Overexpression of microRNA-211 in Functional Dyspepsia via Downregulation of the Glial Cell Line-Derived Neurotrophic Factor (GDNF) by Increasing Phosphorylation of p38 MAPK Pathway

Background Overexpression of miRNA-211 suppresses the differentiation of bone marrow stem cells into intestinal ganglion cells via downregulation of GDNF, a regulator of intestine barrier function. The study aimed to investigate the interaction between miR-211 and GDNF on intestinal epithelial cells. Methods The expression levels of miR-211 and GDNF in duodenal biopsy specimens from FD patients and healthy controls were compared. Enteric glia cell (EGCs) cell line transfected with miR-211 mimics and inhibitors were used to clarify the expression levels of GDNF were analyzed by qRT-PCR and ELISA. Intestine epithelial cell (IECs) cell line cultured in medium from ECGs in different transfection conditions were used in wound healing assay, cell proliferation assay, and western blotting for evaluation of p38 MAPK phosphorylation level. Results MiR-211 expression was significantly upregulated in the duodenal tissue of patients with FD compared to healthy subjects, whereas GDNF expression was significantly downregulated (both p < 0.05). Transfection with miR-211 mimics significantly decreased GDNF mRNA expression and protein secretion (p < 0.001). An inhibited intestinal epithelial cell wound healing (p < 0.05) and increased expression levels of phosphorylated p38 MAPK (p < 0.05) were found in IECs cultured with medium from EGCs transfected with miR-211 mimics. Conclusions MiR-211 may downregulates GDNF mRNA and protein expression via activation of the pp38 MAPK signaling pathway. Targeting miR-211 or the MAPK pathway may be a potential intervention for FD.


Introduction
Functional dyspepsia (FD) is a chronic gastrointestinal (GI) disorder defned by upper abdominal symptoms considered to originate from the gastroduodenal region with no structural disease on routine investigation. FD is one of the most common GI disorders, with a prevalence of up to 30% [1,2]. Studies have found GI mucosal alterations in some conditions of FD, and the changes take the form of increased permeability and are considered the result of mucosal infammation and neural activation [2][3][4][5]. Especially, enteric glia cells (EGCs) are important for maintaining the integrity of the intestinal epithelial barrier [6,7]. Dysregulation of the intestinal epithelial barrier is known to play a major role in the development, perpetuation, and severity of infammatory bowel disease (IBD) [8].
Helicobacter pylori infection plays a pivotal role in gastric disorders, including dyspepsia and peptic ulcers [9]. Te consequences of Helicobacter pylori-caused infammation destroy the gastric mucous membranes, increasing the risk of gastric cancer. Some patients may develop a clonal hyperplasia of lymphocytes and lymphatic tissues, leading to gastric lymphoma [9]. TNF-α is known for the association with Helicobacter pylori-cased infammation, and its different polymorphisms are associated with the risk of gastric cancer in diferent ethnic populations [10]. In addition, dyspepsia is also correlated with low intake of protein in community-dwelling adults older than 65 years [11].
Glia cell line-derived neurotrophic factor (GDNF) is secreted by EGCs and has been found to be a regulator of intestinal barrier function [12]. Studies showed that GDNF attenuated gut barrier infammation in IBD caused by p38 MAPK-dependent phosphorylation [8,13]. Terefore, deep investigations of the mechanisms underlying infammation-induced intestinal epithelial barrier breakdown in IBD are needed to identify novel therapeutic approaches [12].
MicroRNAs (miRNAs) are small, noncoding RNAs that mediate posttranscriptional gene expression of the target gene. Studies have shown interactions between miRNAs and GDNF. Overexpression of miRNA-211 suppresses the differentiation of bone marrow stem cells into intestinal ganglion cells via downregulation of GDNF expression [14]. Te purposes of this study were to (1) investigate miR-211 expression in duodenal biopsy specimens of patients with FD; (2) investigate the role of miR-211 on the expression of GDNF in EGCs; and (3) examine the efect of GDNF on wound healing and cell proliferation and migration in small intestine epithelial cells.

