Association Between Single-Nucleotide Polymorphisms in Pre-miRNAs and the Risk of Asthma in a Chinese Population

Introduction

A sthma is a common serious health problem throughout the world that affects all ages. The precise etiopathogenesis of asthma remains uncertain. Several reports have suggested that exposure to potentially harmful environmental factors may contribute to asthma (Vazquez Nava et al., 2008; Chen et al., 2009; Juhn et al., 2010; Midodzi et al., 2010; Uthaisangsook, 2010). However, more people in the same environment do not suffer from asthma indicating that genetic factors play important roles in its pathogenesis (Skadhauge et al., 1999; Moffatt, 2008; van Beijsterveldt and Boomsma, 2008; Vercelli, 2008).

microRNAs (miRNAs) known as non–protein-coding or nonmessenger RNAs are diverse molecules with structural, enzymatic, and regulatory functions (Ambros, 2004). miRNAs can regulate gene expression by translational inhibition and destabilization of mRNAs (Kloosterman and Plasterk, 2006). Some miRNAs regulate remodeling of human airway smooth muscle cells in severe asthma (Mohamed et al., 2010). Expression changed through TLR4- or MyD88-dependent mechanisms when the airway was exposed to dust mites. However, changes in miRNA expression did not appear to be involved in the development of a mild asthmatic phenotype or in the anti-inflammatory action of the corticosteroid budesonide (Williams et al., 2009). On the other hand, studies have demonstrated correlation of specific miRNAs with survival of lung adenocarcinoma (Yanaihara et al., 2006).

Regulation of miRNA expression and maturation remains poorly defined. However, previous studies suggest that single-nucleotide polymorphisms (SNPs) may regulate the processing of mature miRNA (Duan et al., 2007). Small changes in the amount of miRNAs may have an effect on thousands of target mRNAs and result in diverse functional consequences. SNPs in pre-miRNA sequences, therefore, may also be functional and may represent ideal biomarkers (Hu et al., 2008) for related diseases. Recently, several reports showed that SNPs in pre-miRNA sequences, especially the four SNPs rs11614913, rs3746444, rs2910164, and rs229283 in pre-miRNAs hsa-mir-196a2, hsa-mir-499, hsa-mir-146a, and hsa-mir-149, were associated with the risk of respiratory system disease (Hu et al., 2008; Tian et al., 2009; Wang et al., 2010) and other diseases (Xu et al., 2009, 2010; Christensen et al., 2010; Dou et al., 2010; Qi et al., 2010). However, the association between these SNPs and asthma has not been explored.

Materials and Methods

The present study was performed with the approval of the ethics committee of Sichuan University and all the participants provided written informed consent. The study included 220 unrelated asthma patients and 540 healthy controls. The demographics of the patients and controls enrolled in this study are shown in Table 1.

All the patients were recruited from West China Hospital, Sichuan University, from July 2005 to March 2010. Diagnosis of asthma was based on at least a 1-year duration of wheeze, shortness of breath, cough, or chest tightness, together with lung function measurements showing significant reversibility to bronchodilator (increase of ≥12% [and 200 mL] in 1-s forced expiratory volume after delivering 400 μg albuterol by a metered dose inhaler with a spacer) (Ko et al., 2010). The controls were from local unrelated populations and screened for a history of asthma or other pulmonary diseases. The controls were frequency matched with cases according to sex and age. Blood was taken from all participants by peripheral venous puncture and stored at −20°C with the anticoagulating agent ethylenediaminetetraacetic acid (EDTA) until analysis.

Genomic DNA of each individual was extracted from 200 μLEDTA–added peripheral blood samples using a DNA isolation kit (Bioteke, Peking, China) according to the manufacturer's instructions. The DNA concentration was determined using a NanoDrop™ 1000 Spectrophotometer (Thermo Fisher Scientific, Copenhagen, Denmark). Genotyping was performed using polymerase chain reaction (PCR)–restriction fragment length polymorphism analysis. The PCRs were performed in a total volume of 25 μL containing 50 ng genomic DNA, 20 pM of each primer, 0.2 mM dNTPs, 2.5 μL of 10 × PCR buffer, 1.5 mM MgCl₂, and 0.3 unit of Taq polymerase (Tiangen Biotech, Peking, China). PCR products were digested with specific restriction enzyme according to the manufacturer's instructions. Primer sequences, reaction conditions, and restriction enzymes used have been previously described (Hu et al., 2008; Zhou et al., 2010). To confirm the genotyping results, PCR products were examined by DNA sequencing. The results were in 100% concordance.

