Association Study of Mir-146a Rs2910164 with Susceptibility to Allergic Rhinitis

Summary

The present study aims to clarify the relationship between the miR-146a rs2910164 polymorphism and allergic rhinitis (AR) susceptibility. Lower plasma miR-146a levels and the rs2910164 GG genotype are linked to elevated AR risk, enhanced inflammatory responses, and more severe clinical manifestations in AR patients.

Abstract

Allergic rhinitis (AR) does not pose a threat to patients' lives, but it seriously impairs their quality of life. MicroRNA-146a (miR-146a) is critical for immune regulation and inflammatory responses; however, the relationship between the miR-146a rs2910164 polymorphism and susceptibility to AR remains incompletely understood. This case-control study was designed to investigate the influence of the miR-146a rs2910164 polymorphism on susceptibility to AR. This study included a total of 180 non-AR volunteers and 211 AR patients. The expression of plasma miR-146a was measured by RT-qPCR; The rs2910164 genotype was detected using the TaqMan-qPCR method. ELISA was used to detect the levels of serum IgE, IL-4, IL-6, and IL-10. Pearson correlation analysis evaluated the linear correlations between the expression of miR-146a and the TNSS score, RQLQ score, as well as the concentration of serum factors. The risk factors of AR were identified through logistic regression. The GG genotype and G allele of miR-146a rs2910164 were significantly more frequent in AR patients than in healthy controls. The plasma miR-146a level was reduced in AR patients. The plasma miR-146a level was lower in AR patients carrying the GG genotype, exhibiting more active allergic and inflammatory responses. The miR-146a expression was closely related to allergic symptoms and the severity of clinical manifestations. Logistic regression indicates that the levels of serum IgE and IL-4 were independent risk factors for AR, IL-10 and miR-146a expression were independent protective factors. Low plasma miR-146a levels are observed in AR patients, and the GG genotype of miR-146a rs2910164 increases susceptibility to AR.

Introduction

Allergic rhinitis (AR) is an allergen-mediated non-infectious inflammatory disorder of the nasal mucosa. The typical clinical symptoms of AR include an itchy nose, nasal congestion, clear nasal discharge, and frequent sneezing¹. In more severe cases, patients may also experience a reduced sense of smell, and even affect the quality of sleep. Epidemiological data indicate that the global prevalence rate of AR is increasing year by year, reaching 10% to 25%². Although AR does not endanger patients' lives, it seriously disrupts sleep, learning, and work, significantly reduces the quality of life, and imposes a heavy economic burden on individuals and society³. The study verified that AR serves as a risk factor for asthma, and as the disease progresses, patients are more likely to develop sinusitis or nasal polyps, further increasing the burden of the disease^4,5. Therefore, prevention and control of AR become a key task in the field of respiratory diseases. However, the pathogenesis of AR has not been fully elucidated. In-depth exploration of the pathophysiological mechanism of AR and the development of targeted treatment strategies hold crucial significance. It is also the current scientific issues that need to be urgently addressed.

The onset of AR is related to environmental and genetic factors⁶. Current research generally holds that the synergistic effect of individual genetic susceptibility and allergen exposure environment is the core driving factor for the onset of AR. The Brozek team further confirmed that the interplay between genetic susceptibility of the body and environmental exposures is the fundamental cause of AR². In the field of AR genetic mechanism research, genetic polymorphism has gradually become the focus of attention for scholars. Single-nucleotide polymorphisms (SNPs), as the prevalent form of genetic polymorphism, are the primary cause of genetic information variation in organisms⁷. Accumulating evidence indicates that the TT genotype at the IL-33 rs7044343 may serve as a potential target for the prevention and treatment of AR⁸. Additionally, the TT genotype at the CD14 promoter C-159T site acts as a predictive marker for the risk of AR⁹. This indicates that the key gene SNPs can serve as important molecular targets for assessing the risk of AR, providing early warnings, and achieving precise prevention and treatment. This provides crucial clues for a deeper understanding of the genetic susceptibility mechanism of AR.

Numerous studies have revealed that abnormal expression of microRNAs (miRNAs) is one of the core driving factors for the occurrence and development of a wide spectrum diseases¹⁰. miRNAs possess the characteristic of stable existence in body fluids, making them highly sensitive molecular markers for disease diagnosis. miR-146a is a type of non-coding RNA molecule with extensive biological functions¹¹. The rs2910164 polymorphism is located within the stem-loop region of the precursor miR-146a sequence. Variation at this site can affect the processing and maturation efficiency of pre-miR-146a, thereby altering the expression level of mature miR-146a and influencing downstream immune and inflammatory responses. Previous studies have confirmed that overexpression of miR-146a can significantly reduce airway hyperresponsiveness in asthma mouse models and inhibit the activation of sensitized lymphocytes¹². By targeting and inhibiting the activity of the epidermal growth factor receptor (EGFR), miR-146a can effectively alleviate the inflammatory damage of sinusitis¹³. Furthermore, overexpression of miR-146a can also alleviate the pathological symptoms of the AR mouse model by blocking the Toll-like receptor 4 (TLR4) signaling pathway¹⁴. These observations highlight the vital role of miR-146a in modulating respiratory system diseases.

