Advances in microRNA and inflammatory bowel disease and their related mechanisms

CHEN Mu-li, TIAN Yuan-yuan, SUN Xiao-ning

1. Department of Gastroenterology, Hainan Affiliated Hospital of Hainan Medical University, Haikou 570311, China

2. Hainan Medical University, Haikou 571199, China

Keywords:

ABSTRACT Inflammatory bowel disease (IBD) is a complex and multifactorial disease characterized by chronic inflammation of the gastrointestinal tract, mainly manifested by the accumulation of immune cells and pro-inflammatory cytokines in the intestinal mucosa.It is a kind of immune digestive system disease with high incidence in humans and can be divided into ulcerative colitis (UC) and Crohn's disease (CD).The pathogenesis of IBD is complex, and numerous studies have shown that genetic, environmental, microbial, immune, autophagy and other factors may be involved in the pathogenesis of IBD.MicroRNAs (miRNAs) play an important role in the pathophysiology of IBD.Studies have confirmed that miRNA play an important role in the targeted regulation of intestinal barrier homeostasis, immune response, and intestinal epithelial autophagy.MiRNA have not only been confirmed as important diagnostic biomarkers for IBD.It also shows new prospects for treatment strategies for IBD.This article mainly describes the differences in miRNA expression between UC and CD, summarizes the relationship between miRNA and intestinal barrier, immune homeostasis and autophagy mechanism in the pathogenesis of IBD, and the research progress of miRNA involved in the diagnosis and treatment of IBD, so as to provide new insights for the development of IBD.

Inflammatory bowel disease (IBD) is a complex multifactorial disease characterized by chronic gastrointestinal inflammation,which is mainly manifested by the accumulation of intestinal mucosal immune cells and proinflammatory cytokines.It is a highly prevalent immune digestive system disease in humans.There are two main types of IBD: Crohn’s disease (CD) and ulcerative colitis (UC).The lesion of CD is characterized by an unevenly distributed transmural inflammation pattern, which can affect multiple intestinal walls in the gastrointestinal tract and is usually limited to the terminal ileum and colon.The lesions of UC are limited to the mucosa and submucosa and may be accompanied by signs such as cryptitis and crypt abscess.IBD has a high incidence in young people and can cause bloody diarrhea, abdominal pain,malabsorption, fatigue, and decreased quality of life[1].Prolonged inflammation will also increase the risk of colorectal cancer in IBD patients, and the mortality of IBD related colorectal cancer(CRC) accounts for 10～15% of the annual mortality of IBD[2].The pathogenesis of IBD is complex, including genetic, environmental,microbial, immune, autophagy and other factors may be involved in the pathogenesis of IBD, among which immune response disorders,intestinal microbiota and intestinal barrier destruction play a dominant role.Studies have shown that microRNAs (miRNAs)play an important role in the pathogenesis of IBD, and can even distinguish between UC and CD.As a small RNA molecule that regulates gene expression, it has different expression in IBD and plays an important role in targeting the signaling pathways that regulate intestinal barrier homeostasis, inflammatory response and intestinal epithelial autophagy.This review mainly describes the differences in miRNA expression between UC and CD, summarizes the relationship between miRNA and intestinal barrier, immune homeostasis and autophagy mechanism in the pathogenesis of IBD,and the research progress of miRNA involved in the diagnosis and treatment of IBD, so as to provide new insights for the development of IBD.

1.Molecular biological mechanisms of miRNA

miRNA is a kind of evolutionarily conserved single-stranded noncoding RNA with a length of about 18～24 nucleotides.By cutting specific target mRNA and binding to the 3’ untranslated region(UTR), 5’ untranslated region or part of translated region of target mRNA, miRNA inhibits mRNA translation and blocks its expression[3], and is the main regulator of cell function and homeostasis.The canonical biosynthetic pathway begins with pri-miRNAs,which are produced by transcription of miR genes in the nucleus by RNA polymerase II.The RNA-binding protein DGCR8 binds to ribonuclease III called Drosha to form a protein complex that further processes pri-miRNAs.Precursor miRNAs (pre-miRNAs)are generated, which are brought to the cytoplasm via exportin-5/RanGTP complex, processed by the endonuclease Dicer, and cleaved into miRNA duplexes[4,5].One of the single strands combines with RNA-induced silencing complex to become a mature miRNA,which acts on the 3’-UTR of the target gene and plays a role in post-transcriptional regulation, participating in cell differentiation,proliferation, metabolism, death and other processes, and playing an important role in inflammation and tumor development[6].Many literatures have reported the correlation between miRNA and cancer,proving that it can affect the occurrence, proliferation, invasion and metastasis of cancer[7-9].The occurrence of most diseases is related to the interference of gene expression caused by miRNA dysregulation, especially in chronic inflammatory diseases (such as IBD and cancer)[10].

