Conceived and designed the experiments: MR VM SL. Performed the experiments: MR KC. Analyzed the data: SL MR KC VM CS AB SM. Contributed reagents/materials/analysis tools: SM NS CS AB. Wrote the paper: MR VM SL KC.
The authors have declared that no competing interests exist.
The characterization of miRNAs and their target mRNAs involved in regulation of the immune process is an area of intense research and relatively little is known governing these processes in allergic inflammation. Here we present novel findings defining the miRNA and mRNA transcriptome in eosinophilic esophagitis (EoE), an increasing recognized allergic disorder.
Esophageal epithelial miRNA and mRNA from five paired biopsies pre- and post-treatment with glucocorticosteroids were profiled using Taqman and Affymetrix arrays. Validation was performed on additional paired biopsies, untreated EoE specimens and normal controls. Differentially regulated miRNAs and mRNAs were generated, within which miRNA-mRNA target pairs with high predicted confidence were identified.
Compared to the post-glucocorticoid treated esophageal mucosa, of all the 377 miRNA sequences examined, 32 miRNAs were significantly upregulated and four downregulated in the pre-treated biopsies. MiR-214 was the most upregulated (150 fold) and miR-146b-5b, 146a, 145, 142-3p and 21 were upregulated by at least 10 fold. Out of 12 miRNAs chosen for validation by qRT-PCR, five (miR-214, 146b-5p, 146a, 142-3p and 21) were confirmed and 11 shared the same trend. When the expression of the 12 miRNAs in the EoE mucosa was compared to unrelated normal mucosa, six (miR-214, 146b-5p, 146a, 21, 203, and 489) showed similar significant changes as in the paired samples and 10 of them shared the same trend. In the same five pairs of samples used to profile miRNA, 311 mRNAs were down-regulated and 35 were up-regulated in pre-treated EoE mucosa. Among them, 164 mRNAs were identified as potential targets of differentially regulated miRNAs. Further analysis revealed that immune-related genes, targeted and non-targeted by miRNAs, were among the most important genes involved in the pathogenesis of EoE.
Our findings add to the accumulating body of data defining a regulatory role for miRNA in immune and allergic processes.
Eosinophilic esophagitis (EoE) is an increasingly recognized antigen-driven disorder of the esophagus occurring in children and adults
MicroRNAs (miRNAs) are small, non-coding 19–25 nucleotide long RNAs that constitute the most abundant class of regulators of gene expression
The characterization of miRNAs and their target mRNAs involved in regulation of the immune process is an area of intense research
This goal of this study was to characterize the miRNA profile of well documented EoE mucosal biopsies, before and after successful treatment with glucocorticosteroids, and to correlate this profile with dysregulated mRNA identified using the same cohort. These findings were further verified on an additional cohort of biopsies from patients with EoE as opposed to normal mucosa.
Archival esophageal biopsies from seven pediatric patients before and after treatment for EoE were retrieved from the Department of Pathology at Rhode Island Hospital (Providence, RI). The first five pairs were used for both miRNA and mRNA profiling and all seven were used for validation (
Patient Group | No. | Age at Diagnosis | Gender | Presenting Complaint | Food Allergy | Steroid Used |
Paird EoE patients | 1 | 11 | M | Ab pain | Yes | Bud |
2 | 5 | M | Vomiting, Food refusal | Yes | Bud | |
3 | 2 | M | Regurgitation | Yes | Bud | |
4 | 13 | F | Dysphagia, Chest pain | Yes | Bud | |
5 | 2 | F | Vomiting, FTT | No | Bud | |
6 | 9 | M | Vomiting, Food refusal | No | Flu | |
7 | 1.3 | M | FTT, Food refusal | Yes | Flu | |
Untreated EoE | 1 | 5 | M | Dysphagia | Yes | |
2 | 16 | M | Dysphagia | Yes | ||
3 | 10 | M | Dysphagia, Reflux symptoms | No | ||
4 | 2 | M | Food refusal | Yes | ||
5 | 10 | M | Reflux symptoms, Ab pain | No | ||
6 | 1.5 | M | Dysphagia | Yes | ||
7 | 15 | F | Diarrhea | No | ||
8 | 14 | F | Ab Pain, Diarrhea | No | ||
Normal Control | 1 | 13 | F | Ab pain, Diarrhea, Wt loss | No | |
2 | 14 | F | Ab pain, Fatigue | No | ||
3 | 15 | F | Ab pain | No | ||
4 | 5 | M | Ab Pain | No | ||
5 | 6.5 | M | Dysphagia, | Yes | ||
6 | 17 | F | Nausea | Yes | ||
7 | 17 | F | Hematochezia | No | ||
8 | 3 | F | Food refusal, Wt loss | No | ||
9 | 16 | M | Ab pain | No | ||
10 | 8 | F | Ab pain | No |
Abbreviations used: Ab, Abdominal; Wt, weight; FTT, Failure to Thrive; Bud, Budesonide; Flu, Fluticasone.