Participants. Te FD patients included in this study
were recruited from the Digestive Endoscopy Center and Gastroenterology Outpatient of Jinshan Hospital, the First Afliated Hospital of Chongqing Medical University, between November 2017 and December 2018. Te patient's chief complaint is upper abdominal dyspepsia diagnosed according to Rome IV (RC-IV) criteria. Patients were diagnosed as having FD with one or more than one symptom: (1) postprandial fullness after a meal; (2) early satiation; (3) upper middle abdominal pain; and (4) a burning sensation in the upper abdomen without any evidence of structural disease that can explain the above symptoms including gastroscopy. In addition, FD patients were subclassifed as postprandial distress syndrome (PDS) and epigastric pain syndrome (EPS).
Te inclusion criteria were: (1) aged 18-68 years (mainly exclude those with immature or degraded gastrointestinal tract development function to avoid afecting the analysis); (2) routine blood, urine, stool, stool occult blood, liver function, kidney function, blood sugar, ECG, and abdominal ultrasound examinations were normal; (3) electronic gastroscopy revealed no abnormalities or chronic superfcial gastritis, or no organic lesions that could explain the patient's dyspeptic symptoms were found; and (4) FD symptoms appeared for at least 6 months before diagnosis and met the above diagnostic criteria in the past 3 months. Exclusion criteria: (1) gastroscopy showed organic diseases of the upper digestive tract; (2) abdominal ultrasound showed organic diseases of the digestive tract; (3) severe primary diseases in heart, liver, kidney, hematopoietic systems; (4) history of pelvic or abdominal surgery; (5) taking drugs that may afect the absorption of gastrointestinal function with the past 7 days; (6) history of drug or food allergy; (7) pregnant or breastfeeding women; (8) depressive and anxiety patients with signifcant mental disorders and a clear diagnosis; (9) chronic kidney disease (CKD), diabetes, chronic hepatitis, pneumonia, and other chronic diseases that may cause digestive symptoms; and (10) recent history of high-dose smoking (≥10 cigarettes per day) or high-dose drinking (≥75 g of alcohol per day).
Healthy volunteers recruited from physical examination center or health medical stafs of the same period serve as the control group of this study. Teir routine blood, urine, stool, stool occult blood, liver function, kidney function, blood sugar, ECG, and abdominal ultrasound examinations were normal. Electronic gastroscopy revealed no abnormalities.
To investigate the association between miR-211 and FD, duodenal biopsy tissue specimens from 20 patients with FD and from 20 healthy controls were obtained during routine endoscopy. For PCR analysis, total RNA, including miRNAs, was extracted from biopsy specimens using the mirVana RNA Isolation Kit (Applied Biosystem, CA) according to the manufacturer's instructions. Te quality of total RNA was evaluated by the Bioanalyzer RNA Nano Kit (Agilent, Santa Clara, CA), and the RNA was quantifed using a Nanodrop spectrophotometer.
Te project was carried out in accordance with the Declaration of Helsinki. Te study was reviewed and approved by Institutional Review Board of our hospital (Approval number: 2017-457). Informed consents were obtained from all participants.

qRT-PCR.
Te cDNA synthesis of miRNA was performed using the All-in-One First-Strand cDNA Synthesis Kit (AORT-0060; GeneCopoeia). Real-time PCR was performed using the All-in-One TM miRNA qRT-PCR Detection Kit (QP015; GeneCopoeia). Te conditions for qPCR were: 42°C for 5 min, 95°C for 10 sec, 20 cycles of 95°C for 5 sec, 55°C for 30 sec, and 72°C for 30 sec. A primer sequence of cDNA was obtained from total RNA using a SMART ® MMLV Reverse Transcriptase Kit (Clontech, Mountain View, CA). qPCR was performed using a FastStart Universal SYBR Green Master Kit (Sigma-Aldrich, St. Louis, MO), and all reactions were run in triplicate on a real-time PCR system (Applied Biosystems). Te cycle passing threshold (Ct) values were recorded for GDNF, GAPDH, miR-211, and a small RNA (RNU48). MiR-211 was normalized by RNU48, and GDNF was normalized by GAPDH. Te relative expressions of miR-211 and GDNF were calculated using 2 −ΔΔCt method. Te specifc primers for miR-211, RNU48, GAPDH, and GDNF were purchased from Ribobio (Shanghai, China). Primer sequences for GDNF and GAPDH/RNU48 are listed in Supplementary Table 1.