2.Differential expression of miRNAs in IBD

In 2008, WU et al.[11] confirmed that miR-192 was down-regulated in active UC intestinal mucosa compared with healthy intestinal mucosa, and was involved in the pathogenesis of UC by targeting macrophage inflammatory peptide-2α, which was the first discovery of abnormal regulation of miRNA in IBD.He pioneered the field of miRNA expression research in IBD.There are abnormal expressions of miRNAs in the intestinal tissue, peripheral blood, feces and saliva of IBD patients.UC and CD can be distinguished by specific miRNA profiles.

Guz et al.[12] used RT-PCR to detect five microRNAs (miRs-155-5p, -21-3p, -31-3p, -146a-3p, -125b-1-3p) and E-cadherin (CDH1)gene in the intestinal mucosa of IBD patients and normal people.The results showed that the expression of the above five miRNAs was significantly up-regulated in the inflammatory bowel tissues of UC, while miRs-21-3p, -146a-3p and miR-155-5p were highly expressed in the inflammatory mucosa of CD patients, while the expression of CDH1, a transmembrane glycoprotein essential for maintaining intestinal epithelial homeostasis, was down-regulated.The up-regulation of miR-155-5p in CD affects the stability of intestinal mucosa by down-regulating the expression of CDH1,and is involved in the pathogenesis of CD.The expression of miR-146a-3p in UC patients is negatively correlated with the duration of the disease, indicating that the main role of miR-146a-3p is to monitor the progression of the disease, and has no significant role in distinguishing UC from CD.Chen Hanwen[13] used density gradient centrifugation to isolate mononuclear cells in peripheral blood of 164 experimental subjects (CD=87, UC=38, healthy subjects =39),extracted RNA from them for miRNA sequencing, and found that the expression of miR-542-5p and miR-582-5p was significantly increased in IBD.It is suggested that it is of great significance for the diagnosis of IBD.However, miR-96-5p plays a major role in monitoring the disease activity of UC and CD.miR-21 is closely related to UC.It has been found that the expression of Mir-21 is upregulated in the blood, feces and colon tissue of UC patients, and promotes the progression of intestinal inflammation[14-16].The upregulation of miR-21 in UC is also related to disease activity[16].When UC develops into CRC, the expression of plasma miR-21 is further increased, and its sensitivity and specificity to distinguish UC from CRC are 93.5 and 100%, respectively.It indicates that the measurement of miR-21 expression level plays an important role in the clinical evaluation of whether UC lesions are cancerous[14].More and more studies at home and abroad have proved the importance of miRNAs expressed in blood for assessing the activity of IBD lesions.Chen et al.[17] found that the expression of serum miR-146b-5p in UC and CD was 2.87 times and 2.72 times higher than that in healthy control group, which can evaluate IBD disease activity and is more specific than C-reactive protein.Cordes et al.[18] found that compared with IBD patients in remission and healthy people, the expression of miR-320a in blood samples of UC and CD in active stage was increased, and the expression of miR-320a was closely related to the activity of endoscopic lesions, which could detect the activity of IBD.From the above studies, it is not difficult to see that the expression of miRNA in IBD intestinal mucosa and blood is specific.The abnormal expression of miRNA in intestinal mucosa is usually involved in the pathogenesis of IBD by targeting gene expression, while most miRNAs in blood are found to be involved in the monitoring of disease activity.If it is applied to clinical practice, it can reduce unnecessary invasive examination and has considerable development prospects.

