by acetic acid instillation of on the of diarrhea irritable bowel syndrome by 2012; Google Scholar, L. visceral in with diarrhea-predominant irritable bowel syndrome through of the signaling pathway via PubMed Scopus Google Scholar). for the were with the model were with acetic acid The model was instillation for The stool and gastrointestinal of were and were for visceral in were using experiments were the by the and of to of and the of The mouse model was L. and role of receptor in and in with irritable bowel Med. 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