ELISA Assay.
Te GDNF level released into CM was quantifed by the GDNF ELISA Kit (Cat. No. ab213901, Abcam, Cambridge, UK). Te OD value of EGC-CM in 450 nm was measured.
2.6. Wound Healing Assay. Wound healing assays were performed using 6-well dishes. A standardized scratch was made on an IEC-6 monolayer using the tip of a 100-µl pipette. Te IEC-6 monolayers were cultured with EGC-CM from each group prior to wounding, and were maintained for 48 hours after wounding. Te wounded areas were observed by a Motic AE31 inverted microscope (Motic China Group Co., Ltd.) and photographed at 24 and 48 hours. Wound closure was determined by the ratio of the wounded area at each time point compared with the initial scratch area using ImageJ software. Te software attached to the camera marked the scale bar item when taking the photo, there is a basis for the relative length in the subsequent scientifc image software analysis.

Cell Proliferation Assays.
Cell proliferation was determined using the CCK-8 assay kit (Dojindo Laboratories, Kumamoto, Japan). In brief, WST-8 (a water-soluble tetrazolium salt) is reduced by dehydrogenases in cells to produce a yellow-colored product (formazan), which is soluble in the tissue culture medium. Te amount of the formazan generated by the activity of dehydrogenases in cells is directly proportional to the number of living cells. Cells were trypsinized and replated for the CCK-8 proliferation assay at 24 and 48 hours after EGC-CM treatment. Briefy, the IEC-6 cells were seeded (5 × 10 3 cells/well) on 6well plates and cultured for 24 hours and 48 hours. At the indicated times (24 hours and 48 hours), 10 μL of the CCK-8 solution was added to each well, cells were cultured for an additional 2 hours, and then the absorbance was measured at 450 nm using a microplate reader.

Western
Blotting. Cultured IEC-6 cells were grown in 6well plates; the seeding concentration and further siRNA transfection were described in the previous "Cell Transfection." After 24 hours, the cells were subjected to Western blotting. Briefy, the IEC-6 cells were lysed using SDS lysis bufer, followed by SDS gel electrophoresis and immunoblotting. Te immunoblots were treated with antibodies against phosphorylated p38 MAPK (Cat. No. #9211) and p38 MAPK (Cat. No. #9212) at a dilution of 1 : 500, and then, secondary antibodies, the respective horseradish peroxidase-labeled goat antirabbit IgG at a dilution of 1 : 3,000 in PBS. β-actin was used as loading control. Chemiluminescence signal detection and quantifcation were performed by densitometry. All antibodies were purchased from cell signaling (Danvers, MA).

Statistical
Analysis. Continuous data are reported as mean ± standard deviation. Data of 2 groups were compared using an unpaired two-tailed Student's t test. When comparing MiR-211 and GDNF expression in normal subjects and FD patients, the Student's t-test and Mann-Whitney U test were used, respectively. All the statistical analyses were carried out using GraphPad Prism v6 software. A value of p < 0.05 is considered to indicate statistical signifcance.

