通过检测红细胞特异性微小RNA标志物进行粪便潜血检测的新方法。
Novel Approach to Fecal Occult Blood Testing by Assay of Erythrocyte-Specific microRNA Markers.
作者信息
Wu Chung Wah, Cao Xiaoming, Berger Calise K, Foote Patrick H, Mahoney Douglas W, Simonson Julie A, Anderson Bradley W, Yab Tracy C, Taylor William R, Boardman Lisa A, Kisiel John B, Ahlquist David A
机构信息
Division of Gastroenterology and Hepatology, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA.
Division of Biomedical Statistics and Informatics, Mayo Clinic, 200 First Street SW, Rochester, MN, 55905, USA.
出版信息
Dig Dis Sci. 2017 Aug;62(8):1985-1994. doi: 10.1007/s10620-017-4627-6. Epub 2017 Jun 28.
BACKGROUND
Fecal occult blood testing (FOBT) has historically relied on methods to detect hemoglobin with no fundamental innovations in decades.
AIM
To examine microRNA (miRNA) as a new marker class for FOBT.
METHODS
Candidate miRNA markers were identified by small RNA sequencing of human whole blood compared to colorectal epithelia. Markers were tested in human blood cell subsets and blood from non-human species. We assessed assay linearity in blood spiking and marker stability in stool over incubation experiments. Levels of candidate erythrocyte markers were explored in stools from colorectal cancer (CRC) cases and controls.
RESULTS
Based on small RNA sequencing and validation RT-qPCR, expression level of each of the top blood-enriched markers (hsa-miR-144-3p, 144-5p, 451a, 486-5p, 363-3p, 20b-5p) could perfectly discriminate blood from colorectal epithelia. All six markers arose from and showed specificity to human erythrocytes. Marker levels increased linearly with erythrocyte concentration in saline or stool and demonstrated a broader dynamic range than did immunochemical test for hemoglobin. Degradation of markers occurred in stool but was reduced with preservative buffers. Erythrocyte marker candidates for stool testing were selected in an exploratory set of stools (20 CRC, 40 normal). Candidates were then further tested in a feasibility set (29 CRC, 31 advanced adenoma, and 115 normal); a miRNA panel (hsa-miR-451a, 144-5p, and 200b-3p as normalizer) yielded an AUC of 0.89 (95% CI 0.82-0.95, P < .0001) for CRC.
CONCLUSIONS
A novel miRNA-based approach accurately quantifies fecal blood levels over a broad, clinically relevant range.
背景
粪便潜血检测（FOBT）长期以来一直依赖于检测血红蛋白的方法，数十年来没有根本性的创新。
目的
研究将微小RNA（miRNA）作为FOBT的一种新型标志物类别。
方法
通过对人类全血与结直肠上皮进行小RNA测序来鉴定候选miRNA标志物。在人类血细胞亚群和非人类物种的血液中对标志物进行检测。我们在血液加样实验中评估了检测的线性，并在粪便孵育实验中评估了标志物的稳定性。在结直肠癌（CRC）病例和对照的粪便中探索了候选红细胞标志物的水平。
结果
基于小RNA测序和验证性逆转录定量聚合酶链反应（RT-qPCR），每种血液富集程度最高的标志物（hsa-miR-144-3p、144-5p、451a、486-5p、363-3p、20b-5p）的表达水平能够完美地区分血液与结直肠上皮。所有六种标志物均来自人类红细胞并对其具有特异性。标志物水平随生理盐水或粪便中红细胞浓度呈线性增加，并且与血红蛋白免疫化学检测相比显示出更宽的动态范围。标志物在粪便中会发生降解，但使用保存缓冲液可减少降解。在一组探索性粪便样本（20例CRC、40例正常样本）中选择了用于粪便检测的红细胞标志物候选物。然后在一组可行性样本（29例CRC、31例高级别腺瘤和115例正常样本）中对候选物进行进一步检测；一个miRNA检测组合（以hsa-miR-451a、144-5p和200b-3p作为标准化物）对CRC的曲线下面积（AUC）为0.89（95%可信区间0.82 - 0.95，P <.0001）。
结论
一种基于miRNA的新方法能够在广泛的临床相关范围内准确量化粪便中的血液水平。
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