细粒棘球蚴病患者外周血微小RNA的差异表达分析及其特异性诊断标志物的筛选

摘要/Abstract

摘要：

目的对细粒棘球蚴病（cystic echinococcosis,CE）患者外周血微小RNA（microRNA,miRNA）进行差异性表达分析,并筛选新型潜在诊断标志物。方法提取3例临床确诊的CE患者血清和3名健康者血清miRNA,采用芯片Affymetrix miRNA4.0对血清中miRNA进行高通量筛选,通过生物信息学分析,找出差异性的miRNA。采用实时荧光定量PCR（qRT-PCR）对21例CE患者血清与25名健康者血清的差异miRNA进行验证分析。采用Pearson’s相关性分析法分析miRNA与包囊重量或子囊个数的相关性。采用IBM SPSS 19.0软件分析不同miRNA的受试者工作特征曲线（receiver operating characteristic curve,ROC）与曲线下面积（area under curve,AUC）。结果芯片对比分析结果显示,与健康者血清比较,CE患者血清中有172个miRNA上调2倍以上,35个下调1/2以下。qRT-PCR结果表明,hsa-miR-762、hsa-miR-3536、hsa-miR-6125、hsa-miR-6821-5P、hsa-miR-663a、hsa-miR-24-3P、hsa-miR-191-5P、hsa-miR-320a 等8个上调miRNA的表达水平分别为10.28 ± 0.38、5.11 ± 0.34、11.02 ± 0.55、11.87 ± 0.58、5.31 ± 0.36、5.75 ± 0.51、5.75 ± 0.51、4.96 ± 0.36,均高于其对应的健康组（P < 0.05）。ROC曲线分析结果表明,8个miRNA的AUC均大于0.8,提示这些分子具有潜在的临床诊断价值。相关性分析结果显示,miR-24-3p与包囊重量和子囊数量均呈正相关（r = 0.525 0,P < 0.05;r = 0.617 5,P < 0.05）。结论筛选出的8个宿主miRNA在CE患者血清中有差异性表达,具有潜在的临床诊断价值,其中miR-24-3p还可能与CE的发生发展相关。

关键词: 细粒棘球蚴病, miRNA, 生物标志物, miR-24-3p

Abstract:

Objective To analyze the differential expression of circulating microRNAs (miRNAs) in patients with cystic echinococcosis (CE) and screen for potential diagnostic biomarkers for CE. Methods Serum miRNAs were extracted from 3 CE patients and 3 healthy controls (HCs) and analyzed with miRNA microarray using an Affymetrix miRNA4.0 chip to find miRNAs with differential expression. Results were verified on sera from 21 CE patients and 25 HCs using real-time PCR (qRT-PCR). Correlation between the miRNA and hydatid cyst weight or the number of daughter cysts was analyzed with Pearson’s correlation analysis. The IBM SPSS 19.0 software was used to generate the receiver operating characteristic curve (ROC) and analyze the area under curve (AUC). Results of microarrays revealed 172 miRNAs with an over 2-fold increase and 35 miRNAs with an over 50% decrease in CE sera as compared to the HC. qRT-PCR further quantitated the expression of hsa-miR-762, hsa-miR-3536, hsa-miR-6125, hsa-miR-6821-5P, hsa-miR-663a, hsa-miR-24-3P, hsa-miR-191-5P and hsa-miR-320a to be 10.28 ± 0.38, 5.11 ± 0.34, 11.02 ± 0.55, 11.87 ± 0.58, 5.31 ± 0.36, 5.75 ± 0.51, 5.75 ± 0.51 and 4.96 ± 0.36, respectively, all significantly higher than their HC counterparts (P < 0.05). ROC analysis showed that the AUC values of these 8 miRNA candidates were all above 8, implying their diagnostic potential for CE. Moreover, correlation analysis indicated that only miR-24-3p was positively correlated with both cyst weight (r = 0.5250, P < 0.05) and the number of daughter cysts (r = 0.6175, P < 0.05). Conclusion Eight circulating miRNAs have been identified as potential biomarkers for diagnosis of CE. MiR-24-3p may correlate with the progression and pathogenesis of CE.

Key words: Cystic echinococcosis, miRNA, Biomarker, MiR-24-3p

中图分类号:

R532.32

任梦迪, 宁玉烨, 黎明, 王浩, 赵巍. 细粒棘球蚴病患者外周血微小RNA的差异表达分析及其特异性诊断标志物的筛选[J]. 中国寄生虫学与寄生虫病杂志, 2017, 35(5): 423-428.

Meng-di REN, Yu-ye NING, Ming LI, Hao WANG, Wei ZHAO. Differential expression of circulating microRNAs in patients with cystic echinococcosis and screening for specific diagnostic biomarkers for the disease[J]. Chinese Journal of Parasitology and Parasitic Diseases, 2017, 35(5): 423-428.

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引物名称 Primer name	序列 (5′→3′) Sequence (5′→3′)	长度/nt Length/nt
hsa-miR-762（S）	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCTCGG	50
hsa-miR-762（F）	TATTGAGGGGCTGGGGCC	18
hsa-miR-762（R）	CCAGTGCAGGGTCCGAGGTA	20
hsa-miR-3536（S）	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCTCCC	50
hsa-miR-3536（F）	TATATCGGCGGGTGCGGG	18
hsa-miR-3536（R）	CCAGTGCAGGGTCCGAGGTA	20
hsa-miR-6125（S）	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCCGCC	50
hsa-miR-6125（F）	CACACAGCGGAAGGCGGA	18
hsa-miR-6125（R）	CCAGTGCAGGGTCCGAGGTA	20
hsa-miR-6821-5P（S）	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCCCCGC	50
hsa-miR-6821-5P（F）	TACACAGTGCGTGGTGGC	18
hsa-miR-6821-5P（R）	CCAGTGCAGGGTCCGAGGTA	20
hsa-miR-663a（S）	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACGCGGTC	50
hsa-miR-663a（F）	CTTCTAAGGCGGGGCGCC	18
hsa-miR-663a（R）	CCAGTCCAGGGTCCGAGGTA	20
hsa-miR-24-3P（S）	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCTGTTC	50
hsa-miR-24-3P（F）	CGCGCATGGCTCAGTTCA	18
hsa-miR-24-3P（R）	CCAGTGCAGGGTCCGAGGTA	20
hsa-miR-191-5P（S）	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACCAGCTG	50
hsa-miR-191-5P（F）	CACAGTCAACGGAATCCC	18
hsa-miR-191-5P（R）	CCAGTGCAGGGTCCGAGGTA	20
hsa-miR-320a（S）	GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCGCCC	50
hsa-miR-320a（F）	CGCAGCAAAAGCTGGGTT	18
hsa-miR-320a（R）	CCAGTGCAGGGTCCGAGGTA	20