Expression of miR-211 and GDNF in Duodenal Tissue of Patients with FD.
A total of 20 FD patients (8 males and 12 females) were included, with a mean age of 48.75 ± 10.55 (ranging from 28 to 66) years. According to the FD symptom classifcation criteria, 11 cases were simple PDS, 7 cases were PDS + EPS, and 2 cases were simple EPS. Among the symptoms of FD, 8 patients thought that postprandial fullness after a meal were the most obvious, 5 patients thought that early satiation were the most obvious, 4 cases were mainly belching, 2 cases were mainly epigastric pain, and 1 case was nausea.
qPCR analysis indicated that miR-211 expression was upregulated in duodenal tissue of patients with FD ( Figure 1(a), p < 0.05), whereas GDNF expression was signifcantly downregulated (Figure 1(b), p < 0.01). Te raw data of QPCR results for miR-211 and GDNF expression in human duodenal biopsy tissue specimens from 20 controls and FD patients each is shown in Supplementary Table 2. Histopathological examination of duodenal biopsy specimens showed the eosinophil count (94.40 ± 75.28 vs. 64.45 ± 51.95, p < 0.05, Figure S1A) and mast cell count (8.95 ± 8.92 vs. 5.85 ± 4.53, p < 0.05, Figure S1B) in patients with FD were signifcantly higher than healthy controls.

MiR-211 Suppresses GDNF mRNA Expression in EGCs.
To examine the efect of miR-211 on the transcriptional regulation of GDNF, EGCs were transfected with miRNA inhibitors or miRNA mimics, and the mRNA expressions of miR-211 and GDNF were examined at 48 hour with realtime PCR. Signifcantly increased miR-211 and decreased the level of GDNF mRNA expression were found in EGCs transfected with miR-211 mimic (Figure 2(a)), and vice versa in EGCs transfected with miR-211 inhibitors (Figure 2(b)), as compared with respective controls.

MiR-211 Suppresses GDNF Secretion.
Because increased GDNF mRNA expression does not directly indicate the level of secreted GDNF, GDNF levels in the culture media at 24 hours after transfection were quantifed using an ELISA assay according to the manufacturer's protocol.

Inhibition of miR-211 Increases GDNF Levels. Small intestinal epithelium cells (IEC-6) were cultured with conditioned medium (CM) collected from the cultures of
EGCs treated in 6-well plates, as described in the methods. GDNF level in the diferent CM was measured by ELISA ( Figure 4). Te level of GDNF in CM collected from EGCs transfected with miR-211 mimic was signifcantly lower than the control group (p < 0.001). Te level of GDNF in CM collected from EGCs transfected with GDNF siRNA was signifcantly lower than that in the control group (p < 0.01).

GDNF Level Has No Efect on Wound Healing and
Proliferation. Results of the wound healing assay are shown in Figure 5. No signifcant diference was displayed at 24 hr after transfection. At 48 hr, the level of healing in the miR-211 mimic group was signifcantly lower than the mimic control group (p < 0.05). Results of the cell proliferation assays are summarized in Figure 6. Te data indicate that GDNF has no efect on cell proliferation (all p > 0.05).

GDNF Level Is Inversely Correlated with p38 MAPK
Level. GDNF has been shown to afect p38 MAPK signaling in small intestinal epithelial cells [15]. Mechanical wounding of IEC-6 cell monolayers results in rapid activation of the extracellular signal-regulated kinase (ERK) and p38 MAP kinase pathways, and intestinal epithelial cell wound signal transduction is, at least in part, mediated by activation of the ERK and p38 MAP kinase signaling cascades [13,15]. Terefore, we examined whether the p38 MAPK signaling pathway is activated in EGC-CM-treated IEC-6 cells. p38 MAPK were investigated in IEC-6 cells following EGC-CM application. IEC-6 cells cultured with CM collected from diferent experimental groups were assayed to determine p38 MAPK and pp38 MAPK levels (Figure 7). Te p38 MAPK level was comparable in IEC-6 cells cultured with CM collected from the experiment groups (Figure 7(b)). Te pp38 MAPK level was signifcantly decreased in the miR-211 inhibitor group and increased in the miR-211 mimics group as compared with respective controls (Figure 7(c), p < 0.05).