The expression of miRNA in fecal tissues is also different in IBD.Zhou et al.[19] used microarray analysis and in situ hybridization to find that five miRNAs(miRs-15a-5p, -16-5p, -21-5p, -338-5p and miR-483-5p) were overexpressed in feces of IBD patients.Two miRNAs (let-7i-3p and miR-326) were down-regulated, miR-16-5p was up-regulated in UC and CD fecal tissues, while only miR-21-5p was up-regulated in UC patients, which has clinical diagnostic significance.Verdier et al.[20] used NanoString technology to detect fecal miRNAs in IBD patients, including fecal tissues of Clostridium difficile infection (CDI), and detected a total of 150 miRNAs, among which miR-223 and miR-1246 expression levels were increased in active UC and CD groups.In active UC, the level is increased more significantly.However, the level of miR-1246 was higher and miR-223 was not in the feces of CDI patients; this is the first study to comprehensively screen fecal miRNAs in IBD.Schonauen et al.[15]also found that the expression level of miR-233 was higher in the fecal tissues of UC and CD patients than in the control group, and miR-223 was up-regulated by more than 67 times in the feces of active UC patients.Therefore, miR-223 was considered as a potential fecal biomarker to identify disease activity and remission with a sensitivity of 80%.The specificity was 93%.Wohnhaas et al.[21]detected 9 miRNAs up-regulated and 8 miRNAs down-regulated in the fecal tissues of CD patients.miRs-192-5p, -375 and miR-141-3p were correlated with clinical activity index (CDAI) and endoscopic severity index (CDEIS) of CD.These results indicate that fecal miRNA is involved in the pathophysiological mechanism of CD and can detect disease progression.Stool can be in direct contact with intestinal epithelium, and miRNA can affect the progression of IBD by regulating the proliferation, differentiation and apoptosis of intestinal epithelial cells.Therefore, the diagnostic accuracy of IBD diagnosis by detecting miRNA in stool may be higher, and stool samples are easy to collect, as a non-invasive means, which can greatly reduce the suffering of patients.As a new biomarker of inflammatory bowel disease, miRNA has great clinical potential.Understanding the molecular mechanism of its regulation of gene expression is crucial for the diagnosis and treatment of IBD.The application of miRNA expression regulation research in IBD may provide a new way for its disease progression.

3.Role of miRNA in the pathogenesis of IBD

In the first study to explore the changes of miRNA expression in canine IBD, it was found that the expression of miRNA in canine IBD colonic mucosa and serum was also significantly different,which was similar to that in human IBD, and played a regulatory role through different mechanisms, further indicating that miRNA was involved in the pathogenesis of IBD[22].Combined with the current literature reports, miRNA can regulate the differentiation and secretion of immune cells, affect the intestinal mucosal barrier function and the composition of intestinal flora, and regulate the autophagy response in the pathogenesis of inflammatory bowel disease, and can be used as an important biomarker in the diagnosis process of IBD, which is closely related to IBD[6,23,24].

3.1 miRNA and intestinal barrier function

The destruction of intestinal mucosal barrier is one of the most important factors in the pathogenesis of IBD.The tight junction between intestinal epithelial cells protects the body from antigen invasion, and miRNA can maintain the integrity of intestinal mucosa by affecting the tight junction of intestinal epithelial cells.Epithelial-mesenchymal transition (EMT) is not only involved in the pathogenesis of IBD, but also involved in tumor invasion and metastasis, which can cause the loss of tight junction between epithelial cells.A variety of miRNAs have been found to be involved in this mechanism, and then affect intestinal homeostasis.The miR-200 family is a typical miRNA that inhibits the formation of EMT, and miR-200b/c is closely related to the differentiation and proliferation of EMT[25].In addition, Mehta et al.[26] found that the miR-200 family was abnormally regulated in the process of CD intestinal fibrosis, and miR-200b showed a stronger anti-EMT function than miR-200a and miR-200c.By promoting the proliferation of intestinal mucosal epithelium (IECs) and inhibiting EMT, it can maintain the integrity of intestinal epithelial cells and improve intestinal fibrosis.Sun et al.[27] also found that miR-200b could increase ZO-1 levels by targeting HMGB3 and p-JNK pathways in IECs, leading to reduced apoptosis of IECs and protection of TJs structures.ZO-1 was found to be the first epithelial tight junction protein, which can regulate the integrity of TJs structure and effectively protect the intestinal barrier function[28].Therefore,miR-200b is extremely important in maintaining the stability of intestinal mucosal barrier function, mainly by regulating key structures of the intestinal mucosal epithelium.miR-21 is also involved in the regulation of EMT, and in contrast to miR-200b,it plays a role in weakening the intestinal barrier function.Guo Xiaogang et al.[29] found that the up-regulation of miR-21 in IBD colon tissue aggravated intestinal inflammation and could participate in the regulation of intestinal barrier function.Subsequently, Wang et al.[30] further studied its mechanism and found that miR-21 upregulated mTOR signaling by reducing the expression of PTEN in NCM460 cells.miR-21 antagonist was applied to the CD chronic intestinal fibrosis model, and the results showed that EMT was inhibited, intestinal inflammation was alleviated, and intestinal fibrosis was alleviated, indicating that this is a potential target for the treatment of CD intestinal fibrosis.

