Human miR‐1228 as a stable endogenous control for the quantification of circulating microRNAs in cancer patients

• Published in: International journal of cancer Vol. 135; no. 5; pp. 1187 - 1194
• Main Authors: Hu, Jie, Wang, Zheng, Liao, Bo‐Yi, Yu, Lei, Gao, Xue, Lu, Shaohua, Wang, Shuyang, Dai, Zhi, Zhang, Xin, Chen, Qing, Qiu, Shuang‐Jian, Wu, Ying, Zhu, Hongguang, Fan, Jia, Zhou, Jian, Wang, Jiping
• Format: Journal Article
• Language: English
• Published: Hoboken, NJ Wiley-Blackwell 01.09.2014; Wiley Subscription Services, Inc
• Subjects: Adult; Biological and medical sciences; Biomarkers; Biomarkers, Tumor - blood; Biomarkers, Tumor - genetics; Breast cancer; Cancer; circulating microRNA; Cohort Studies; Control Groups; endogenous control; Female; Gene Expression Profiling; Genes, Essential; Hematology; Hepatitis B, Chronic - blood; Hepatitis B, Chronic - genetics; Humans; Male; Medical research; Medical sciences; MicroRNAs; MicroRNAs - blood; MicroRNAs - genetics; MicroRNAs - metabolism; Middle Aged; Multiple tumors. Solid tumors. Tumors in childhood (general aspects); Neoplasms - blood; normalization; Oligonucleotide Array Sequence Analysis; qRT‐PCR; Real-Time Polymerase Chain Reaction; Signal Transduction; Tumors; Human; RNA interference; Micro RNA; qRT-PCR; Quantization; Malignant tumor; endogenous control; Gene silencing; Cancerology; Endogenous; normalization; circulating microRNA; Cancer; Real time polymerase chain reaction; cancer
• ISSN: 0020-7136; 1097-0215; 1097-0215
• DOI: 10.1002/ijc.28757

Circulating microRNAs are promising biomarkers for non‐invasive testing and dynamic monitoring in cancer patients. However, no consensus exists regarding the normalization of circulating microRNAs in the quantification, making the results incomparable. We investigated global circulating microRNA profiles to identify a stable endogenous control for quantifying circulating microRNAs using three cohorts (n = 544), including 168 control individuals (healthy subjects and those with chronic hepatitis B and cirrhosis) and 376 cancer patients (hepatocellular, colorectal, lung, esophageal, gastric, renal, prostate, and breast cancer patients). GeNorm, NormFinder, and coefficient of variability (CV) were used to select the most stable endogenous control, whereas Ingenuity Pathway Analysis (IPA) was adopted to explore its signaling pathways. Seven candidates (miR‐1225‐3p, miR‐1228, miR‐30d, miR‐939, miR‐940, miR‐188‐5p, and miR‐134) from microarray analysis and four commonly used controls (miR‐16, miR‐223, let‐7a, and RNU6B) from literature were subjected to real‐time quantitative reverse transcription‐polymerase chain reaction validation using independent cohorts. MiR‐1228 (CV = 5.4%) with minimum M value and S value presented as the most stable endogenous control across eight cancer types and three controls. IPA showed miR‐1228 to be involved extensively in metabolism‐related signal pathways and organ morphology, implying that miR‐1228 functions as a housekeeping gene. Functional network analysis found that “hematological system development” was on the list of the top networks that associate with miR‐1228, implying that miR‐1228 plays an important role in the hematological system. The results explained the steady expression of miR‐1228 in the blood. In conclusion, miR‐1228 is a promising stable endogenous control for quantifying circulating microRNAs in cancer patients. What's new? While circulating microRNAs (miRNAs) are promising cancer biomarkers, a standard control for the normalization of serum/plasma miRNA levels is yet to be established. Without such a control, data from different studies and different cancers remains incomparable. Here, analysis of global circulating microRNA profiles in healthy individuals and cancer patients suggests that miR‐1228, one of seven candidates identified from microarray analysis, is a stable endogenous control for the quantification of circulating miRNAs in cancer patients. MiR‐1228 allows for the comparison of circulating miRNA expressions in the same cancer across different studies and in different cancers of the same study.