The Mechanism Of MiRNA In The Regulation Of Function Of Duodenal Mucosal In Patients With Functional Dyspepsia

Introduction:Functional dyspepsia(FD)is one of the most common gastrointestinal(GI)disorders and has a prevalence of up to 30%.The pathophysiology of FD is considered to be complex and multifactorial.Remarkably,duodenal immune activation plays a role in the pathogenesis of FD.Recent research is providing evidence of structural gut alterations,at least in certain subsets,mainly in functional dyspepsia(FD)and irritable bowel syndrome(IBS).These alterations are associated with increased permeability,which seems to reflect mucosal inflammation and neural activation.Enteric glial cells(EGCs)have been demonstrated to play a key role in maintaining intestinal epithelial barrier integrity.The glial cell line-derived neurotrophic factor(GDNF),which is usually secreted by EGCs,was identified to be a novel regulator of intestinal barrier functions.GDNF was shown to affect gut barrier properties in vivo directly or indirectly by largely undefined processes in a model of inflammatory bowel disease(IBD).GDNF signals through a multicomponent receptor system,composed of the RET(rearranged during transfection)receptor and one of the four GFR?(?1–?4)(GDNF family)receptors.Activation of the receptor complex in neurons can lead to modification of various signaling events,including p38 MAPK,ERK,protein kinase B(AKT),and PKA.Enterocytes express the GDNF receptor GFR?and RET receptor(GR).GDNF has effects on proliferation of enterocytes and thereby promotes epithelial wound healing.GDNF promotes tight junction differentiation and has anti-apoptotic in enterocytes.Additional barrier-protective effects can be assumed from the immunomodulatory effects by GDNF.A significant reduction of inflammation in a model of chronic inflammatory bowel disease after application of GDNF had been reported.It might partially associate with the role that GDNF blocked the secretion of various proinflammatory cytokines,such as TNF-and interleukin-1?.Micro RNAs(miRNAs)are non-coding small RNAs that mediate post-transcriptional gene regression by binding to complimentary sequences on to the 3?-untranslated region(3?-UTR)of the mRNA of a specific gene.Expression of GDNF can be regulated by microRNA(miRNA).Suppression of miR-383 may increase the therapeutic potential of human bone-marrow-derived MSCs in treating spinal cord injury via augmentation of GDNF protein levels.miR-383 inhibited protein translation of GDNF,through binding to the 3'-UTR of the GDNF mRNA..Overexpression of miR-211 suppresses the differentiation of bone marrow stem cells into intestinal ganglion cells by directly down-regulating the expression of GDNF.Recently,concentration of GDNF is reported to positively correlate with symptoms in the functional dyspepsia.Increased expression of duodenal GDNF might be involved in FD pathophysiology and symptom perception.GDNF was localized in enteric glial cells,eosinophils,and epithelial cells.Since the number of eosinophils was significantly greater in FD patients than in controls,the eosinophil count in duodenal biopsy specimens were measured in this study,in parallel with assay for GDNF and miR-211expression.Objective:The main purpose of this research plan is to identify the potential miRNA candidates involved in GDNF or p38 MAPK signaling during the pathogenesis of functional dyspepsia.In addition,the molecular mechanism of miRNA-mediated GDNF or SERT signaling will be elucidated in this study.Thus,the first step of the study will focus on the clinical identification of miRNA candidates involved in the pathogenesis of functional dyspepsia.The main study will clarify the molecular mechanism of miRNA-mediated GDNF or p38 MAPK signaling.Furthermore,the association between the specific miRNA and the risk of functional dyspepsia will be investigated.Methods:Duodenal biopsies from FD patients and control subjects(N=6 in each group)will be used to identify potential miRNA candidates in FD.Duodenal biopsies were obtained during routine endoscopy.In each person,biopsy specimen was fixed in 10%neutral buffered formalin,embedded in