Zonulin, as a regulator that maintains tight junctions between cells, is highly associated with miRNAs.Overexpression of miR-122a increases the expression of NLRP3 mRNA and protein and down-regulates Zonulin level by targeting EGFR activity, thereby enhancing intestinal epithelial mucosal permeability, leading to increased antigen exposure and inflammatory response.In addition,it has been found that miR-122a has a negative effect on aggravating intestinal ischemia/reperfusion injury[31].The expression of HsamiR-375 is down-regulated in the intestinal mucosa of IBD patients.Down-regulation of Hsa-miR-375 induces the activation of NFκB and stimulates the production of pro-inflammatory cytokines by targeting the TLR4 pathway.It also reduces TJ proteins (such as ZO-1 and occludin) and destroys the integrity of the intestinal mucosal barrier.It promotes the disease process of IBD[32].

In summary, miRNA plays a key role in the mechanism of regulating intestinal barrier function in IBD and shows great clinical application prospects.However, most of the current studies focus on the mechanism, and a few studies have been applied to cell or animal models.Although considerable results have been achieved, larger sample size studies are still needed for clinical trials.

3.2 MiRNAs and immune homeostasis

The immune dysregulation of IBD is characterized by intestinal epithelial injury.Intestinal flora disorder and the infiltration of a large number of immune cells into the lamina propria lead to inflammation.Under the state of stress, the activation of lamina propria cells leads to immune cell overreaction, and the secretion of proinflammatory cytokines TNF, IL-1β, IFN-γ increases, which aggravates the expansion of inflammatory response[33].MiRNAs play an important role in cell development of the innate and adaptive immune systems and in regulating immune responses[34].When pathogens invade the body and are captured by immune cells using Toll-like or NOD-like receptors, the signal transduction pathway is activated, inflammatory cytokines are released, and inflammation occurs[35].MiRNAs can participate in this pathway to regulate the stability of intestinal immune function.

Nucleotide-binding oligomerization domain 2 (NOD2) is the first identified susceptibility gene associated with CD, and is frequently mutated in about one-third of CD patients.NOD2 is a member of the cytosolic NOD-like receptor (NLR) family, which is one of the important detection systems for sensing microbial invasion[36].Chuang et al.[37] found that miRs-192, -495, -512 and miR-671 in colon epithelial cell HCT116 could bind to the 3’utr site of NOD2 mRNA and regulate NOD2 expression to varying degrees.This is the first study to report that miRNAs regulate the expression of NOD2 and its signaling pathway.miR-192 is a key miRNA regulating the innate immune response of intestinal epithelial cells in the pathogenesis of IBD, mainly by inhibiting NOD2 activity and causing down-regulation of NF-κB expression in colitis cells.Inhibition of IL-8 and CXCL3 mRNA expression can improve intestinal inflammation.miR-146a also plays an important role in the pathogenesis of IBD, and its mechanism is related to TLR signaling cascade and NOD2 signaling pathway.Wang et al.[38]established a UC rat model by rectal injection of TNBS to explore the potential mechanism of miR-146a in regulating the pathogenesis of UC, and found that miR-146a was up-regulated in UC rats.After the intervention of miR-146a inhibitor, the colon surface of rats was smoother and tissue adhesion was reduced.Further studies suggest that its mechanism of action is mainly through inhibiting the expression of TLR4, MyD88 and NF-κB proteins, reducing the secretion of pro-inflammatory factors and relieving the symptoms of UC.In addition, Garo et al.[39] found that miR-146a could target RIPK2 to regulate NOD2 signaling pathway, inhibit IL-17 inducible factor production, and reduce IL-17 secretion in myeloid cells, and could target TRAF6 to inhibit tumorigenic IL-17R signaling pathway in intestinal epithelial cells.Through these two mechanisms, it can prevent colon inflammation and colorectal cancer, but the specific regulatory mechanism is still unclear.Based on the protective role of miR-146a in colon inflammation and tumors, administration of miR-146a mimics to DSS-induced colitis mice and CRC mice showed that the severity of intestinal inflammation and colon tumors in the mice was improved.In conclusion, miRNA can regulate the innate immune process of IBD by targeting Toll-like or NOD-like receptors, causing the interaction between pro-inflammatory and anti-inflammatory cytokines, thereby affecting the pathogenesis of IBD.miRNA as a target drug administration has a certain prospect in clinical practice, but its drug delivery mode and clinical effectiveness still need to be confirmed by more in-depth research.

