Evaluating normalization approaches for the better identification of aberrant microRNAs associated with hepatocellular carcinoma

Abstract

Aim Dysregulated microRNAs (miRNAs) have been identified in hepatocellular carcinoma (HCC), but only a small proportion have been confirmed. An appropriate normalizer is crucial to determining the accuracy and reliability of data from miRNA studies. Methods Different normalization strategies were used to validate genome-wide miRNA profiles in HCC tumor and non-tumor tissues, and to determine the consistency and discrepancy of data on dysregulated miRNAs. Results Two sets of stable miRNAs (miR-30c/miR-30b and miR-30c/miR-126) were identified in HCC tissues by geNorm and NormFinder tools, respectively. The mean of global miRNAs also showed good stability for ranking the top 1-2 miRNAs, but the stabilities of the manufacturer-recommended ncRNAs controls were poor. Four panels of miRNAs were significantly associated with HCC by separately using various normalizers, and 14 miRNAs were consistently identified by three normalization strategies. Although fewer miRNAs (17-26) were dysregulated in HCC using the global mean or the 2 stable miRNAs as normalizers, perfect clustering of tissues was also obtained with only 1 to 2 misclassifications, suggesting the efficiency of the miRNA panels. Using global mean as the normalizer, the authors identified 7 miRNAs, including 2 novel (miR-324-5p and miR-550) significantly upregulated in HCC that were omitted when using 3 endogenous controls as the normalizer. Conclusion An optimal normalization strategy to identify biologically important miRNAs in HCC tissue studies of miRNA may be the combination of global mean and 2 stable miRNAs. Selection of appropriate normalization strategies to adjust miRNAs levels is particularly important for epidemiological studies dealing with large data sets and covering multiple experimental batches.

Role of microRNAs in the main molecular pathways of hepatocellular carcinoma

“…A recent study of our group showed how the miRNA profile of HCCs changed using a pool of cirrhotic tissues or a pool of healthy livers as non-tumor controls[ 121 ]. Other authors suggested to employ stable miRNAs as controls for the study of the expression of other miRNAs[ 122 ]. In the light of the available data, it would be useful to elaborate, based on the most representative miRNA in in vivo model ( e.g ., rat, mouse), to better understand the possible role of these molecules as theragnostic markers in HCC.…”

Reduced expression of miR-30c-5p promotes hepatocellular carcinoma progression by targeting RAB32

“… 13 , 14 , 16 , 17 It was reported that aberrant expression of miR-30c was a potential early diagnostic marker in HCC. 18 , 24 Meanwhile, miR-30c downregulation was associated with HCV core protein-induced EMT in HCC cells. 25 In our study, we demonstrated that the expression of miR-30c-5p is downregulated in HCC, and reduced miR-30c-5p expression predicts poor prognosis in HCC patients.…”

Evaluation of endogenous miRNA reference genes across different zebrafish strains, developmental stages and kidney disease models

“…Dre-miR-30c-5p is known as a key player in pronephric development in Xenopus laevis and Danio rerio, where it shows a pronephros specific expression within the first days of development 34,35 . It has also been used as an endogenous reference gene before 36 . Unfortunately, there were significant dre-miR-30c-5p expression differences between different developmental stages, meaning that it had to be rejected as a suitable normalizer for that kind of experimental setups.…”