Patient records were reviewed by a pediatric gastroenterologist (V.M.) to ensure that the clinical characteristics of the patients fit the diagnosis of EoE. The slides were reviewed by a pathologist (M.R.) to confirm the presence or absence of EoE. Classic features of EoE consist of numerous intraepithelial eosinophils greater than 15 per high powered field (HPF) (ranging from 35 to over 100 per HPF), superficial eosinophilic microabscesses, basal layer hyperplasia and subepithelial fibrosis. The posttreatment biopsy material consisted of normal appearing squamous mucosa with only rare intraepithelial eosinophils numbering less than 2 per HPF.
A summary of the seven EoE patients’ clinical information is presented in
The study was performed according to a protocol approved by the institutional review board (IRB) of Lifespan/Rhode Island Hospital. The requirement of consent was specifically waived by the IRB committee.
Eight 10 µm sections were cut from each block and mounted onto plain glass slides. If the biopsy section contained only epithelium, the sections were scraped from the slides and were ready for total RNA extraction. If biopsy sections contained sub-epithelium, the sections were deparaffinized, stained, dehydrated through graded alcohols using the Paradise FFPE reagent System (Applied Biosystems, Foster City, CA) and subjected to LCM within 2 hours of deparaffinization. About 20,000 epithelial cells were captured on LCM Macro CapSure caps (Applied Biosystems) using the Arcturus XT LCM instrument (Applied Biosystems) and the captured cells in the caps were used to extract total RNA. In either case, only the RNA from the epithelial mucosa was extracted. RecoverAll Total Nucleic Acid Extraction Kits for FFPE tissues (Ambion, Austin, TX) were used for extraction. RNA was further purified and concentrated using the RNEasy Minelute Cleanup Kit (Qiagen, Valencia, CA), and then evaluated by the Agilent Bioanalyzer using an RNA 6000 Nano or Pico LabChip (Agilent Technologies Santa Clara, CA) as described previously
Five nanograms of total RNA was reverse-transcribed using the Taqman MicroRNA Reverse Transcription Kit and the Megaplex RT primer Human Pool A (Applied Biosystems). The reverse-transcribed cDNA was then pre-amplified in 12 cycles of PCR using Taqman PreAmp Master Mix and the Megaplex PreAmp primers, Human Pool A (Applied Biosystems). The cDNAs were then diluted and loaded onto a Taqman Human miRNA Array card A (Applied Biosystems), which contains probes for 377 distinct miRNAs and a housekeeping gene (MammU6). The Array cards were run on an ABI HT7900 quantitative PCR (qPCR) instrument. Ct values were obtained for all miRNAs represented on the cards and fold changes in expression were calculated using the delta delta Ct (ddCt) method. Expression levels of MammU6 on the Array card were used as control for the purpose of ddCt calculation. The expression array data have been deposited in Gene Expression Omnibus (GEO) of National Center for Biotechnology Information (NCBI) and are accessible through GEO series accession number GSE36726.