. Discussion
Our results show that miR-211 downregulates GDNF expression through increased phosphorylation of MAPK-38 in FD, suggesting a potential of targeting miR-211 for treating FD. FD is a chronic disorder of sensation and movement in the upper digestive tract. It is well known that the enteric nervous system (ENS) plays an important role in the regulation of intestinal epithelial barrier function [16]. Moreover, GDNF is largely studied in neuromuscular pathologies, as it can prevent motor neuron degeneration in animal models of amyotrophic lateral sclerosis (ALS) and it represents a possible biomarker to predict ALS development [17]. In search of new therapeutic agents for neurodegenerative disorders, special interest has been devoted to neurotrophins because of their potential to promote survival and neurotics' growth as well as infuence the diferentiation of several neuronal populations. GDNF has received lots of attention because it has been shown to be a potent survival factor for dopaminergic midbrain and spinal cord neurons [17][18][19]. Tey identifed astrocytes as a possible endogenous source of GDNF and suggested that astrocyte-derived GDNF can protect from neurodegeneration through inhibition of neuroinfammation. Terapeutic inhibition of miRNAs using antisense oligomers (called anti-miRs) has also been shown to reduce tumor growth [20]. Zhang et al. reported that miRNAs mediate GDNF-induced proliferation and migration of glioma cells [21]. Recent studies revealed a strong connection between the regulatory mechanism of miRNA and disease etiology [22,23]. A number of miRNAs have been associated with diferent intestinal disorders, and targeting miRNAs is a promising method of treatment for gastrointestinal disorders [24].
Te main mediator secreted by enteric glial cells is the GDNF, which has efects on proliferation on glial cells and neurons in the embryonic development of the enteric nervous system [25]. Moreover, it has been consistently reported that reduced levels of GDNF lead to morphological and functional abnormalities of intestinal barrier function in mice similar to IBD [26]. EGCs can be classifed into 4 main types (I-IV) according to their shapes and locations in the gut wall, but the precise role of each type of EGCs in gastrointestinal physiology and pathophysiology is not well understood [13,27]. EGCs have been traditionally considered as a mechanical support for enteric neurons, and they are able to release a wide range of factors responsible for the development, survival, and diferentiation of peripheral neurons [13,27,28]. Nowadays, besides their supportive role, it has been demonstrated that EGCs possess a more complex nature, playing a leading role in the maintenance of intestinal homeostasis [28]. Te results of this study demonstrated that miR-211 expression is inversely related with GDNF mRNA expression and protein levels in EGCs. Tis association was also demonstrated in duodenal biopsy tissue specimens of patients with FD. Tese results suggest that miR-211 may play a role in the pathogenesis of FD by regulating GDNF.
In the present study, GDNF level was positively correlated with the wound-healing capacity of intestinal epithelium cells (IEC-6 cells) but has no efect on cell proliferation. While the length of time of treatment can afect results, the treatment time of 48 hours used in this study should have been sufcient as it is similar to the time used in other studies [29]. We cannot exclude the possibility that GDNF is inactivated during the process of collecting the CM. It is also possible that other factors in the unpurifed medium may also adversely afect the activity of GDNF.
Accumulating evidence shows that many gastrointestinal disorders are associated with intestinal permeability defects, epithelial barrier dysfunction, and infammation [3,30,31]. GDNF is an important regulator of intestinal barrier function [25], and studies have shown that GDNF reduces infammation and promotes wound healing in intestinal epithelial cells [15,32,33]. Te results of the current study are consistent with those of prior studies, which indicate that GDNF has a protective/healing role with respect to intestinal epithelial cells. GDNF predominantly binds to GFRα1, NRTN to GFRα2, ARTN to GFRα3, and PSPN to GFRα4. Various signaling pathways are