Establish The Detection Methods Of MiRNA In Tissue/Serum Of NSCLC By Real-time Fluorescence Quantitative PCR And XMAP Bead-based Suspension Array And Their Application Research

Objective Establish the detection methods of microRNA(miRNA) in tissue/serum of NSCLC by Real-time fluorescence quantitative PCR and xMAP bead-based suspension array. Detect the expression of miRNA in tissue/serum of NSCLC by these methods and analyze the expression change of miRNA. Discuss the miRNA's role in the formation and development of NSCLC and its value in NSCLC diagnosis, treatment and prognosis.Methods First part Establish the detection method of miRNA in tissue/serum of NSCLC by Real-time fluorescence quantitative PCR. Detect the expression of miR-21, miR-20a, miR-31, miR-222, miR-223, miR-145, miR-126 in 40 cases of NSCLC cancer tissues and adjacent tissues. Detect the expression of miR-21, miR-20a and miR-222 in 40 cases of NSCLC patients serums and 40 cases of normal controls serums. Analyze the correlation of miR-21, miR-20a and miR-222 in 20 paired cases of NSCLC serums and tissues.Second part Establish the detection method of miRNA in tissue of NSCLC by xMAP bead-based suspension array. Detect the expression of miR-21, miR-20a, miR-31, miR-222, miR-223, miR-145, miR-126 in 40 cases of NSCLC cancer tissues and adjacent tissues. Analyze and evaluate the application of Real-time fluorescence quantitative PCR and xMAP bead-based suspension array in miRNA detection.Results First part The detection method of miRNA by Real-time fluorescence quantitative PCR can do effective quantitative analysis for miRNA not only in NSCLC tissue but also in NSCLC serum. Expression of miR-21, miR-20a, miR-31, miR-222 is higher in NSCLC cancer tissue than in adjacent tissue (P<0.05), in which miR-31, miR-20a are significantly higher expressed and their higher multiples are greater than 2 (P <0.01). Expression of miR-223, miR-145, miR-126 is lower in NSCLC cancer tissue than in adjacent tissue (P<0.05). Expression of miR-21 increases gradually with the progress of disease. MiR-222 expression increases with the decrease degree of differentiation and with the increase tumor size.MiR-126 expression decreases with disease progression and with the decrease degree of differentiation. Expression of miR-21, miR-20a, miR-222 is higher in NSCLC patient serum than in normal control serum, in which miR-222 is significantly higher expressed and its higher multiple is greater than 2. MiR-222 expression increases with disease progression and with the decrease degree of differentiation and with the increase tumor size. Expression of miR-21, miR-20a, miR-222 in NSCLC cancer tissue is positive correlated with those in NSCLC patient serum. Correlation coefficients is 0.898, 0944, 0.946 separately.Second part The detection method of miRNA in tissue of NSCLC by xMAP bead-based suspension array is initially established successfully. Expression of miR-21, miR-20a, miR-31, miR-222 is higher in NSCLC cancer tissue than in adjacent tissue(P<0.05). Expression of miR-145, miR-126 is lower in NSCLC cancer tissue than in adjacent tissue (P<0.05). Expression of miR-223 is no significant difference (P> 0.05). The expression of miR-21, miR-20a, miR-31, miR-222, miR-145 and miR-126 detected by Real-time fluorescence quantitative PCR and by xMAP bead-based suspension array correlate well. Correlation coefficients are 0.631, 0.717, 0.604, 0.810, 0.601, 0.702 separately. MiR-223's correlation coefficient is only 0.105, it may not relevant.Conclusion (1) The detection methods of miRNA in tissue/serum of NSCLC by Real-time fluorescence quantitative PCR and xMAP bead-based suspension array have high specificity and good sensitivity. xMAP bead-based suspension array for miRNA has additional advantages such as less sample required (only 2μl), no sample amplification, high throughput, flexible combination of multiple detection targets and so on. (2) The significant expression of miR-21, miR-20a, miR-31, miR-222, miR-145, miR-126 in NSCLC cancer tissue and adjacent tissue has a very important diagnostic value. (3) Expression of miR-21, miR-20a, miR-222 in NSCLC serum and tissue increase significantly and correlate positively. MiR-21, miR-20a, miR-222 are expected to be serum diagnostic markers of NSCLC. (4) MiR-21, miR-20a, miR-31, miR-222 expression may be high and miR-145, miR-126 expression may be low in the formation and development of NSCLC.