Identification of suitable endogenous controls for gene and miRNA expression studies in irradiated prostate cancer cells

Lawlor, H.; Meunier, Armelle; McDermott, Niamh; Lynch, Thomas; Marignol, Laure

doi:10.1007/s13277-015-3278-5

Cited by 3 publications

(3 citation statements)

References 37 publications

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“…SNORD48 also known as RNU48, was shown to be significantly upregulated in ALS patients, adding to mounting evidence that this commonly used qPCR internal control should be used with care 96 , 97 . While the role of SNORD84 and SNORD97 are unknown, SNORD95 is expressed from the intron of the RACK1 (GNB2L1) gene, which is involved in translational repression and ribosomal quality control 98 , 99 .…”

Section: Discussionmentioning

confidence: 99%

Differential expression of microRNAs and other small RNAs in muscle tissue of patients with ALS and healthy age-matched controls

Kovanda

Leonardis

Zidar

et al. 2018

Sci Rep

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Amyotrophic lateral sclerosis is a late-onset disorder primarily affecting motor neurons and leading to progressive and lethal skeletal muscle atrophy. Small RNAs, including microRNAs (miRNAs), can serve as important regulators of gene expression and can act both globally and in a tissue-/cell-type-specific manner. In muscle, miRNAs called myomiRs govern important processes and are deregulated in various disorders. Several myomiRs have shown promise for therapeutic use in cellular and animal models of ALS; however, the exact miRNA species differentially expressed in muscle tissue of ALS patients remain unknown. Following small RNA-Seq, we compared the expression of small RNAs in muscle tissue of ALS patients and healthy age-matched controls. The identified snoRNAs, mtRNAs and other small RNAs provide possible molecular links between insulin signaling and ALS. Furthermore, the identified miRNAs are predicted to target proteins that are involved in both normal processes and various muscle disorders and indicate muscle tissue is undergoing active reinnervation/compensatory attempts thus providing targets for further research and therapy development in ALS.

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Section: Discussionmentioning

confidence: 99%

Differential expression of microRNAs and other small RNAs in muscle tissue of patients with ALS and healthy age-matched controls

Kovanda

Leonardis

Zidar

et al. 2018

Sci Rep

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“…Starting from total RNA (10 ng), miRNAs were converted to cDNA using reverse transcriptase and miRNA-specific stem-loop primers: hsa-miR-29a (002112), hsa-miR-29b (000413), hsa-miR-29c (000587). For relative miRNA quantification, snoRNA U48 (RNU48; 001006) was used as a reference gene 37,56 . The PCR reaction (20 µl), containing 1.33 µl of cDNA, 10 µl of TaqMan 2X Universal PCR Master Mix, 1 µl of TaqMan MicroRNA Assay (20X) containing probes specific for the miRNAs of interest, was incubated at 95°C for 10 min, and then at 95°C for 15 s and 60°C for 60 s for 40 cycles.…”

Section: Mirna Analysismentioning

confidence: 99%

DNA methylation variations are required for epithelial-to-mesenchymal transition induced by cancer-associated fibroblasts in prostate cancer cells