Expression of miRNAs that demonstrated high differential expression by array card in five matched EoE cases before and after treatment were confirmed again in those same matched cases as well as in two additional matched cases. In addition, the expression of these differentially expressed miRNAs was determined in eight new samples of EoE biopsy material and compared to ten biopsies from unrelated normal esophageal epithelium (
Fifty nanograms of total RNA was amplified and reverse-transcribed into cDNA using Ovation FFPE WTA system (Nugen Technologies, San Carlos, CA). Five to ten micrograms of amplified cDNA from the amplification above was fragmented and labeled for Affymetrix array analysis using Encore Biotin Module (Nugen Technologies). Array hybridization and analysis were performed by Genomics Core Facility at the Center for Genomics and Proteomics (Brown University, Providence, RI) according to Affymetrix protocols (Affymetrix Inc, Santa Clara, CA).
Affymetrix Gene 1.0 ST Array data was analyzed by Genomics Core Facility using Affymetrix Command Console and Expression Control. The expression array data have been deposited in GEO of NCBI and are accessible through GEO series accession number GSE36725.
Functional annotation was performed using Partek Genomics Suite 6.0, Partek Inc, St. Louis, MI). Differentially regulated genes are selected based on the fold-change (FC), false discovery rate (FDR), and statistical significance (P-value of Student’s paired t-test). Target genes for miRNA were predicted using Ingenuity Pathway Analysis (IPA, Ingenuity Systems, Inc. Redwood City, CA). The miRNA and mRNA in a pair have to be regulated in opposite directions and their interactions are either “Experimentally observed” or exhibit “High (predicted) Confidence” as defined by IPA. Functional pathway analysis and network analysis were performed using modules within IPA.
Total RNA from five pairs of EoE biospies before and after treatment was extracted and miRNA expression levels were quantified by Taqman Low Density Array. Of all the miRNAs tested by the array, 36 of them were differentially regulated based on a paired t-test P-value of less than 0.05. Five transcripts were down-regulated after treatment and 33 were up-regulated. The absolute fold changes range from 1.6 to 151. Some miRNAs share the same target sites so that a total of 31 target sites were identified from all the miRNAs. MiR-373 and miR-520e share the same target sites while miR-373 is up-regulated and miR-520e is down-regulated post treatment and thus are excluded for further analysis. The differentially regulated miRNAs are listed in
The heat map illustrates miRNA expression in five paired esophageal epithelium samples of EoE purified before and after treatment. Each column represents one sample. MiRNA expression levels were stratified individually for each miRNA and each square was colorized based on the level of its expression. The overall fold changes pre- and post-treatment are listed in
miRNA ID | Overlapping target sites | P-value |
Fold change |
hsa-miR-214 | 0.019 | 151.46 | |
hsa-miR-146b-5p | 0.0015 | 47.15 | |
hsa-miR-146a | Same as miR-146b-5p | 0.00045 | 39.22 |
hsa-miR-145 | 0.028 | 35.95 | |
hsa-miR-142-3p | 0.0017 | 26.55 | |
hsa-miR-21 | 0.0010 | 11.51 | |
hsa-miR-29b | 0.015 | 9.43 | |
hsa-miR-339-3p | 0.018 | 7.79 | |
hsa-miR-100 | 0.025 | 7.27 | |
hsa-miR-10a | 0.048 | 6.70 | |
hsa-miR-223 | 0.0028 | 5.44 | |
hsa-miR-324-5p | 0.014 | 5.34 | |
hsa-miR-99a | Same as miR-100 | 0.024 | 5.20 |
hsa-miR-34a | 0.030 | 4.99 | |
hsa-miR-195 | 0.0041 | 4.87 | |
hsa-miR-125a-5p | 0.019 | 4.75 | |
hsa-miR-140-5p | 0.017 | 4.64 | |
hsa-miR-502-3p | 0.0020 | 4.62 | |
hsa-miR-150 | 0.0043 | 4.53 | |
hsa-miR-29a | Same as miR-29b | 0.0060 | 4.28 |
hsa-miR-99b | Same as miR-100 | 0.033 | 4.13 |
hsa-miR-139-3p | 0.020 | 3.86 | |
hsa-miR-126 | 0.010 | 3.82 | |
hsa-miR-342-3p | 0.0019 | 3.22 | |
hsa-miR-181a | 0.012 | 3.19 | |
hsa-miR-93 | 0.019 | 2.95 | |
hsa-miR-27a | 0.0038 | 2.93 | |
hsa-miR-152 | 0.0058 | 2.87 | |
hsa-miR-27b | Same as miR-27a | 0.043 | 2.05 |
hsa-miR-29c | Same as miR-29b | 0.036 | 1.97 |
hsa-miR-222 | 0.026 | 1.85 | |
hsa-miR-155 | 0.035 | 1.58 | |
hsa-miR-361-5p | 0.038 | −2.22 | |
hsa-miR-147 | 0.0064 | −2.45 | |
hsa-miR-203 | 0.036 | −2.68 | |
hsa-miR-489 | 0.037 | −2.76 |
Paired Student’s T-test; comparing the expression levels before and after treatment.