Regulatory T (Treg) cells have the main function of inhibiting active T cells, and play an important role in regulating immune homeostasis and controlling excessive immune response[40].The expression of miR-155 in peripheral Treg cells of IBD patients is increased.The up-regulation of miR-155 leads to the reduction of follicular regulatory T cells and the activation of B cells by targeting CTLA-4 and inhibiting CTLA-4 expression, so that the overexpression of autoantibodies damages intestinal epithelial cells.This may be the cause of intestinal mucosal damage in patients with autoimmune IBD[41].T helper cells include Th1 and Th2 cells, which regulate cellular and humoral immunity by secreting different cytokines, respectively.Under normal circumstances, Th1 and Th2 cells are both antagonistic and synergistic with each other.The pathogenesis of autoimmune diseases is characterized by the imbalance of Th1 and Th2 cells.Wang Haiying et al.[42] confirmed that the level of miR-145 was negatively correlated with Th1/Th2 ratio.Compared with the control group, the expression of miR-145 was down-regulated in IBD patients, while the proportion of Th1 cells and Th1/Th2 ratio showed an upward trend, and the secretion of inflammatory cytokines increased.Therefore, miR-145 may promote the production of inflammatory factors by regulating Th1/Th2 immune balance in IBD, and further affect the progression of IBD.miR-223 has been shown to be a key regulatory molecule involved in innate immunity.It can inhibit the activation and function of immune cells in inflammatory bowel disease, and participate in the regulation of immune response and cytokine production[43].After administration of miR-223 mimics to mice with DSS-induced colitis, the expression of miR-223 was increased, while the levels of IL-6, gp130 and p-STAT3 were decreased, resulting in decreased secretion of the pro-inflammatory cytokines TNF-α, IL-6 and IL-17 and increased secretion of the anti-inflammatory factor IL-10.Furthermore, the intestinal inflammation of experimental mice was reduced, indicating that its anti-inflammatory mechanism may be related to the inhibition of IL-6/STAT3 signaling pathway[44].Although many studies have proved that miRNA can participate in the development of IBD by affecting the homeostasis of the immune system, the specific mechanism is still not clear, which not only needs more in-depth research to verify the theory, but also needs to further overcome the interference of related cytokines.

3.3 miRNA and autophagy

Autophagy is a cellular response, which belongs to a type II programmed cell death.It can degrade and recycle inteRNAl structures, play a protective role in regulating cellular homeostasis and inflammatory response, and has also been found to be associated with anti-tumorigenesis and inhibition of tumor progression[45,46].Defects in autophagy can lead to epithelial cell dysfunction and immune destruction, and play an important role in the pathogenesis of IBD[47].Macrophages play an important role in the inflammatory and immune cell layer of the intestinal submucosa.When the intestinal tract is invaded by foreign pathogens, macrophages play a phagocytic role and present the phagocytic pathogens to immune cells for the next step of clearance.However, under pathological conditions, macrophages frequently stimulate autophagy response,and excessive immune response will cause intestinal dysfunction.Two autophagy regulators, including receptors, receptor regulators and inflammasome regulators, have been widely studied in the treatment of IBD, and have achieved good results in animal experiments.However, no clinical studies have reported their successful use in clinical practice, and their mechanism of action needs to be further explored[48].