The PCR and digestion products were analyzed directly by vertical nondenaturing 5% polyacrylamide gel electrophoresis with 1 × tris boronate and EDTA buffer (TBE) continuous buffer system and observed by 1.0 g/L argent nitrate staining.

The data analysis was carried out using SPSS 11.5 statistical software. Allele and genotype frequencies of these four SNPs were obtained by directed counting. The odds ratio (OR) and 95% confidence intervals (CI) were calculated to assess the relative risk conferred by a particular allele and genotype. A p-value of <0.05 was considered statistically significant.

Result

A few samples failed to genotype at a given locus even after repeating three times. Therefore, the numbers of samples were different for each of the polymorphisms. The genotype and allele frequencies of these four polymorphisms in the two study groups are listed in Tables 2 and 3. There were significant differences in the genotype and allelic distribution of rs2910164G/C and rs2292832C/T polymorphisms among cases and controls. The CC genotype and C allele of rs2910164G/C were significantly associated with a decreased risk of asthma (CC vs. GG, OR = 0.51, 95% CI: 0.31–0.82; C vs. G, OR = 0.74, 95% CI: 0.59–0.93). Similarly, the TT genotype and T allele of rs2292832C/T were significantly associated with a decreased risk of asthma (TT vs. CC, OR = 0.56, 95% CI: 0.33–0.95; T vs. C, OR = 0.71, 95% CI: 0.53–0.95). However, no significant association between the other two polymorphisms (i.e., rs11614913C/T and rs3746444C/T) and the risk of asthma was observed. The statistical power in the present study was more than 80%, with an estimated OR of 1.8 (rs11614913C/T: 89%; rs3746444C/T: 93%; rs2910164G/C: 87%; and rs2292832C/T: 89%).

Discussion

We found that hsa-mir-146a rs2910164 and hsa-mir-149 rs2292832 but not hsa-mir-196a2 rs11614913 and hsa-mir-499 rs3746444 were associated with a decreased risk of asthma in a Chinese population.

In recent years, several studies focused on the association between miRNA and the increased risk of respiratory system disease. Stretch induces human airway smooth muscle cell hypertrophy through miRNA upregulation and alteration of specific miRNA expression, which has been demonstrated using an miRNA array screen (Mohamed et al., 2010). Also, using lung cell lines such as A549, PC9, and H1299 of lung cancer patients, 39 miRNAs were identified with significantly different expression levels (Wang et al., 2009). For allergic airway disease, exposure of the airway to house dust mites increases the expression of specific miRNAs in the airway through TLR4- or MyD88-dependent mechanisms (Mattes et al., 2009). Also, Lu et al. (2009) reported that 131 miRNAs were highly expressed following IL-13 induction in IL-13 transgenic mice. Additionally, it has been noted that miRNA transcription remains unchanged during lung aging and that stable expression of miRNAs might instead buffer age-related changes in the expression of protein-encoding genes (Williams et al., 2007). In the present study, we hypothesized that the SNPs in pre-miRNAs could alter miRNA expression levels or their maturation process and, thus, contribute to asthma pathogenesis. All four SNPs in the present study were located in pre-miRNA regions (Hu et al., 2008). The allele frequencies of the four SNPs in other ethnic populations are listed in Table 4. Previous studies investigated the correlation of these four SNPs with head and neck squamous cell carcinoma (Christensen et al., 2010), dilated cardiomyopathy (Zhou et al., 2010), glioma (Dou et al., 2010), median survival time of lung cancer (Hu et al., 2008), and other diseases. However, there was no report about the correlation of these four SNPs and asthma.

In conclusion, the present study suggests that genetic factors played important roles in the pathogenesis of asthma and provides evidence that hsa-mir-146a rs2910164 and hsa-mir-149 rs2292832 but not hsa-mir-196a2 rs11614913 and hsa-mir-499 rs3746444 may be associated with asthma. Although the present study had considerable statistical power, weak associations may have been missed. In addition, the present study did not determine the specific mechanism of how these mutations decrease the risk of asthma. Further larger-scale studies will be needed to explore the complicated interaction between pre-miRNAs polymorphisms and the susceptibility to asthma in different populations.