activated, such as RAS/mitogen activated protein kinase (MAPK), RAS/extracellular signalregulated kinase (ERK), phospholipase C gamma (PLCc), phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT), and c-Jun N-terminal kinase (JNK) pathways, which contribute to cell adhesion, migration, proliferation, differentiation, and survival. Recent studies indicate that GDNF also derives from smooth muscle cells and enterocytes in signifcant amounts [34]. Loss of GDNF in experimental models leads to morphological and functional abnormalities in patients with IBD [33]. Although little is known about the role of p38 MAPK signaling in the regulation of intestinal barrier properties, phosphorylation of p38 MAPK was shown to activate the myosin-light chain kinase [35]. Tis leads to contraction of the actin-myosin ring to which the junctional complex is tethered via adaptor proteins and thereby induces destabilization of the epithelial barrier [34]. In line with these observations, the application of SB-202190 on immature Caco2 cells to mimic the inhibition of p38 MAPK signaling led to epithelial barrier stabilization. Activation of p38 MAPK signaling using anisomycin led to increased permeability and blocked  Figure 7: Levels of p38 MAPK and pp38 MAPK. (a) IEC-6 cells precultured with CM collected from diferent experimental group were lysed, and assayed for p38 MAPK and pp38 MAPK using SDS gel electrophoresis followed by immunoblotting. Chemiluminescence signal of (b) p38 MAPK and (c) pp38 MAPK was quantifed by densitometry. Each assay was conducted in triplicate. * p < 0.05.
GDNF-induced barrier stabilization [12]. Terefore, it can be concluded that GDNF-induced inhibition of p38 MAPK signaling contributes to barrier maturation. Te predominant role of p38 MAPK signaling in the context of intestinal epithelial barrier regulation is also supported by previous study in which TNF-α-induced loss of intestinal barrier integrity was mediated by activation of p38 MAPK [12]. In a second step, we investigated the efects of GDNF on p38 MAPK signaling. Intestinal epithelial wound signal transduction is, at least in part, mediated by activation of p38 MAP kinase signaling cascades [12]. Our results showed that GDNF level is inversely correlated with pp38 MAPK. It suggests that GDNF may improve wound healing via pp38 MAPK suppression. GDNF also has immunomodulatory efects, and GDNF has been shown to signifcantly reduce infammation in a study using a model of chronic infammatory bowel disease [36]. It has been suggested that the efect is due to GDNF blocking the secretion of various proinfammatory cytokines, such as TNF-α and interleukin (IL)-1β [19]. We also found that both eosinophils and mast cells increased signifcantly in numbers in FD patients, which is consistent with previous studies [37]. In the study conducted by Tanaka et al., GDNF level is higher in FD patients as compared to controls [37]. However, our study demonstrated a signifcant decrease in GDNF level in FD patients. Such discrepancy may be due to diferences in the sample size, where our study had a sample size of 26 patients and 26 controls, while it was 5 patients and 9 controls of Tanaka's. Indeed, we have found some FD patients are with noticeable higher levels of GDNF, but the mean value of GDNF of all patients is reduced. We suggested that other factors except miR-211 may play a role in the regulation of GDNF, but further studies are required to evaluate this hypothesis. And how miRNAs regulate the expression of GDNF in the nervous system has signs to follow. In the Parkinson diseases (PD) model, GDNF specifcally increases the expression of miR-182-5p and miR-183-5p in primary midbrain neurons [35]. Moreover, miR-9, miR-96, miR-133, and miR-146a negatively regulate GDNF level during kidney development [18]. Interestingly, Xia et al. describe miR-211 was upregulated in ganglion cell dysplasia. Whether GDNF expression was decreased in these patients needs further elucidation [14].

Conclusions
MiR-211 may downregulate GDNF via upregulation of the p38 MAPK signaling pathway, suggesting a potential intervention for FD. Studying the relationship between miR-211 and p38 MAPK is the future plan.

Data Availability
Te data used to support the fndings of this study are available from the corresponding author upon request.