No abstract

Circulating miRNAs are intensively evaluated as promising blood-based biomarkers. This growing interest in developing assays for circulating miRNAs necessitates careful consideration of the effects of preanalytical and analytical parameters on the isolation, stability, and quantification of circulating miRNAs. By using quantitative stem-loop RT-PCR, we compared the relative efficiencies of four miRNA isolation systems and different storage conditions. The effect of the data normalization procedure on the quantification of circulating miRNA levels in plasma from 30 healthy individuals and 30 patients with non-small cell lung carcinoma was estimated by measuring endogenous hsa-miR-21 and hsa-miR-16 and exogenous cel-miR-39 that was spiked in all samples at the same concentration. Silica column-based RNA extraction methods are more effective and reliable with respect to TRIzol LS. Endogenous circulating miRNA levels are unstable when plasma is stored at 4°C, and samples should be kept at -70°C, where the extracted miRNAs remain stable for up to 1 year. When normalization is based on combined endogenous and exogenous control miRNAs, differences in miRNA recovery and differences in cDNA synthesis between samples are compensated. Using this normalization procedure and hsa-miR-21 as a biomarker, we could clearly discriminate healthy individuals from patients with cancer. Experimental handling and the use of exogenous and endogenous controls for normalization are critical for the reliable quantification of circulating miRNA levels in plasma.

BACKGROUND Circulating tumor cells (CTCs) and microRNAs (miRNAs) are important in liquid biopsies in which peripheral blood is used to characterize the evolution of solid tumors. We evaluated the expression levels of miR-21, miR-146a, miR-200c, and miR-210 in CTCs of breast cancer patients with verified metastasis and compared their expression levels in corresponding plasma and primary tumors. METHODS Expression levels of the miRNAs were quantified by quantitative reverse transcription PCR (RT-qPCR) in (a) 89 primary breast tumors and 30 noncancerous breast tissues and (b) CTCs and corresponding plasma of 55 patients with metastatic breast cancer and 20 healthy donors. For 30 of these patients, CTCs, corresponding plasma, and primary tumor tissues were available. RESULTS In formalin-fixed, paraffin-embedded tissues, these miRNAs were differentially expressed between primary breast tumors and noncancerous breast tissues. miR-21 (P < 0.001) and miR-146a (P = 0.001) were overexpressed, whereas miR-200c (P = 0.004) and miR-210 (P = 0.002) were underexpressed. In multivariate analysis, miR-146a overexpression was significantly [hazard ratio 2.969 (1.231–7.157), P = 0.015] associated with progression-free survival. In peripheral blood, all miRNAs studied were overexpressed in both CTC and corresponding plasma. There was a significant association between miR-21 expression levels in CTCs and plasma for 36 of 55 samples (P = 0.008). In plasma, ROC curve analysis revealed that miR-21, miR-146a, and miR-210 could discriminate patients from healthy individuals. CONCLUSIONS Metastasis-related miRNAs are overexpressed in CTCs and corresponding plasma; miR-21 expression levels highly correlate in CTCs and plasma; and miR-21, miR-146a, and miR-210 are valuable plasma biomarkers for discriminating patients from healthy individuals.

Introduction: The efficiency and reproducibility of plasma miRNAs extraction protocol consists a critical step concerning the quantification of circulating miRNAs in plasma. Plasma is a specimen with high concentrations of lipids and proteins, so specific handling is required in order to achieve an enriched fraction of small RNAs at the end of the procedure. In the present study, we conducted a comparative investigation regarding RNA yielding among different commercially available experimental approaches. Our evaluation approach relied on the comparative extraction yielding of endogenous human miR-21 and exogenous spiked-in C.elegans miR-39 from pooled plasma samples from healthy individuals. Materials and Methods: In the present study we evaluated the extraction potential of small RNAs using three different commercially available kits for miRNAs extraction from plasma samples according to the manufacturer's protocols: a) miRNeasy mini Kit (Qiagen), b) mirVana PARIS kit (Ambion), c) microRNA purification Kit (Norgen Biotek) and d) Trizol LS Reagent (Invitrogen). We firstly pooled plasma samples from healthy individuals and used them for spiking experiments. In all cases, the initial plasma volume was 200μL and in each sample we spiked 25fmol of the exogenous standard C.elegans cel-miR-39. The isolation of miRNAs from plasma was conducted as per kit protocols and all extraction methods were performed three independent times from the same initial pooled plasma sample. A fixed volume of the eluted RNA sample was used as an input for the reverse transcription reaction. After isolation, quantification of cel-miR-39 was performed using real-time PCR. Recovery of cel-miR-39 in each case was estimated in respect to the levels of an equivalent amount of C.elegans copies added to total RNA after each extraction method (representing 100% recovery). In parallel, in all these samples the endogenous hsa-miR-21 was also quantified by real-time qPCR. The results were compared between the different isolation protocols concerning this with the greatest miRNAs yield for expression levels of endogenous miRNAs. Results: We found that the phenol:chloroform extraction and ethanol precipitation of RNA protocol (Trizol LS) recovers small RNAs with some loss whereas silica-column based RNA extraction methods provide a more effective and reliable mechanism for the isolation of small RNAs. Although all column protocols have indications to be very effective, in our hands the mirVana PARIS (Ambion) protocol appeared giving higher yields of circulating miRNAs from plasma samples. This was the case after quantifying for both the exogenous (cel-miR-39) and endogenous (miR-21) less abundant levels of miRNAs. Conclusion: According to our results, the mirVana PARIS kit (Ambion) was the most efficient among different commercially available kits for the isolation of circulating plasma miRNAs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5043. doi:1538-7445.AM2012-5043

Introduction: MicroRNA-21 is a well characterized cancer associated miRNA as it has been found overexpressed in nine types of solid tumors and in other malignancies. The role of miR-21 is related with diverse carcinogenesis processes. The downregulation of tumor suppressor genes through the overexpression of miR-21 has been demonstrated for PTEN and TPM1. Very recently, it was revealed that programmed cell death 4 (PDCD4) is another target of miR-21 in colon and breast cancer cell lines. As it has not been yet studied the correlation between miR-21 and PDCD4 levels in clinical samples, we conducted the present study to investigate the possible regulation of pdcd4 protein levels by mature miR-21. Materials and Methods: Forty pairs of NSCLC fresh-frozen tissues and their corresponding noncancerous tissues were analyzed for the expression of mature miR-21 using quantitative real-time RT-PCR, as previously described (Markou et al., 2008). In parallel, PDCD4 protein levels were evaluated by immunohistochemistry. Deparaffinized sections cut from paraffin-embedded tissue samples were stained with a specific anti-PDCD4 antibody (1:100 dilution) (Ozaki et al., 2006) using HRP DAB kit (DAKO) for the detection. The tumor types and stages were determined according to the WHO classification. All samples were analyzed histologically to access the amount of tumor component (at least 70% of tumor cells) and the quality of material. Results: Among the 40 NSCLC tissue specimens studied, suppresion of miR-21, in respect to their adjacent non-neoplastic tissues, was detected in 24 samples (60 %). In 15 out of these 24 patients (62.5%), we observed that the supression of miR-21 was accompanied by increase of PDCD4 protein levels. Mature miR-21 was overexpressed in 16 out of 40 patients (40%), and in 8 out of these 16 patients (50%) we detected reduced PDCD4 protein levels. Totally, in 23 out of 40 samples (57.5%), the altered miR-21 expression levels correlated with changed PDCD4 protein levels. Conclusion: Our data indicate for the first time that PDCD4 protein expression levels are regulated by miR-21 in non small cell lung cancer tissues. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2075.