Existing evidence from genetic polymorphism studies indicates that the G allele frequency at the miR-146a rs2910164 locus is closely related to airway hyperresponsiveness¹⁵. This SNP is also regarded as a potential biomarker of asthma risk in the Korean population¹⁵. In addition, the Jimenez-Morales team further revealed that the CC genotype at the miR-146a rs2910164 locus conferred protection against asthma¹⁶. However, there were no relevant studies reporting on the association between miR-146a rs2910164 polymorphism and AR susceptibility. Therefore, this study aims to verify the influence of the polymorphism of miR-146a rs2910164 on AR, providing a new theoretical basis for the pathogenesis, clinical diagnosis, and treatment of AR.

Protocol

Ethical statement:
All the participants were fully informed of the purpose of our experiment and signed the informed consent form. This research was approved by the ethics committee of Longyan First Affiliated Hospital of Fujian Medical University (Approval number. LYREC2023-k007-01). All procedures were performed in accordance with relevant guidelines, regulations, and the Declaration of Helsinki.

Participant Recruitment, Eligibility Criteria, and Sample Collection
A total of 211 individuals diagnosed with AR and 180 healthy participants (non-AR) were recruited from the otolaryngology outpatient department of Longyan First Affiliated Hospital of Fujian Medical University between January 2023 and May 2025. The diagnosis criteria for AR were adopted from the Chinese Society of Allergy Guidelines for Diagnosis and Treatment of Allergic Rhinitis¹⁷. Patients were required to exhibit no fewer than two clinical symptoms of AR, including nasal itching, sneezing, nasal congestion, or runny nose. In addition, nasal mucosal examination was required to demonstrate pallor and edema, and either the Skin Prick Test (SPT) result or the serum IgE test result had to be positive. The SPT was performed by dedicated staff in the outpatient allergen testing room to guarantee the accuracy and consistent results. All patients with AR underwent the Rhino conjunctivitis Quality of Life Questionnaire (RQLQ) and Total Nasal Symptom Score (TNSS) assessments with assistance from medical staff, and the scores were recorded for subsequent analysis.

Healthy volunteers undergoing routine physical examinations at the hospital were recruited as the non-AR group. Eligible participants had no personal or family history of AR, no history of asthma or other allergic diseases, no chronic sinusitis or other inflammatory diseases of the nasal cavity, and no respiratory tract infection within the previous month. In addition, individuals with a history of serious cardiac, hepatic, pulmonary, or renal diseases, or any malignant tumors, were excluded from the study.

The peripheral venous blood samples of the two groups of volunteers were collected. A portion of the anticoagulated venous blood was used for DNA extraction and for extracting plasma RNA. Another portion of the venous blood was separated to obtain serum, which was used for the detection of serum-related factors (immunoglobulin E (IgE), interleukin-4 (IL-4), IL-6, and IL-10).

DNA Extraction:
200 µL of peripheral venous blood samples were extracted for DNA using the whole blood genomic DNA extraction kit (Refer to Table of Materials). The blood samples were collected into lithium heparin anticoagulation tubes and then aliquoted and frozen at -20 °C for storage. The specific operation procedures should strictly follow the instructions of the kit. Finally, 100 µL of elution buffer was used to elute the DNA. The purity of the extracted DNA sample was determined by the nucleic acid/protein analyzer (Spectrophotometer) (the OD₂₆₀/OD₂₈₀ ratio was between 1.8 and 2.0). The DNA samples after the detection were stored at -20 °C for future use in the subsequent genotyping experiments.

Genotype Detection
The TaqMan SNP genotyping kit was available for detecting the distribution of miR-146a rs2910164 genotypes. The kit information has been entered into the consumables list. The PCR total reaction volume was 10 µL, containing Master Mix 5 µL, typing probe primer mixture 0.5 µL, template DNA 10 ng, and RNase-free water. DNA amplification was carried out with the Roche LightCycler 480 II real-time fluorescence quantitative PCR instrument. Reaction conditions: Pre-denaturation at 95 °C for 10 min; 40 cycles (95 °C for 15 seconds, 60 °C for 1 min), and the FAM/VIC fluorescence was read at 60 °C. After the amplification was completed, the collected FAM/VIC dual fluorescence signals were analyzed using LightCycler 480 Software, and the genotype data were statistically processed. A single FAM signal represents a homozygous genotype, a single VIC signal represents another homozygous genotype, and the presence of both signals indicates a heterozygous genotype. Samples with abnormal signals or inconsistent results upon repetition will be re-tested. Each experiment was conducted with both template-free control and positive control for quality control. All samples were subjected to three technical replicates.