Fold change = expression levels before/after treatment.
To validate the expression of the differentially regulated miRNAs discovered by Taqman Low Density Array in
qRT-PCR Verification | ||||||
miRNA | Taqman Array | Pre- vs post treatment | EoE vs Normal | |||
Fold Change | p-value | Fold Change | p-value | Fold Change | p-value | |
hsa-miR-214 | 151.45 | 0.019 | 26.62 | 0.02 | 2.02 | 0.078 |
hsa-miR-146b-5p | 47.15 | 0.0015 | 18.79 | 0.0031 | 5.4 | 0.004 |
hsa-miR-146a | 39.22 | 0.00045 | 9.89 | 0.0022 | 3.1 | 0.033 |
hsa-miR-145 | 35.95 | 0.028 | 3.15 | 0.079 | 1.73 | 0.1 |
hsa-miR-142-3p | 26.55 | 0.0017 | 8.61 | 0.019 | 1.3 | 0.12 |
hsa-miR-21 | 11.51 | 0.001 | 6.67 | 0.006 | 4.39 | 0.003 |
hsa-miR-29b | 9.43 | 0.015 | 13.04 | 0.09 | 0.97 | 0.24 |
hsa-miR-339-3p | 7.78 | 0.018 | 1.92 | 0.31 | 1.21 | 0.28 |
hsa-miR-100 | 7.27 | 0.025 | 1.58 | 0.38 | 0.93 | 0.25 |
hsa-miR-10a | 6.7 | 0.048 | 1.03 | 0.49 | 0.38 | 0.062 |
hsa-miR-203 | 0.37 | 0.036 | 0.237 | 0.072 | 0.19 | 0.0006 |
hsa-miR-489 | 0.36 | 0.037 | 5.27 | 0.21 | 0.13 | 0.042 |
Delta Ct values are used to represent selected miRNA expression levels in esophageal epithelium of 7 paired EoE patients pre- vs post-treatment (5 pairs used in miRNA profiling and 2 additional pairs;
Paired biopsies from the same patient were used to profile the miRNA expression for the purpose of eliminating the inter-individual variation. However, as glucocorticosteroids were the major treatment component in our patient’s cohort, miRNA expression following treatment may be related in part to the treatment effect. To test this hypothesis, we compared the miRNA expression of the mucosal biopsies from a new set of eight EoE patients with those from normal mucosa of ten additional pediatric esophageal biopsy samples from patients without any history of EoE. As shown in
Delta Ct values are used to represent selected miRNAs expression levels in esophageal epithelium of 8 untreated EoE patients versus 10 normal epithelium of control patients (
The major role of miRNA is to regulate mRNA expression at the transcriptional and translational level. We wished to determine how much of the mRNA expression in EoE is influenced by miRNAs. To profile the global mRNA changes in EoE, an Affymetrix GeneChip based mRNA gene profiling was conducted on five paired EoE biopsies before and after treatment. Total RNA was extracted from FFPE tissue sections and hybridized to Human Gene ST 1.0 Genechips. The Principle Component Analysis of data demonstrated that gene expression data from biopsies of the same status are highly correlated with each other (
In this 3-dimentional plot of the first three principle components of all ten samples, samples of the same treatment status were grouped together. Five pre-treatment samples (Blue) and five post-treatment samples (Red) were clearly separated from each other.
