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MicroRNAs荧光定量PCR检测方法的建立及其在急性肺损伤中的表达分析
Establishment of stem-loop qRT-PCR method to test microRNAs expression level in LPS-induced lung injury

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DOI:
作者:
才志刚
CAI Zhi-Gang*, ZHANG Shao-Ming, ZHANG Heng, ZHOU Y
作者单位:
解放军455医院心胸外科
Department of Cardio-Thoracic Surgery, Number 455 Hospital of The Chinese People’s Liberation Army, Shanghai 200052, China
关键词:
microRNA；A549；LPS；急性肺损伤
acute lung injury; microRNA; stem-loop; PCR
摘要:
MicroRNA (miRNA) 是近几年来新被发现的一类具有转录后调节活性的18-22nt的小分子RNA, 通过在转录后水平负性调节蛋白质表达，miRNA参与了许多重要的细胞活动和生理病理过程。本研究旨在探讨miRNA荧光定量PCR检测方法的建立条件，并运用这一方法对急性肺损伤中miRNAs的表达进行分析。通过设计miRNA特异的颈环结构反转录引物，我们顺利得到miRNA的cDNA，从而实现了miRNA长度的扩大；通过对miRNA cDNA定量PCR过程中的标准曲线和溶解曲线进行分析，表明我们建立的miRNA的荧光定量PCR检测方法具有较高的精确性和特异性；通过对心肌特异表达的miR-1进行检测，表明我们建立的miRNA 反转录-荧光定量检测方法整体具有较高的检测灵敏性和特异性。为了探讨miRNAs在急性肺损伤中的异常表达，我们通过miRNA的荧光定量PCR检测方法对LPS诱发的急性肺损伤小鼠中miRNA的表达进行了分析，结果表明miR-23a、miR-155等多种miRNA在LPS诱发的急性肺损伤异常表达，提示了miRNA可能在急性肺损伤中发挥重要功能。
microRNA (miRNA) was a class of highly conserved, single strand, non-coding small RNA that can regulate the expression of target mRNAs post-transcriptionally either through translational inhibition or destabilization of target mRNAs. A novel miRNA quantification method has been developed using stem-loop RT followed by real time PCR analysis. To investigate the expression level of miRNAs in acute lung injury, we established the miRNA quantification method using Real-time PCR. Verification of the dynamic range and melt curve of qRT-PCR indicated that the method we established here showed highly specificity and precision. By analysis of the expression of miR-1, an muscle-specific miRNA, using our quantification method, we showed, in the four tissues examined here, miR-1 was only detected in heart, suggesting our method is sensitivity and specifity to observe the tissue miRNA expression level. To gain insights into the expression profile of miRNAs in acute lung injury, mouse lung injury model was established using LPS intratracheal administration. Using real-time PCR analysis , we found that a collection of miRNAs was dynamically regulated in LPS induced lung injury, indicating that miRNAs played an important role in the processes of acute lung injury.

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