miRNA is closely related to the regulation of autophagy mechanism in inflammatory bowel disease.Related autophagy susceptible genes include ATG16L1, NOD2, IRGM, etc., which can control autophagy response to regulate intestinal inflammatory response in IBD.miRNA can also cause activation or inhibition of intestinal autophagy by regulating NF-κB and mTOR signaling pathways,thereby affecting the release of inflammatory factors[49].ATG16L1 is an autophagy-related gene that forms autophagosomes during autophagy and is associated with an increased risk of IBD[50].It has been reported that the target gene of miR-142-3p is ATG16L1, and up-regulation of miR-142-3p reduces ATG16L1 mRNA and protein expression[51].Another study found that, The expression of miR-142-3p is up-regulated in the ileum tissue of vitamin D deficient mice and the colon tissue of IBD with low vitamin D expression, and the autophagy response is weakened mainly by reducing the level of ATG16L1.This is the first in vitro experiment confirming that miR-142-3p targets ATG16L1 to regulate the autophagy pathway[52].ATG2B is also an autophagy-related gene, and its expression in intestinal tissues is negatively correlated with miR-143.miR-143 can target ATG2B to reduce the level of LC3-II in CD, thereby inhibiting the activation of autophagy in intestinal epithelial cells.Further studies showed that overexpression of miR-143 or lack of ATG2B in intestinal tissues activated the NF-κB pathway, resulting in the release of pro-inflammatory cytokines (IFN-γ, TNF-α and IL-8)and the reduction of anti-inflammatory cytokines (IL-10), indicating that miR-143 could regulate autophagy and participate in the pathogenesis of CD.It has important enlightenment for the treatment of CD[53].

Intestinal microbiota is the main environmental driver of IBD,which maintains the health of the body by regulating the balance of the intestinal environment.Pathogenic microbiota can invade intestinal epithelial cells to destroy the permeability of intestinal mucosa, change the composition of intestinal flora, and increase the expression of pro-inflammatory factors to induce the occurrence of inflammatory response.It can also participate in the pathogenesis of IBD by regulating autophagy in combination with miRNA[54].Fusobacterium nucleatus (Fn), a gram-negative anaerobes, is a risk factor for UC.Fn can promote intestinal inflammation by releasing miRNA in exosomes.miR-574-5p has been confirmed to be downregulated in the colon tissues of DSS-induced colitis mice and UC patients.Fn will further down-regulate the expression of miR-574-5p, mainly by participating in the miR-574-5p/CARD3 axis, leading to the up-regulation of CARD3 and enhancing the autophagy response, thereby promoting colitis.Therefore, the autophagy response by inhibiting this process or inhibiting miR-574-5p/CARD3 axis may be a therapeutic target for UC in the future[55].In the intestinal mucosa of CD patients infected with adhesive-invasive Escherichia coli (AIEC), elevated miR-30c and miR-130a were transferred to recipient cells through exosomes secreted by intestinal epithelial cells, targeting ATG5 and ATG16L1 and reducing their expression, resulting in the inhibition of autophagy.In turn, it promotes further intracellular replication of AIEC[56].Although many studies have found that many miRNAs have the function of autophagy regulation and can participate in the pathogenesis of IBD through a certain mechanism, the development of specific autophagy regulators for different mechanistic pathways has theoretical support for the treatment of IBD, but further research is needed to confirm the importance of autophagy pathway in the pathogenesis of IBD.And the clinical utility of autophagy modulators.