miRNAs and Target mRNAs | Fold Change |
|
− |
ALOX15 | 10.30 |
|
− |
CYP2S1, | 2.03 |
|
|
ZFAND5, CNST, RAP2A, ACTG1, TIAM1 | −2.78 ∼ −2.00 |
|
|
ETNK2, IL1RN, PRDM1, B4GALT1, GRHL1, TRPS1, FAM129B, RIT1, DOCK3, MEGF9 | −4.88 ∼ −2.08 |
|
|
TOM1 | 2.67 |
|
|
PHACTR2, STRADB, SNX16, RALA, RIOK3, C15orf29, PRDM1, MYO6, CAMK2N1, MRPS10 | −4.49 ∼ −2.24 |
|
|
MGLL, C18orf25, USP6NL, CCDC6, RAB2A, TIPARP, HECTD1, KAT2B, WASL, TWF1, C1orf9 | −3.00 ∼ −2.00 |
|
|
PADI1, BNIP3, PHACTR2, SNX24, KIF21A, TSPAN6, SAMD5, SRGAP1, TMOD3, AIM1 | −6.97 ∼ −2.99 |
|
|
S100A12, ZNF117, MYO6, CCDC6, IL1RAP, IL1RL2 | −6.70 ∼ −2.1 |
|
|
SYNPO2L, PDIA6, EREG, TOM1, CNST, TRPS1, ADIPOR2, CAST, AIFM2, FOPNL | −3.63 ∼ −2.20 |
|
|
RMND5A, PHACTR2, EMP1, MXD1, STRADB, B4GALT5, MOSPD1, C18orf25, CLOCK, KIAA0232 | −4.82 ∼ −2.61 |
|
|
ETNK2, ZNF431, PPL, WNK1, TRPS1, CAB39, PEA15, PTN, ZNF714, TWF1 | −4.88 ∼ −2.03 |
|
|
PHACTR2, SASH1, PHLDA1, MOSPD1, SRGAP1, C12orf29, UBL3, PHACTR4, EREG, RALA | −4.49 ∼ −2.83 |
|
|
ALOX12, PHACTR2, CGNL1, KIF21A, STRADB, RASGEF1B, TUFT1, PDIA6, SNX16, TRIP10 | −8.65 ∼ −2.92 |
|
|
IL12A, RMND5A, SASH1, PELI1, TOR1AIP2, SECISBP2L, HIPK3, RFFL, TIAM1 | −6.25 ∼ −2.00 |
|
|
ABLIM3, DOCK9, ARHGAP10, PIM1, TRPS1, CPEB4, RAB5B, CAPN5, TWF1, KIF1B | −5.08 ∼ −2.01 |
|
|
PHACTR4, C18orf25, INPP4B, ZFAND5, KIAA1370, TRPS1, LYPLA1, TIPARP, TP53BP2, MEGF9 | −2.89 ∼ −2.08 |
|
|
NAMPT, SNX24, MSMO1, PRDM1, CDS1, SECISBP2L, TWF1, ZNF365 | −4.01 ∼ −2.02 |
|
|
ENDOU, BNIP3, RMND5A, CRISP2, LPIN1, CCNYL1, CAB39L, CPPED1, CNN3, WNK1 | −6.94 ∼ −2.81 |
|
|
PHACTR2, TUBB2A, EMP1, SNX24, MXD1, AMFR, AIM1, RIOK3, WDR26, DSC2 | −4.49 ∼ −2,53 |
|
|
RMND5A, SYNPO2L, CAMK2N1, CPEB4, C20orf11, TIAM1 | −7.19 ∼ −2.00 |
|
|
ABLIM1,TOM1,CLOCK,NCOA1,AREG, PEA15, MARCH5, DOCK3,RNF169, B4GALR5, AIM1 | −2.92 ∼ −2.99 |
|
|
B4GALT5,AIM1 | −3.36 ∼ −2.99 |
|
|
MXD1, SASH1, NPAS2, HIF1A, SLC16A9, MSMO1, SNX9, MGLL, SNX16, TRIP10, EREG | −3.96 ∼ −2.99 |
|
|
GRHL1, PPP1CB | −2.58 ∼ −2.18 |
Only up to 10 mRNAs with the highest fold change are listed under each miRNA.
