Quantitative RT-PCR-based miRNA profiling of blastemal Wilms’ tumors from formalin-fixed paraffin-embedded samples

Buglyó, Gergely; Magyar, Zsófia; Görbe, Éva; Bánusz, Rita; Csóka, Monika; Micsik, Tamás; Berki, Zsanett; Varga, Péter Pál; Szepes, Zoltán; Nagy, Bálint

doi:10.1016/j.jbiotec.2019.04.001

Cited by 9 publications

(24 citation statements)

References 23 publications

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“…The role of miR-218 in WT is unknown. In WT, there are few studies that have reported it; one of them evaluated its expression in eight serum samples from WT patients and in eight predominantly blastemal FHWT tumors, and in both cases, no changes in its expression were observed, likely due to a low sample number (3,31). In other tumors, its expression has been reported low as in renal cell carcinoma (32), lung cancer (33), and hepatocellular carcinoma (23); moreover, overexpression of this miRNA in these tumors inhibits cell proliferation, migration, and invasion, leading to it being characterized as a tumor suppressor.…”

Section: Discussionmentioning

confidence: 99%

“…Approximately 90% of WT patients survive and are associated with favorable histology (FHWT); however, this percentage diminished to 75% in metastatic cases. On the other hand, the remaining 10% of WT present unfavorable histology; they are characterized by cellular diffuse anaplasia (DAWT) and poor prognosis (2,3). Histologically, WT presents three types of cells: blastemal, mesenchymal, and epithelial, with the blastemal type being the most frequent, followed by triphasic, which is characterized by the presence of the three cell types in the same tumor (4).…”

Section: Introductionmentioning

confidence: 99%

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MicroRNA Profiling in Wilms Tumor: Identification of Potential Biomarkers

et al. 2020

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Wilms tumor (WT) is the most frequently diagnosed malignant renal tumor in children. With current treatments, ∼90% of children diagnosed with WT survive and generally present with tumors characterized by favorable histology (FHWT), whereas prognosis is poor for the remaining 10% of cases where the tumors are characterized by cellular diffuse anaplasia (DAWT). Relatively few studies have investigated microRNA-related epigenetic regulation and its relationship with altered gene expression in WT. Here, we aim to identify microRNAs differentially expressed in WT and describe their expression in terms of cellular anaplasia, metastasis, and association with the main genetic alterations in WT to identify potential prognostic biomarkers. Expression profiling using TaqMan low-density array was performed in a discovery cohort consisting of four DAWT and eight FHWT samples. Relative quantification resulted in the identification of 109 (48.7%) microRNAs differentially expressed in both WT types. Of these, miR-10a-5p, miR-29a-3p, miR-181a-5p, miR-200b-3p, and miR-218-5p were selected and tested by RT-qPCR on a validation cohort of 53 patient samples. MiR-29a and miR-218 showed significant differences in FHWT with low (P = 0.0018) and high (P = 0.0131) expression, respectively. To discriminate between miRNA expression FHWTs and healthy controls, the receiver operating characteristic (ROC) curves were obtained; miR-29a AUC was 0.7843. Furthermore, low expression levels of miR-29a and miR-200b (P = 0.0027 and P = 0.0248) were observed in metastatic tumors. ROC curves for miR-29a discriminated metastatic patients (AUC = 0.8529) and miR-200b (AUC = 0.7757). To confirm the differences between cases with poor prognosis, we performed in situ hybridization for three microRNAs in five DAWT and 17 FHWT samples, and only significant differences between adjacent tissues and FHWT tumors were found for miR-181a, miR-200b, and miR-218, in both total pixels and nuclear analyses. Analysis of copy number variation in genes showed that the most prevalent alterations were WTX (47%), IGF2 (21%), 1q (36%) gain, 1p36 (16%), and WTX deletion/1q duplicate (26%). The five microRNAs evaluated are involved in the Hippo signaling pathway and participate in Wilms tumor development through their effects on differentiation, proliferation, angiogenesis, and metastasis.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

MicroRNA Profiling in Wilms Tumor: Identification of Potential Biomarkers

et al. 2020

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“…218160) at 42˚C for 60 min. cDNA was stored in a -20˚C refrigerator until subsequent analysis using RT-qPCR, which was performed according to a previously described method to amplify miRNAs (18). Reactions were conducted using SYBR Green PCR Master Mix (Thermo Fisher Scientific, Inc.; cat.…”

Section: Extraction Of Mirnas and Detection Using Reverse Transcriptimentioning

confidence: 99%

Detection of microRNA expression levels based on microarray analysis for classification of idiopathic pulmonary fibrosis

Zheng

et al. 2020

Exp Ther Med

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The etiology and pathophysiological mechanisms of idiopathic pulmonary fibrosis (IPF) are yet to be fully elucidated; however, mining of disease-related microRNAs (miRNAs/miRs) has improved the understanding of the progression of IPF. The aim of the current study was to screen miRNAs associated with IPF using three mathematical algorithms: One-way ANOVA, least absolute shrinkage and selector operation (LASSO) and support vector machinerecursive feature elimination (SVM-RFE). Using ANOVA, three miRNAs and two miRNAs were selected with opposite expression patterns in moderate and severe IPF, respectively. In total, two algorithms, LASSO and SVM-RFE, were used to perform feature selection of miRNAs. miRNAs from patients were also extracted from formalin-fixed paraffin-embedded tissues and detected using reverse transcription-quantitative PCR (RT-qPCR). The intersection of the three algorithms (ANOVA, LASSO and SVM-RFE) was taken as the final result of the miRNA candidates. Three miRNA candidates, including miR-124, hsa-miR-524-5p and hsa-miR-194 were therefore used as biomarkers. The receiver operating characteristic model demonstrated favorable discrimination between IPF and control groups, with an area under the curve of 78.5%. Moreover, RT-qPCR results indicated that miR-124, hsa-miR-524-5p, hsa-miR-194 and hsa-miR-133a were differentially expressed between patients with IPF and age-matched men without fibrotic lung disease. The target genes of these miRNAs were further predicted and Kyoto Encyclopedia of Genes and Genomes enrichment analysis was performed. Collectively, the present results suggested that the identified miRNAs associated with IPF may be useful biomarkers for the diagnosis of this disease.

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“…MicroRNAs (miRNAs) are small non-coding RNA molecules (18-25 nucleotides) that regulate gene expression by the degradation of target mRNAs or the inhibition of protein translation. 1,2 To date, plenty of miRNA biomarkers have been screened for diagnosis and prognosis of cancers. [3][4][5][6][7][8][9][10][11][12][13] However, the inherent characteristics of miRNAs, such as instability, high similarities among family members and low abundance, increase the difficulties of miRNA single-plex detection accuracy.…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, miRNA multiplex detection is benecial to overcome the clinical sensitivity and specicity limitations. Although many miRNA detection technologies have been developed including northern blotting, 14 microarrays, 15 reverse-transcription polymerase chain reaction (RT-PCR), 2,16,17 Isothermal amplication technology 18,19 and next-generation sequencing technology, 20 these methods have some drawbacks, such as requiring target amplication, being poorly exible and reproducible, and not being easily multiplexed. 21 In contrast, suspension array technology has been widely applied in multiplex detection with excellent exibility and strong multiplex ability.…”

Section: Introductionmentioning

confidence: 99%