RT-qPCR
Total plasma miRNA extraction was performed from 100 µL plasma using a dedicated miRNA extraction kit. The kit information has been entered into the consumables list. The RNA concentration was determined using a spectrophotometer. The OD₂₆₀/OD₂₈₀ ratio of the extracted qualified miRNA samples ranged from 1.8 to 2.0. 200 ng of RNA was used for reverse transcription to synthesize cDNA, and the reaction procedure followed the standard instructions of the kit. Reverse transcription was performed in a total volume of 20 µL, including incubation at 42 °C for 60 min and inactivation at 85 °C for 5 min. The qPCR reaction was prepared following the established protocol, with a total volume of 20 µL. The reaction mixture was configured as follows: 10 µL of qPCR pre-mix, 0.4 µL of forward primer, 0.4 µL of reverse primer, 1 µL of cDNA template, and 8.2 µL of RNase-free water. The specific primers for miR-146a and the internal reference gene U6 were used. The amplification was completed using the Roche LightCycler 480 II real-time fluorescence quantitative PCR instrument. The qPCR thermal cycling conditions were: 95 °C for pre-denaturation for 30 s, followed by 40 cycles of denaturation at 95 °C for 5 s and annealing and extension at 60 °C for 30 s. All samples were subjected to three technical replicates. The experimental data were analyzed using the LightCycler 480 Software. Using U6 as the internal reference to correct the inter-sample variation, first calculate ΔCt (the Ct value of miR-146a - the Ct value of U6), then calculate ΔΔCt (the ΔCt of the experimental group - the ΔCt of the control group), and finally the relative expression level of miR-146a was obtained through 2^⁻ΔΔCt.

ELISA
The serum levels of IgE, IL-4, IL-6, and IL-10 were detected by ELISA kits. The experimental procedures were referenced in the instruction manual. Specific steps were as follows: Firstly, the concentrated washing solution was diluted, and the standards were gradiently diluted to prepare working solutions for the standard curve. Secondly, the 50 µL diluent, standard solution, and the sample to be tested were added to the wells and incubated at 37 °C for 60 min. After incubation, the wells were washed three times with washing buffer. Subsequently, 50 µL of enzyme-conjugated antibody working solution was added, and the plates were incubated at 37 °C for 30 min. After the incubation was completed, the liquid was discarded, and the wells were washed again, followed by the addition of chromogenic solution for a 15-min incubation at 37 °C in the dark. After adding the stop solution, the absorbance was measured at 450 nm using a microplate reader. Sample concentrations were calculated according to the standard curve. Each sample was tested three times. Data with a coefficient of variation less than 10% were considered valid. Blank wells and control wells were set up during the experiment for quality control. The detection range and sensitivity of the kit complied with the regulations specified in the manufacturer's manual.

Data statistics
The experimental data were analyzed using SPSS and GraphPad Prism 9 softwares. The data were presented as mean ± standard deviation (mean ± SD). The Shapiro-Wilk test was used to assess the normality of continuous variables, and Levene’s test was applied to evaluate homogeneity of variance. Comparison of continuous variables between the two groups was analyzed using the independent samples t-test. One-way ANOVA followed by Tukey’s post-hoc test was performed for multiple group comparisons, and Two-way ANOVA was applied to evaluate the main effects of two independent factors and their interaction effect. Outliers were identified and excluded based on the 3-standard deviation rule. No missing data were detected in this study. The receiver operating characteristic (ROC) curve was used to analyze the diagnostic efficacy of miR-146a for AR. Relevant ROC parameters and 95% confidence intervals were calculated using built-in statistical algorithms. Pearson correlation analysis was used to measure the linear correlation between miR-146a expression and the levels of serum-related factors. Univariate and multivariate logistic regression analyses were conducted to screen risk factors for AR. Variables with p < 0.05 in univariate analysis were included in the multivariate logistic regression model. p < 0.05 was considered statistically significant.

Results

Participant Characteristics and Genotype Distribution
The clinical baseline data of the two groups of subjects were statistically analyzed. The results indicated that the serum levels of IgE, IL-4, IL-6, and IL-10 showed significant differences. Compared with the non-AR volunteer group, the concentration of serum IgE, IL-4, and IL-6 in the AR patient group was increased, while the IL-10 level was decreased (Table 1). The genotype distribution of miR-146a rs2910164 was detected using ...