et al. 2017

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Widespread genome hypo-methylation and promoter hyper-methylation of epithelium-specific genes are hallmarks of stable epithelial-to-mesenchymal transition (EMT), which in prostate cancer (PCa) correlates with castration resistance, cancer stem cells generation, chemoresistance and worst prognosis. Exploiting our consolidated 'ex-vivo' system, we show that cancer-associated fibroblasts (CAFs) released factors have pivotal roles in inducing genome methylation changes required for EMT and stemness in EMT-prone PCa cells. By global DNA methylation analysis and RNA-Seq, we provide compelling evidence that conditioned media from CAFs explanted from two unrelated patients with advanced PCa, stimulates concurrent DNA hypo- and hyper-methylation required for EMT and stemness in PC3 and DU145, but not in LN-CaP and its derivative C4-2B, PCa cells. CpG island (CGI) hyper-methylation associates with repression of genes required for epithelial maintenance and invasion antagonism, whereas activation of EMT markers and stemness genes correlate with CGI hypo-methylation. Remarkably, methylation variations and EMT-regulated transcripts almost completely reverse qualitatively and quantitatively during MET. Unsupervised clustering analysis of the PRAD TCGA data set with the differentially expressed (DE) and methylated EMT signature, identified a gene cluster of DE genes defined by a CAF+ and AR- phenotype and worst diagnosis. This gene cluster includes the relevant factors for EMT and stemness, which display DNA methylation variations in regulatory regions inversely correlated to their expression changes, thus strongly sustaining the ex-vivo data. DNMT3A-dependent methylation is essential for silencing epithelial maintenance and EMT counteracting genes, such as CDH1 and GRHL2, that is, the direct repressor of ZEB1, the key transcriptional factor for EMT and stemness. Accordingly, DNMT3A knock-down prevents EMT entry. These results shed light on the mechanisms of establishment and maintenance of coexisting DNA hypo- and hyper-methylation patterns during cancer progression, the generation of EMT and cell stemness in advanced PCa, and may pave the way to new therapeutic implications.

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“…The researchers showed that expression levels were associated with a low coefficient of variation after irradiation (6 Gy). Scientists have noticed the effect of radiation on changes in endogenous SNORD48 [ 41 ].…”

Section: Introductionmentioning

confidence: 99%

Absolute Quantification of Selected microRNAs Expression in Endometrial Cancer by Digital PCR

Bogaczyk,

Potocka,

Paszek

et al. 2024

IJMS

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MicroRNAs (miRNA) are involved in the process of carcinogenesis, including the development of endometrial cancer (EC). This study aimed to investigate the association between the expression of three miRNAs (miR-21-5p, miR-205-5p, and miR-222-3p) in endometrial cancer tissues. In addition, the stability of expression of SNORD48 and U6, which were initially planned to be used as reference miRNAs for normalization, was investigated. Endometrial tissue was obtained from 111 patients with EC during hysterectomy and from 19 patients undergoing surgery for uterine fibroids or pelvic organ prolapse as a control group without neoplastic changes. Our study was based on calculations made with a digital PCR method (Qiagen, Hilden, Germany) to measure the absolute expression. In the endometrial cancer tissue, miR-205-5p was upregulated, while miR-222-3p and SNORD48 were downregulated compared to the control group. We detected statistically significant correlation of miR-205-5p, U6, and SNORD48 expression with different histological grades; the expression of miR-205-5p increases with the histopathological grade advancement (intraepithelial neoplasia- EIN = 1590, G1 = 3367.2, G2 = 8067 and G3 = 20,360), while U6 and SNORD expression decreases from EIN to G2 and increases again in the G3 grade (U6: EIN = 19,032, G1 = 16,482.4, G2 = 13,642.4, G3 = 133,008; SNORD48: EIN = 97,088, G1 = 59,520, G2 = 43,544, G3 = 227,200). Our study suggests that upregulation of miR-205-5p and downregulation of miR-222-3p and SNORD48 may influence development of endometrial cancer. Moreover, miR-205-5p, U6, and SNORD48 expression changes may be associated with progression of endometrial cancer. The results also indicate that SNORD48 and U6, commonly used as internal references, may influence endometrial cancer development and progression; therefore, they should not be used as references. However, it is important to note that further research is required to understand their role in endometrial cancer.

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Identification of suitable endogenous controls for gene and miRNA expression studies in irradiated prostate cancer cells

Cited by 3 publications

References 37 publications

Differential expression of microRNAs and other small RNAs in muscle tissue of patients with ALS and healthy age-matched controls

Differential expression of microRNAs and other small RNAs in muscle tissue of patients with ALS and healthy age-matched controls

DNA methylation variations are required for epithelial-to-mesenchymal transition induced by cancer-associated fibroblasts in prostate cancer cells

Absolute Quantification of Selected microRNAs Expression in Endometrial Cancer by Digital PCR

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