paraffin wax,sectioned at 5?m.Another set of biopsy specimen was snap-frozen and stored at-80°C until used for real time PCR.To examine the effect of miR-211 on the transcriptional regulation of GDNF,EGCs were transfected with miRNA inhibitors or miRNA mimics and the expression of miR-211,and GDNF were examined by using real-time PCR.Cells were divided into four groups,including control for mimic,miR-211 mimic,control for inhibitor,mimic inhibitor.Enteric glial cells(1×10~5cells)were plated onto 6 well plates in culture medium overnight.The following day,media were removed and cells were transfected with 10 n M mimic non-targeting control,and the relevant miRNA mimics or 50 n M inhibitor non-targeting control and the relevant miRNA inhibitors using lipofectamine 3000 transfection reagent according the manufacturer's protocol(Life Technologies).Cells were harvested for real-time PCR experiments at 48 h after transfection.The conditional media were collected and stored in-80°C or liquid nitrogen.The conditional media were used for ELISA assay.Increased GDNF mRNA expression does not directly indicate the level of secreted in a sustained GDNF.Next above conditional media were collected and GDNF were quantified by ELISA assay.To quantify secreted GDNF,24 h-conditioned media were obtained from each group of EGCs.The GDNF ELISA Immunoassay were purchased from abcam and were used as indicated by the manufacturer's booklet.To clarify the roles of GDNF on the wound healing and cell proliferation,and migration in small intestinal epithelium cells,EGCs were divided into six groups,including control for inhibitor,miR-211 inhibitor,control for mimic,miR-211 mimic,control for small interfering RNA(siRNA),GDNF siRNA.In GDNF siRNA group,expression of GDNF should be reduced.EGCs were cultured in DMEM containing 10%FBS until the cells reach 80%confluence.Transient transfection of GNDF siRNA,and miRNA inhibitors were performed using lipofectamie 3000.All transfections were carried out for24 hours and following the manufacturer's protocol.The culture medium was then replaced by serum-free DMEM and incubate for an additional 48h.The EGC conditioned medium(EGC-CM)were collected,centrifuged at165g for 5 min and filtered through a 0.22-?m syringe filter.The EGC-CMs were stored at-20°C.The IEC-6 monolayers were cultured with or without EGC-CM.To quantify secreted GDNF,EGC-CM were obtained from each group of EGCs.The GDNF ELISA Immunoassay were purchased from abcamand were used as indicated by the manufacturer's booklet.Wound-healing assays were performed on six-well dishes to measure cell migration in vitro.A standardized scratch were applied on an IEC-6 monolayer using the tip of a 100-?l pipette.The IEC-6 monolayers were cultured with EGC-CM from each group prior to wounding and were maintained an additional 72 h in culture.The wounded areas were photographed after 24,48,and 72 h.Wound closure were determined by the ratio of wounded area compares with the initial scratch area using Image J Software.Cell proliferation was determined using the CCK-8 Kit according to manufacturer protocols.Cells were trpsinized and replated for CCK-8 proliferation assay at 24 and 48 h after EGC-CM treatment.Briefly.we examined whether p38 MAPK is the upstream signaling to regulate p38activation in EGC-CM-treated IEC-6 cells.p38 MAPK were investigated in IEC-6 cells following EGC-CM application.Cells were divided into six groups as described.The cultured IEC-6 cells were grown in 6-well plates and treated as described before.Antibodies against phosphorylated p38MAPK(pp38 MAPK)and p38 MAPK were used at a dilution of 1:500.As secondary antibodies,the respective horseradish peroxidase-labeled goat anti-rabbit Ig G,(Santa Cruz Biotechnology,Heidelberg,Germany)were used(1:3,000 in PBS).Chemiluminescence signal detection and quantification were performed by densitometry.To investigate the clinical association between miR-211and the risk of functional dyspepsia,the samples from biopsy were used.Briefly,duodenal biopsies from 20 patients with functional dyspepsia and 20 controls were obtained during routine endoscopy.In each person,on set of biopsy