4.Clinical application of miRNA in IBD

4.1 MiRNAs are involved in the diagnostic process of IBD

At present, the diagnosis of inflammatory bowel disease mainly depends on the collection of medical history, clinical manifestations,endoscopy, pathology, imaging examination, and can also draw blood to detect CRP, PCT, ESR and other indicators to determine disease activity.It is difficult to diagnose IBD if relying on clinical manifestations, which is because the extraintestinal manifestations are various and most of them are not typical.Endoscopy is still a reliable diagnostic method for distinguishing organic or functional gastrointestinal lesions.However, endoscopy is an invasive procedure, which not only has the risk of bleeding and perforation, but also requires a long time to wait for pathological results after examination, which increases the burden of patients.As an emerging specific target, miRNA has been proved to be an important diagnostic biomarker for IBD.The expression of miRNA in peripheral blood, saliva and feces is relatively stable, and it can also be used as a sensitive and specific marker for the pathogenesis,prognosis and remission of the disease[57].The study of Guo Yan et al.confirmed that miR-146a could be involved in the pathogenesis of inflammatory bowel disease, because they found that compared with healthy people, the level of miR-146a in peripheral blood mononuclear cells of IBD patients was significantly increased, and it was also related to the activity and severity of IBD[58].Later, the diagnostic value of miR-146b-5p in IBD has also been proved,and studies have found that its diagnostic sensitivity is similar to CRP, and its specificity is higher[17].In recent years, a systematic review of clinical studies on miRNA as diagnostic markers for IBD evaluated the diagnostic accuracy of miRNA through metaanalysis, and found that miRs-16, -21, and -192 were up-regulated in the blood of IBD patients, showing high diagnostic accuracy in distinguishing healthy people from IBD patients[59].miRNA can not only help in the diagnosis of IBD, but most importantly, its role in judging disease activity.Studies have found that the expression level of miR-145 in peripheral blood mononuclear cells (PBMCs) of IBD patients is lower than that of normal group, and the expression level of miR-145 in active IBD is lower than that in remission IBD,indicating that Mir-145 may be related to the progression of disease activity[42].Given the high diagnostic accuracy of miRNA in IBD,how to detect it? At present, the traditional methods used to detect miRNA include RT-qPCR, microarray analysis, Northern blot,etc.RT-qPCR is a relatively simple and mature miRNA detection technology, which is widely used in various basic experiments,but its sensitivity and specificity are not optimistic.New detection methods include nano-material detection, nucleic acid amplification technology, fluorescence in situ hybridization technology, rolling circle amplification, enzyme-free amplification, strand displacement amplification, loop-mediated isothermal amplification, etc., which are improved compared with traditional methods, but also have disadvantages such as high cost and complex procedures[59,60].

Although there are many methods to detect miRNA expression, RTqPCR is still the main experimental technique from the perspective of current research at home and abroad.Yao et al.[61] used highthroughput sequencing to identify the differentially expressed miRNAs in UC mice, and the results showed that a total of 95 differentially expressed miRNAs were detected.Then, RT-qPCR was used to verify the expression of miRNA and corresponding mRNA in the intestinal tissues of UC mice.In addition to the RTqPCR method, Chen Hanwen[13] also adopted DNBSEQ technology carried out by rolling circle amplification, which is a secondgeneration sequencing technology and greatly reduces the error rate of detection.Lian et al.[62] isolated exosomes from the plasma of UC patients and healthy people, analyzed plasma exosomal miRNA by next-generation sequencing technology, and finally detected its expression in colonic epithelial cells by RT-qPCR, indicating that detection of plasma exosomal miRNA may become a new way for the diagnosis of IBD.However, there is still a lack of a unified quantitative standard for the detection of miRNA, especially for the detection of samples such as fecal tissue, and more standard and effective methods are needed.Although miRNA in the form of exosomes is more stable, the technical problems of dealing with exosomes are also challenging.

4.2 MiRNAs are involved in the treatment of IBD

As for the treatment of IBD, traditional methods include aminosalicylic acid preparations, glucocorticoids and immunosuppressants, etc.Since then, new drugs have been studied,such as biological agents.Anti-TNF-α preparations have been effective so far, and miRNA therapy has great clinical potential as a new targeted drug[63].It mainly includes miRNA inhibitor/antagomir and miRNA mimics, which will lead to the inhibition or overexpression of miRNA expression and cause different degrees of intestinal inflammation.miRNA agomir is an upgraded product of mimics.After special chemical modification, miRNA Agomir can be directly used in cell and animal experiments, and its expression is more stable and lasting[64].Given the ability of miRNAs to regulate multiple gene targets at the same time, and the importance of miRNAs in the pathophysiology of IBD confirmed by in vitro studies, changing or even controlling miRNA expression may be a new strategy for future therapeutic development.