miR-339-3p, miR-489, miR-361-5p, miR-324-5p, and miR-139-3p do not have predicted targets in combined gene list.
Information regarding the miRNA-mRNA pairs is incorporated into a pathway network analysis on the 346 differentially regulated genes using IPA program. One of the top pathway networks IPA revealed is a gene regulatory network of more than 24 genes involving “inflammatory response”, “cell-to-cell signalling and interaction”, and “hematological system development and function” (
One of the primary pathway networks is associated with network functions of “Inflammatory Response, Cell-To-Cell Signaling and Interaction, Hematological System Development and Function”.
Given the finding that both targeted genes and non-targeted genes are equally represented in the network (
ID | p-value | Fold Change | ID | p-value | Fold Change |
ALOX15 | 0.0012 | 10.30 | B4GALT1 | 0.00098 | −2.59 |
ALOX12 | 3.4E-05 | −8.65 | NCOA1 | 0.00044 | -2.56 |
S100A12 | 0.00014 | −6.70 | DSC2 | 0.00047 | −2.53 |
IL12A | 5.0E-05 | −6.25 | PIM1 | 4.3E-05 | −2.44 |
ETNK2 | 7.7E-05 | −4.89 | CNST | 0.00021 | −2.42 |
LPIN1 | 6.4E-05 | −4.60 | KIAA1370 | 0.00038 | −2.41 |
PHACTR2 | 4.7E-05 | −4.49 | TRPS1 | 0.00013 | −2.39 |
TUBB2A | 0.00036 | −4.19 | CCDC6 | 1.9E-05 | −2.38 |
NAMPT | 0.0011 | −4.01 | ADIPOR2 | 0.00042 | −2.35 |
SNX24 | 0.00017 | −4.00 | PSD3 | 0.00049 | −2.35 |
MXD1 | 0.0012 | −3.96 | CPEB4 | 0.00092 | −2.33 |
NPAS2 | 8.7E-05 | −3.58 | CYP4F3 | 1.2E-05 | −2.35 |
CPPED1 | 0.00096 | −3.53 | AREG | 0.0004 | −2.32 |
HIF1A | 0.00020 | −3.45 | CAST | 0.0012 | −2.31 |
SC4MOL | 0.00023 | −3.33 | RAB2A | 5.1E-05 | −2.30 |
SAMD5 | 0.00028 | −3.24 | ACTG1 | 0.00027 | −2.22 |
IL1RN | 0.00026 | −3.16 | GNG12 | 3.8E-05 | −2.20 |
AMFR | 2.6E-06 | −3.13 | PPP1CB | 0.00078 | −2.18 |
PELI1 | 0.00033 | −3.13 | EPB41L4A | 1.7E-06 | −2.15 |
CNN3 | 0.00064 | −3.09 | MARCH5 | 0.00096 | −2.14 |
PDIA6 | 0.00037 | −3.01 | DOCK3 | 0.00024 | −2.14 |
MGLL | 6.8E-07 | −3.00 | WASL | 0.00025 | −2.12 |
ABLIM1 | 4.2E-05 | −2.92 | GABARAPL2 | 0.00037 | −2.11 |
UBL3 | 0.00044 | −2.90 | IL1RAP | 8.0E-05 | −2.10 |
EREG | 0.00014 | −2.89 | RAB5B | 0.00077 | −2.10 |
C18orf25 | 0.00063 | −2.86 | STK39 | 0.0011 | −2.08 |
PFKFB3 | 7.6E-05 | −2.84 | ZNF714 | 0.00096 | −2.08 |
INPP4B | 0.00014 | −2.84 | PI4K2A | 0.00047 | −2.06 |