specimen was fixed in 10%neutral buffered formalin,embedded in paraffin wax,sectioned at 5?m.Another set of biopsy specimen was snap-frozen and stored at-80°C until used for real time PCR.Statistical analysis:Continuous data were reported as mean±standard deviation.Data of 2 groups were compared using an unpaired 2-tailed Student's t test.All the statistical analyses were carried out using Graph Pad Prism v6 software.A value of p<0.05 was considered to indicate statistical significance.Results:1.In the duodenal mucosa of FD patients,mast cells and eosinophils had a higher tendency than that of healthy controls,and the difference of mast cells was more significant in the tissues of FD patients.GDNF was significantly lower than healthy controls,and the expression of mir-211 in the same tissues was significantly higher than that of healthy controls,which was consistent with the hypothesis.2.miR-211 suppresses GDNF mRNA expression in EGCs Transfection of EGCs with miR-211 mimic significantly increased miR-211 expression,and decreased the level of GDNF mRNA.Transfection of EGCs with miR-211 inhibitor significantly decreased miR-211 expression,and increased the level of GDNF mRNA,as compared with mimic and inhibitor controls.ELISA also indicated that transfection of EGCs with miR-211 mimic significantly inhibited the secretion of GDNF.The OD value at 450 nm decreased from 0.6305 before transfection to 0.6160 after transfection(p<0.05).On the other hand,transfection with miR-211 inhibitor significantly increased EGC GDNF secretion.The OD value at 450 nm increased from 0.6425 to 0.6525(p<0.05).3.Small intestinal epithelium cells(IEC-6)were cultured with conditioned medium(CM)collected from the cultures of EGCs treated in 6ways,as described in the methods.GDNF level in the different CM was measured by ELISA.The level of GDNF in CM collected from EGCs treated with miR-211 inhibitor was higher than in the control group,and the level of GDNF in CM collected from EGCs transfected with miR-211mimic was significantly lower than the control group(p<0.001).The level of GDNF in CM collected from EGCs transfected with GDNF siRNA was significantly lower than the control group(p<0.01).4.Results of the wound healing assay are shown in Figure 3-2.The level of healing in the miR-211 inhibitor CM group was relatively higher than that of the control group(215.0?47.4?m vs.205.8?37.3?m).The level of healing in the miR-211 mimic CM group was relatively lower than that of the control group(163.2?42.1?m vs.196.2?28.0?m).The level of healing in the GDNF siRNA CM group was relatively lower than that in the control group(165.3?34.1?m vs.185.3?54.1?m).5.GDNF level has no effect on IEC proliferation:Results of the cell proliferation assays are summarized in Figure 5.The data indicate that GDNF has no effect on cell proliferation.6.GDNF level is negatively correlated with p38 MAPK level:IEC-6cells cultured with CM collected from different experimental groups were assayed to determine p-38 MAPK and pp38 MAPK levels.IEC-6 cells cultured with CM collected from the miR-211 inhibitor group had a decreased p-38 MAPK level,and a significantly lower pp-38 MAPK level as compared with the control group(p<0.05).IEC-6 cells cultured with CM collected from the miR-211 mimic group had an increased p-38MAPK level,and a significantly higher pp-38 MAPK level(p<0.05)as compared with the control group.Culture of IEC-6 cells with CM collected from GDNF siRNA group exhibited no significant change p-38 MAPK or pp-38 MAPK level.7.miR-211 expression is upregulated,and GDNF expression is downregulated in duodenal tissue of patients with FD:Real-time PCR analysis indicated that miR-211 expression was upregulated in duodenal tissue of patients with FD,whereas GDNF expression was significantly downregulated(p<0.05)(Figure 7).Histopathological examination of duodenal biopsy specimens showed the eosinophil count in patients with FD was significantly higher than in healthy controls(94.40±75.28 vs.64.45±51.95,p<0.05).In addition,the mast cell count in duodenal tissue of patients with FD was significantly higher than in healthy controls(8.95±8.92 vs.5.85±4.53,p<0.05).