Discussion

AR is a chronic allergic inflammatory disease of the respiratory tract. The core mechanism of AR is that after the body is exposed to allergens, inflammatory lesions in the nasal mucosa are mediated by IgE^17,18. The clinical symptoms mainly include nasal itching, nasal congestion, sneezing, and clear nasal discharge, which seriously impair patients’ quality of life. The epidemiological and genetic studies confirmed that genetic susceptibility exerts a vital...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
15mL centrifuge tube	Corning	430791	For plasma separation, sample subpackaging, nucleic acid extraction operation
Applied Biosystems TaqMan Genotyping Master Mix	Thermo Fisher Scientific	4371355	2× concentrated master mix optimized for SNP genotyping, containing hot-start Taq polymerase, dNTPs, magnesium ions, reaction buffer and ROX reference dye. No manual system preparation required. Compatible with the above TaqMan SNP Genotyping Assay, it provides a stable enzymatic reaction system for SNP amplification and fluorescent genotyping with good reproducibility and clear clustering results.
Applied Biosystems TaqMan SNP Genotyping Assay (rs2910164, miR-146a)	Thermo Fisher Scientific	C__15946974_10	Pre-designed primer and probe mix for SNP genotyping of human miR-146a rs2910164 locus. It contains specific PCR primers and two MGB fluorescent probes. VIC labels C allele and FAM labels G allele. It can accurately distinguish three genotypes (CC, CG, GG) in a single reaction tube, suitable for end-point allelic discrimination on qPCR instruments.
BD Lithium Heparin Anticoagulant Blood Collection Tubes	BD Vacutainer	367884	Clinical biochemistry, emergency tests, and hematology research
Graphpad	USA	/	Professional scientific graphing & statistics software, standard for biology/medicine, no coding required. Key strengths: t-tests, ANOVA, survival analysis, nonlinear regression (e.g., dose-response curves); generates publication-ready graphs (bar, scatter, survival curves) for qPCR, Western Blot, cell assays.
Immunoglobulin E (IgE) ELISA Kit	Jiancheng	H108-1-2	Application: Immune response, allergic disease research
Interleukin-10 (IL-10) ELISA Kit	Jiancheng	H010-1-2	Application: Inflammation, immune regulation, clinical biomarker research
Interleukin-4 (IL-4) ELISA Kit	Jiancheng	H005-1-2	Research field: Inflammation, Th2 immune response, allergic reaction
Interleukin-6 (IL-6) ELISA Kit	Jiancheng	H007-1-2	Application: Inflammation, immune regulation, clinical biomarker research
LightCycler 480 Software (v1.5 /v1.6)	Roche Diagnostics	/	Official Roche software that enables one-click qPCR data acquisition, quantitative analysis, melting curve analysis, and SNP genotyping (TaqMan end-point method + HRM).
miRcute Enhanced miRNA cDNA First-Strand Synthesis Kit	TIANGEN	KR211-02	Suitable for all mature miRNA reverse transcription
miRcute Serum/Plasma miRNA Extraction and Isolation Kit	TIANGEN	DP503	Applicable sample: Human serum, plasma, cell supernatant
Roche LightCycler 480 Instrument II (Real-Time PCR System)	Roche Diagnostics	05015278001	qPCR Quantification + SNP Genotyping (including TaqMan) + HRM High-Resolution Melting Curve Analysis
SPSS 27.0	USA	/	All-in-one social science/medical statistics software, menu-driven (no coding), ideal for large samples & complex designs. Core features: descriptive stats, ANOVA, regression, chi-square, survival analysis, reliability, sample size calculation; supports data cleaning & multi-format import (Excel/CSV) for clinical, survey, epidemiological data.
SuperReal PreMix Plus (SYBR Green)	TIANGEN	FP205-02	High-specificity enhanced master mix; optimized buffer with H-Bond factor minimizes primer dimers & non-specifics; hot-start Taq + ROX for LightCycler 480 II; high sensitivity & reproducibility; ideal for low-abundance genes, complex samples, high-throughput genotyping.
Thermo Scientific Multiskan FC Microplate Photometer	Thermo	51119180	High-end multimode plate reader; absorbance (450 nm included) + fluorescence + luminescence; linearity 0–4 Abs (±2%) at 450 nm, precision SD<0.001 Abs; 96/384-well compatible, auto dynamic range; ideal for ELISA, cell viability, immunoassays, molecular interactions.
Thermo Scientific NanoDrop One UV-Vis Spectrophotometer	Thermo	/	Gold-standard microvolume UV-Vis spectrophotometer; 1–2 μL sample, no cuvette needed; directly measures nucleic acids (260 nm), proteins (280 nm), OD600 with concentration & purity ratios (A260/A280, A260/A230); full-spectrum scan 190–850 nm, auto-calibration, touchscreen; ideal for qPCR, NGS, protein purification QC.
Whole blood genomic DNA extraction kit	TIANGEN	DP304-03	Applicable sample: Peripheral whole blood, blood cells, tissue samples