RALA | 0.00070 | −2.83 | PLEKHA6 | 0.00013 | −2.06 |
WNK1 | 5.16E-06 | −2.81 | ZNF365 | 0.00035 | −2.02 |
KRT7 | 8.21E-06 | −2.69 | KIF1B | 0.00051 | −2.01 |
PLEKHM1 | 0.00025 | −2.69 | RNF169 | 0.00080 | −2.00 |
RIOK3 | 0.00038 | −2.65 | IL13RA1 | 0.0010 | −2.00 |
CLOCK | 0.00089 | −2.62 | TIAM1 | 8.0E-05 | −2.00 |
PRDM1 | 0.00063 | −2.59 |
Genes related to “hypersensitivity response”, “immunological disease”, “inflammatory disease”, and “inflammatory response” by IPA annotation are selected to represent immune related genes.
ID | p-value | Fold Change | ID | p-value | Fold Change |
TNFAIP6 | 0.00033 | 39.23 | WNT5A | 0.00099 | −3.32 |
CPA3 | 7.0E-05 | 7.70 | SCNN1B | 5.2E-05 | −3.28 |
HPGDS | 3.0E-05 | 5.87 | TMEM57 | 0.00011 | −3.17 |
SAMSN1 | 0.00017 | 3.82 | GNG4 | 0.0011 | −2.97 |
SERPINE2 | 0.00046 | 3.15 | TAX1BP1 | 0.00025 | −2.94 |
TPSAB1 | 0.00069 | 3.02 | MAPK3 | 1.4E-05 | −2.92 |
TFPI | 0.00019 | 2.82 | UACA | 0.00011 | −2.81 |
LAPTM5 | 0.00091 | 2.6 | ZNF92 | 9.2E-06 | −2.63 |
LCP2 | 0.00016 | 2.41 | ST3GAL4 | 1.5E-06 | −2.62 |
CNTN4 | 0.00020 | 2.30 | ADIPOR1 | 0.00015 | −2.58 |
HIST1H4K | 0.00077 | 2.24 | EFNA5 | 0.00076 | −2.57 |
C1R | 0.00039 | 2.23 | ZDHHC21 | 0.0012 | −2.54 |
HDC | 0.00025 | 2.21 | S100A9 | 0.00089 | −2.36 |
IGFBP3 | 0.00097 | 2.18 | NHSL1 | 0.00016 | −2.34 |
LOX | 0.00016 | 2.15 | DOPEY2 | 0.00031 | −2.33 |
B2M | 0.00088 | 2.08 | CD68 | 0.00046 | −2.29 |
IFITM3 | 0.00043 | 2.03 | GNA15 | 0.00041 | −2.22 |
SLURP1 | 0.000062 | −8.13 | DYNLL1 | 0.00024 | −2.21 |
SFTA2 | 0.00011 | −7.87 | S100A14 | 6.6E-05 | −2.18 |
CRYAB | 0.00032 | −5.74 | DSG1 | 0.00034 | −2.15 |
IL18 | 0.00015 | −5.11 | AHCY | 0.00036 | −2.15 |
CST6 | 0.0012 | −4.25 | RALB | 0.00038 | −2.14 |
PSCA | 0.00029 | −4.14 | GPT2 | 0.0011 | −2.12 |
CXCR2 | 0.000053 | −4.12 | PARD3 | 0.00013 | −2.12 |
ACPP | 0.00017 | −3.98 | LGMN | 0.00082 | −2.11 |
C3orf67 | 1.9E-07 | −3.90 | PLD1 | 0.00055 | −2.08 |
CDA | 0.001 | −3.71 | PCSK5 | 0.00071 | −2.08 |
RHCG | 0.00033 | −3.7 | OSTF1 | 0.00026 | −2.04 |
MTHFD1L | 0.00092 | −3.51 | CDK7 | 0.00082 | −2.01 |
TPRG1 | 0.00081 | −3.43 |
Genes related to “hypersensitivity response”, “immunological disease”, “inflammatory disease”, and “inflammatory response” by IPA annotation are selected to represent immune related genes.
