Analysis Of Circulating MiR-155 In Asthma Serum By Capillary Electrophoresis

MicroRNAs(miRNAs)are a class of non coding microRNAs,which are involved in a variety of biological processes,such as cell proliferation,apoptosis,differentiation,metabolism,development,tumor metastasis and so on.MiR-155 is closely related to the physiological and pathological processes of many diseases.Our previous studies found that smooth muscle derived exosomes rich in miR-155 induced endothelial injury and promoted atherosclerosis.In bone marrow-derived macrophages,miR-155 promoted inflammation and neointimal hyperplasia by forming a positive feedback loop with TNF-α.MiR-155 could also promote inflammation and oxidative stress by influencing the interaction of MEK,Raf-1 and mammalian mst2.In addition,miR-155 also plays a key role in cellular immune response.Asthma is a common childhood disease.There are 235 million asthma patients in the world,and about 3.5 million children have an asthma attack.At least 50 miRNAs are known to be associated with the progression and prognosis of asthma.Allergic asthma is considered to be a chronic inflammatory disease,which is the result of abnormal activation of type 2helper T cells(Th2)to harmless allergens.It was reported that miR-155 played an important role in the development of asthma by reducing Th2 and regulating inflammatory response.Therefore,we can determine the prognosis of asthma by detecting the content of miR-155 in blood.It is well known that northern blot,RT-q PCR and microarray are the commonly used methods to detect miRNA,among them,RT-q PCR is the most common and sensitive method,but it has the disadvantage of indirect detection of complementaryRNA and high diagnostic cost.Therefore,it is very meaningful to find a rapid and noninvasive method to obtain miRNA directly.Capillary electrophoresis(CE)is an efficient technique for the isolation and identification of miRNAs.In this research,we optimized the detection method of miR-155 by capillary electrophoresis,and applied it to evaluate the severity of childhood asthma and the prognosis of drug treatment.Objective: To optimization of capillary electrophoresis for miR-155 detection in body fluid.To apply in detection of asthmatic children in blood and urine.Methods:1.Optimized method for detecting miR-155 in body fluid using capillary electrophoresis by adjusting the buffer solution ammonium acetate,changing the injection time,separation voltage and buffer solution concentration.2.The miR-155 level in blood and urine of childhood asthma was analyzed by optimized capillary electrophoresis andRT-q PCR.3.The asthmatic mouse model was established,and the asthmatic model was confirmed by biological behavior and morphological staining.4.The miR-155 level in blood of asthma in mice was analyzed by optimized capillary electrophoresis andRT-q PCR.The distribution of miR-155 in lung tissue were detected by FISH.Results:1.The optimized detection conditions of CE were 15 ℃ in a 158 cm(LT)× 50 μ m I.D.× 365 μ m O.D,the buffer solution was ammonium acetate and adjusted to p H = 8,the injection time was 15 s,and the separation voltage was15 k V.The repeatability and intraday and interday repeatability were good.2.miR-155 in serum and urine of asthmatic children was significantly higher than that of normal children using capillary electrophoresis.The expression of serum miR-155 was positively correlated with the severity of asthma.3.miR-155 in serum of asthmatic mice was significantly higher than that of normal mice.FISH results verified the above conclusion.