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

Abstract

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 5 544), 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 transcriptionpolymerase chain reaction validation using independent cohorts. MiR-1228 (CV 5 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.MicroRNAs (miRNAs) have drawn significant attention in cancer research after they were linked to oncogenesis and tumor metastasis.1 Owing to their distinct patterns of expression associated with tumor tissues, miRNAs are considered to be highly promising cancer biomarkers. However, miRNAs from tumor tissues can only be obtained after surgery or invasive biopsy, remarkably limiting their clinical values.Recent studies have shown the existence of a large amount of stable miRNAs in human serum/plasma, 2 laying the foundation for studying the role of serum/plasma miRNAs in the diagnosis and prognosis of cancer patients. Studies on the identification of aberrant circulating miRNAs in different diseases are rapidly increasing. Nevertheless, no standard endogenous control exists for normalizing circulating miRNAs.

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“…8A). The amplification of miR-1228 was performed as the negative control because of its stable expression [38]. This is generally consistent with the findings of imaging flow cytometry, which show that LPS promoted miR-590-5p endocytosis (Fig.…”

“…qPCR was performed with miScript SYBR Green qPCR kit (QIAGEN, Dusseldorf, Germany). miR-1228 amplification was performed as was negative control [38]. Beta-actin were amplified as a reference (Fig.…”

microRNA-372 Suppresses Migration and Invasion by Targeting p65 in Human Prostate Cancer Cells

“…The level of miR-372 was quantified by quantitative reverse transcription (RT)-polymerase chain reaction (PCR) using TaqMan MicroRNA Assay Kits (Applied Biosystems, Foster City, CA) with miR-1228 as an internal normalized reference as previously described (Hu et al, 2014). Their relative levels were measured in triplicate on a PRISM 7300 real-time PCR machine (Applied Biosystems).…”

Extracellular Vesicle MicroRNAs: Biomarker Discovery in Various Diseases Based on RT-QPCR

“…The geometric mean of miR-26a, miR-221 and miR-22* Serum TLDA miRNA array geNorm and NormFinder, both [100] Multiple cancer miR-128 Plasma TaqMan geNorm, NormFinder and coefficient of variability (CV), both [101] Lymphoid, colon, prostate, lung and esophagus tumors miR-103, miR-191 Tissue TaqMan geNorm and NormFinder, Figure 1B [102]…”