Zhao et al.[65] found that the expression of miRNA-130a-3p was increased in TNBS-induced colitis in mice.After the administration of miRNA-130a-3p inhibitor, the secretion of pro-inflammatory factors was reduced to improve the degree of intestinal inflammation in CD, and the mechanism was related to the reduction of NLRP3 inflammasome activity by autophagy.In contrast, administration of miR-130a-3p mimics aggravated intestinal inflammation.Mannosemodified trimethylchitosan (MTC) coupled miR-146b nanoparticles(NPs), referred to as mtc-mir146b, is a dual-target molecular targeted immunotherapy drug.After oral administration of mtc-mir146b, the mice with DSS-induced colitis showed weight recovery and colon length increase.MTC-mir146b can selectively target intestinal macrophages to promote intestinal mucosal repair and intestinal epithelial cell regeneration, and also inhibit the occurrence of colitisassociated colon cancer (CAC), which can be used as a potential target for oral administration in the treatment of UC and CAC[66].Pan et al.[67] injected miR-146b agomir into DSS-induced colitis mice through the tail vein and found that miR-146b overexpression could down-regulate its target gene FGL2, resulting in the downregulation of NLRP3 expression and the inhibition of NF-κB and MAPK pathways, thereby improving the symptoms and pathological damage of IBD.Exosomes containing high levels of miR-29b were also injected into IBD mice through the tail vein.The results showed that the expression of brain-derived neurotrophic factor (BDNF)in the heart tissue of IBD mice was inhibited, leading to cardiac dysfunction.BDNF plays an important role in the circulatory system and its expression level is reduced in UC.Reducing the expression of miR-29b may prevent the damage of IBD to cardiac function.This study has proved the mechanism of miRNA in regulating the function between the intestine and the heart in IBD[62].Tian et al.[68] embedded miR-31 mimicry particles into microspheres and administered them through enema into the large intestine of DSSinduced colitis mice.The results showed that the body weight and colon length of mice increased, intestinal epithelial cell proliferation increased, and colon inflammation was reduced.Dendritic cells(DC) are one of immune cells, and immune cell dysfunction is an important mechanism in the pathogenesis of IBD.Lin et al.[69] found that miR-144/451 inhibited DC activation and reduced colon inflammation.More severe intestinal inflammation; They administered chitosan nanoparticle capsules containing either miR-144/451 vector or control vector daily intraperitoneally to mice with DSS-induced colitis.They found that miR-144/451 nanoparticles reduced colonic inflammation in mice compared with the control group.Regulating the expression of miR-144/451 and DC activation in IBD may be a new approach for the treatment of IBD in the future.

In summary, the study of miRNA therapy for IBD has been used in many animal experiments, and its therapeutic effect has been repeatedly confirmed.The administration methods include intravenous, oral, enema and intraperitoneal injection, etc., which can all have an effect on intestinal inflammation.For example,production may be more complicated and lead to high cost.The mechanism of miRNA targeted therapy is not yet fully understood,and more in-depth studies are needed to verify its feasibility,effectiveness and safety from clinical use.We should also focus on how to further reduce the side effects of drugs.

5.Summary

Inflammatory bowel disease (IBD) is a multifactorial disease that is related to environmental, genetic, immune and microbial factors.However, recent studies have shown that dysregulated gene regulation due to miRNA dysregulation plays an important role in the pathophysiology of IBD.Many mirnas are involved in the complex regulatory system of intestinal inflammation.The expression of mirnas in intestinal mucosa, peripheral blood and feces can be used as a means of diagnosis of IBD, which can help to distinguish the type of disease, determine its activity and severity.A number of studies have shown many new therapeutic targets, which are expected to improve the clinical efficacy and prognosis of IBD.Although a number of studies have shown that miRNA is specifically expressed in IBD and can be used as a new biomarker to accurately diagnose IBD, miRNA has a variety of different phenotypes and targets, and its mechanism, pathway and function are complex.The most critical is the high risk of off-target of miRNA therapy.At present, the research is mainly focused on the basic research of cell and animal models.If it is really applied to clinical diagnosis and treatment, it faces a great challenge, and more standardized clinical data are needed to improve the timeness and efficacy.Studying the role of miRNA in IBD will help clarify the mechanism of disease activity, further promote the development of IBD diagnosis and treatment, optimize the prognosis of the disease and improve the quality of life.Larger scale studies are needed to verify the clinical feasibility of miRNA before clinical practice, and we look forward to its better utility in the future.

Author’s contribution

Chen Muli: Read literature and write papers; Tian Yuanyuan: The literature was collected and supplemented; Sun Xiaoning:Thesis guidance and proofreading.

All the authors declare no conflicts of interest.