Reference miRNAs for colorectal cancer: analysis and verification of current data

Abstract

MicroRNAs (miRNAs) hold great promise in cancer research. The use of appropriate reference miRNAs for normalization of qPCR data is crucial for accurate expression analysis. We present here analysis and verification of current data, proposing a workflow strategy for identification of reference miRNAs in colorectal cancer (CRC). We performed a systematic review of studies aimed to identify stable reference miRNAs in CRC through high-throughput screening. Among the candidate miRNAs selected from the literature we excluded those predicted to target oncogenes or tumor suppressor gene. We then assessed the expression levels of the remaining candidates in exosomes, plasma and tissue samples from CRC patients and healthy controls. The expression stability was evaluated by box-plot, ∆Cq analysis, NormFinder and BestKeeper statistical algorithms. The effects of normalisers on the relative quantification of the oncogenic miR-1290 was also assessed. Our results consistently showed that different combinations of miR-520d, miR-1228 and miR-345 provided the most stably expressed reference miRNAs in the three biological matrices. We identified suitable reference miRNAs for future miRNA expression studies in exosomes plasma and tissues CRC samples. We also provided a novel conceptual framework that overcome the need of performing ex novo identification of suitable reference genes in single experimental systems.

Plasma miRNA‐based signatures in CRC screening programs

“…found that the plasma levels of miR‐423‐5p correlated inversely with CRC progression, including adenomas and polyps, suggesting its potential as a biomarker for CRC, particularly in the early stages . Finally, one of the four miRNAs we identified as reference for our analyses, miR‐345‐5p, has already been proposed as reference miRNA for CRC analyses in a plasma circulating setting …”

“…51 Finally, one of the four miRNAs we identified as reference for our analyses, miR-345-5p, has already been proposed as reference miRNA for CRC analyses in a plasma circulating setting. 52 As reviewed by Slaby et al, 53 many studies have examined the feasibility of using circulating miRNAs as biomarkers for the early detection of CRC, but in most cases there is minimal overlap in the identified miRNAs. In fact, there is variability among studies, and the reproducibility of results can be influenced by multiple factors, including the number of samples and the number of miRNA tested, different experimental procedures and methods, normalization approaches, differences in patient populations and instrumentation.…”

TLR-mediated miR-125b-5p downregulation enhances CD248-induced metastasis and drug resistance in colorectal cancer cells

“…MicroRNAs (miRNA), small noncoding RNAs, bind to the 3' untranslated region (3' UTR) of their messenger RNA (mRNA) targets for posttranscriptional gene regulation that inhibits translation or mRNA degradation [9][10][11]. Thus, changing the expression patterns of either oncogenes or tumor suppressor genes via miRNAs plays an important role in carcinogenesis.…”

Free Circulating miRNAs Measurement in Clinical Settings

“…In a recent study by Danese et al, the combination of hsa-miR-1228 and hsa-miR-520d was identified as the best reference to normalize RT-qPCR miRNA analysis in colorectal adenocarcinoma from exosome, plasma, and tissue samples. These two miRNAs were selected among six potential reference genes (hsa-miR-191, hsa-miR-193a, hsa-miR-345, hsa-miR-520d, and hsa-miR-1228) based on their stability assessed trough both Norm-Finder and BestKeeper algorithms [100]. Song et al, in a study on serum miRNAs from gastric cancer patients, have identified hsa-miR-16 and hsa-miR-93 as reference genes.…”