J Cancer 2020; 11(23):7032-7044. doi:10.7150/jca.48903 This issue

Research Paper

Identification of miR-4644 as a suitable endogenous normalizer for circulating miRNA quantification in hepatocellular carcinoma

Jun Zhao1,2, Xin-Chao Zhu1,2, Xiao-Song Wu1,2, Lin Wang1,2, Can-Can Zhu1, Ke Yang1, Guo-Qing Deng1, An Wang1, Yong Liu1, Wei-Dong Jia3✉, Ling Zhu1✉

1. Center of Engineering Technology Research for Biomedical Optical Instrument, Anhui Institute of Optics and Fine Mechanics, Hefei Institutes of Physical Science, Chinese Academy of Sciences, Hefei 230031, China.
2. Science Island Branch of Graduate School, University of Science and Technology of China, Hefei 230026, China.
3. Department of General Surgery, Anhui Provincial Hospital & the First Affiliated Hospital of USTC, Division of Life Science and Medicine, University of Science and Technology of China, Hefei 230001, China.

This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.
Zhao J, Zhu XC, Wu XS, Wang L, Zhu CC, Yang K, Deng GQ, Wang A, Liu Y, Jia WD, Zhu L. Identification of miR-4644 as a suitable endogenous normalizer for circulating miRNA quantification in hepatocellular carcinoma. J Cancer 2020; 11(23):7032-7044. doi:10.7150/jca.48903. Available from https://www.jcancer.org/v11p7032.htm

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Background: Circulating microRNAs (miRNAs) have proved to be promising biomarkers for early diagnosis and therapeutic monitoring in cancers. Particularly for hepatocellular carcinoma (HCC), detection of circulating miRNA biomarkers as a new diagnostic approach has been written into the latest Guidelines for Diagnosis and Treatment of Primary Liver Cancer in China (2019 edition). However, no general consensus on an ideal endogenous normalizer for circulating miRNAs quantification has been reached, so it will affect the accuracy of quantitative results. In this study, we aim to identify a stable endogenous normalizer for analyzing circulating miRNAs.

Methods: Candidate miRNAs were selected by screening dataset GSE104310, as well as data statistics and analysis. Five commonly reference genes were chosen for further comparison and verification. Then, the expression levels of these genes in serum were analyzed by quantitative reverse transcription PCR (RT-qPCR) among four groups, including patients diagnosed with HCC, chronic hepatitis B (CHB), liver cirrhosis, and healthy subjects. Furthermore, the stability of target genes was evaluated using geNorm, NormFinder, comparative ΔCq programs, and validated by database. We also explored the availability of the miRNA combination, and compared the performance difference between combination and individuals, as well as the selectivity of miRNA references in the combinations.

Results: 11 candidate miRNAs were obtained, and miR-4644 stood out among these miRNAs, and proved to be much more stable than other endogenous miRNAs. Further study showed that miR-4644 exhibited higher stability and expression abundance than other commonly miRNA reference controls. Finally, we discovered the combination of miR-4644 and miR-16 revealed high performance in stability when compared to miRNA individuals. Furthermore, the combination consisted of references with closer nature could give rise to amplification effects in stability.

Conclusions: Our findings demonstrated that miR-4644 is an ideal endogenous normalizer for circulating microRNA quantification in hepatocellular carcinoma. Besides, combining miR-4644 with miR-16 into a whole as a reference control would greatly improve the accuracy of quantification.

Keywords: circulating microRNA, endogenous control, normalization in RT-qPCR, hepatocellular carcinoma