Little is known regarding miRNA regulatory pathways in allergic inflammation in general
In our study 36 miRNAs were differentially expressed (32 upregulated and 4 downregulated) in EoE before and following treatment including 25 miRNAs at a level of 3 fold or more. Differential miRNA expression was correlated with differential mRNA expression in the same patients: 311 genes were found to be downregulated and 35 upregulated when comparing the pretreated to posttreated samples. Several of the mRNAs identified have been shown to play a role in EoE or in other allergic disorders. In general the results of this study are more heavily weighted towards miRNAs with increased expression levels and mRNAs with decreased expression levels in the inflammatory state as opposed to the treated samples. The opposing direction of the miRNA and mRNA expression is in line with miRNA’s regulatory role on gene expression
In an attempt to better define how much of the differential regulation of miRNAs in EoE was related to the effect of the glucocorticosteroid treatment on the allergic process as opposed to the allergic process alone we examined the expression levels of the miRNAs originally detected to those in additional material from EoE patients compared to control tissue from histologically normal appearing esophageal mucosa. The fold changes as detected by qPCR were significantly higher in the pre vs posttreatment tissue than in the EoE vs control tissue and not all of the qRT-PCR differential expression reached statistical significance. The differential expression of certain of these miRNAs such as miR-214, miR-142-3p and miR-29b appear to be influenced much more significantly by the treatment as opposed to the allergic process alone.
Another important issue is to determine which cell types are responsible for the differential expression of miRNAs in this study. We purposely dissected areas of epithelium only and avoided submucosal stromal tissue in many cases employing LCM for this purpose. In addition to eosinophils the squamous epithelium in EoE also exhibits increased infiltration by T-cells and mast cells
A number of miRNAs that were found to be differentially regulated in this study were implicated by others to play a role in allergic and inflammatory disorders
Although miR-214 has been implicated in a number of neoplastic disorders, to date a role for miR-214 in inflammatory or allergic disorders has not been described. MiR-214 has been found to be elevated in models of renal injury and may play a role in epithelial mesenchymal transition
Both miR-146a and miR-146b were markedly increased in the epithelium of EoE patients. MiR-146a has been shown to regulate several physiological and pathophysiological pathways in hematopoietic cells as reviewed by Hua et al
Increased expression of miR-21 was detected in both the pretreated EoE group as opposed to the post-treated group and in the EoE group as oppose to normal controls. MiR-21 is involved in the regulation of many different pathways and is considered an “oncomir” due to its widespread role in neoplasia. In allergic disorders miR-21 has been shown to be involved in the regulation of allergic lung inflammation by targeting IL12a (IL-12p35) a cytokine that contributes to polarization of Th cells toward Th2 cells
MiR-203 down regulated three fold targets the ALOX15 gene (upregulated 10 fold) which encodes for arachidonate 15 lipoxygenase (15-LO). The 15-LO protein is upregulated in asthma
As the human genome may encode over 1000 miRNAs, which may target about 60% of mammalian genes, several immune related genes which were dysregulated in our cohort had no complementary regulatory miRNA identified. Of these genes several have been shown to play a role in the pathophysiology of allergic disorders including EoE. TNFAIP6 the most significantly elevated mRNA in the pretreated biopsies as well as CPA a mast cell derived gene were both shown to be significantly upregulated in a previous study of EoE
Our data implicate several miRNAs and miRNA-MRNA gene sets expressed in the epithelial layer in EoE. The identified gene expression changes provide additional diagnostic and therapeutic targets for EoE and other eosinophilic epithelial diseases.
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