Construction Of Dynamic MiRNA Expression Profiling And Identification Of Serum MicroRNA Biomarkers In COPD

Background:Chronic obstructive pulmonary disease (COPD) is a serious health problem. Its incidence is increasing due to cigarette smoking and air pollution. COPD is characterized by chronic bronchitis and emphysema that progressively limit effective airflow and inflammation in the lungs. More importantly, patients with long term COPD and frequent exacerbation have a high rate of morbidity and mortality. Heavy cigarette smoking is the major risk factor for the development of COPD and accounts for95%of cases. However, heavy cigarette smokers usually have no COPD-related specific symptom at the early stage of the COPD process and there is little information about biomarkers for evaluating COPD development. Therefore, the discovery of new biomarkers for the development of COPD in COPD patients and heavy smokers may help in the prevention and delay of COPD development. miRNAs are endogenous small (about22nucleotides) non-coding RNAs and bind to the3’untranslated region (3’UTR) of targeting mRNAs to inhibit translation and promote mRNA degradation. More than thousands of miRNAs have been discovered and they can regulate many biological processes, such as development, apoptosis, cell proliferation, and haematopoiesis.miRNAs are remarkably stable in plasma and serum and can resist endogenous RNase, varietal storage conditions, and repeating freeze-thaw cycles. Recent studies have validated that some circulating miRNAs act as biomarkers for the evaluation of different diseases, such as lung cancer, gastric cancer, nasopharyngeal carcinoma, sepsis, and pulmonary tuberculosis. However, there is no available information about which serum miRNA(s) are valuable for the evaluation the development of COPD in heavy smokers. Currently, the role of miRNAs in regulating the development of COPD remains unclear. A recent study of serum miRNAs were elevated in a Turkey COPD patients, and found some dysregulated miRNAs. However, the role of miRNAs in regulating the development of COPD remains largely unknown. Furthermore,more research need to identify serum miRNAs as new biomarkers in serum of COPD patients.Objectives:We first established Cigarette smoking-induced dynamic emphysema animal model and observed the changes in the lung pathology. The dynamic miRNA expression profiling in rat model of COPD was established and the dysregulated miRNAs were analysed by bioinformatics methods.This study is aimed at evaluating whether the levels of serum miRNAs can serve as biomarkers for predicting the occurrence of COPD.Methods:1The male Wistar rats were randomly divided into six groups:the healthy control group; the cigarette smoking (CS) for two weeks; the CS for4weeks; the CS for6weeks; the CS for8weeks; and the CS for15weeks plus elastase-treated group. Each group had five rats. The Cigarette smoking-induced emphysema animal model was established and histological examination under a light microscope of lung tissues was performed.2The miRNA microarray analysis of the CS for4weeks group and15weeks plus elastase-treated group was performed.3The dysregulated miRNAs were detected by qRT-PCR for the purpose of microarray data confirming.4The miRNA expression profiling was analysed by bioinformatics methods including target prediction unsupervised hierarchical clustering analysis、GO and Pathway analysis.5The objects of study were divided into healthy subjects、heavy smokers and COPD patients. The serum of blood samples and clinic data were collected.6The expression of serum miRNAs(miR-21、miR-181a、 miR-146a) were detected by qRT-PCR.7The ROC analysis of the levels of serum miR-21and miR-181a was performed.Results:1Animal models of emphysema were established. The dynamic changes in the lung pathology in animal model were inflammatory infiltrates in early stage and emphysema in late stage.2. The dynamic miRNA expression profiling in rat model of COPD was established. There were30and37differentially expressing miRNAs in the lungs of rats with4-weeks and15-weeks of cigarette smoking.3Further qRT-PCR indicated that the relative levels of miR-146a, miR-147, miR-21, miR-92a-2*,and miR-206were significantly up-regulated, while the levels of miR-181a expression were dramatically down-regulated in the lungs of rats.The data was according to the microarray data.4The dysregulated miRNAs may play important role of the development and progression of COPD.5A total of40asymptomatic heavy smokers and49COPD patients as well as age-and gender-matched41healthy controls (HC) were recruited for testing the levels of serum miRNAs. we found that the relative levels of serum miR-21in the asymptomatic heavy smokers and COPD patients were significantly higher than that in the HC (p<0.001). In contrast, the relative levels of serum miR-181a in the asymptomatic heavy smokers and COPD patients were significantly lower than that in the HC (p<0.001). The relative levels of serum miR-21and miRNA-181a were associated negatively with GOLD stages of COPD patients.6The AUC was0.910(95%CI:0.846-0.974) with a sensitivity of85.4%and specificity of85.0%for the ratio of serum miR-21to miR-181a in this population. The results suggest that the ratio of serum miR-21to miR-181a may be a potential biomarker of the early diagnosis of COPD.Conclusion:1The dynamic animal models of emphysema and miRNA expression profiling in rat model of COPD were established successfully.2The dysregulated miRNAs(miR-206、miR-146a、miR-147、 miR-21、miR-181a and miR-92a-2*) may play important role in the pathogenesis of COPD.3Detecting the ratio of serum miR-21to miR-181a expression may be a novel biomarkers of early diagnosis of COPD.26figures,7tables,125references...