Development of a robust, low cost stem-loop real-time quantification PCR technique for miRNA expression analysis

Mohammadi‐Yeganeh, Samira; Paryan, Mahdi; Samiee, Siamak Mirab; Soleimani, Masoud; Arefian, Ehsan; Azadmanesh, Kayhan; Mostafavi, Ehsan; Mahdian, Reza; Karimipoor, Morteza

doi:10.1007/s11033-012-2442-x

Cited by 83 publications

(52 citation statements)

References 28 publications

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“…We also used RT step-loop primer design developed by Mohammadi-Yeganeh et al (2013) for miRNAs reverse transcription and elongation which was approved to be as reliable as commercially available primers with much lower price [26]. We used MDA-MB-231 and MCF-10A cell lines representative of TNBC and normal breast cells, respectively.…”

Section: Discussionmentioning

confidence: 99%

miR-141 as potential suppressor of β-catenin in breast cancer

et al. 2015

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Triple negative breast cancer (TNBC) is well known for its heterogeneous features and lack of targeted therapy. A variety of cell signaling pathways have been linked to the initiation and progression of these tumors where canonical Wnt signaling is one of the main candidate pathways. Considering past literatures on this matter and negative reports regarding mutations in β-catenin gene (CTNNB1), we focus our attention to another level of gene expression control level, microRNAs (miRNAs). For proper miRNA target detection, we utilized bioinformatics as a relatively new and reliable tool for miRNA: mRNA prediction. MDA-MB-231 (invasive breast cancer) and MCF-10A (normal breast) cell lines were chosen as models. We used different bioinformatic tools such as TargetScan, miRanda, etc. For miRNA targeting CTNNB1-3´UTR confirmation, luciferase assay was carried out. miRNA expression was induced in cell lines through viral constructs expressing desired miRNA. Quantitative real-time PCR was performed for the measurement of expression levels of selected miRNA and target gene. miR-141 was selected via expanded search among various bioinformatic tools. miR-141 expression level was downregulated in MDA-MB-231 cell line, and CTNNB1 gene expression was upregulated. After transduction with viral construct, miR-141 expression was elevated in both cell lines, and gene expression was notably decreased. β-Catenin can be considered as one of the main players in these tumors' pathogenesis. Also, it is the potential target of miR-141, in which its downregulation was detected in cell lines, and can be considered as a promising new targeted approach toward TNBC.

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Section: Discussionmentioning

confidence: 99%

miR-141 as potential suppressor of β-catenin in breast cancer

et al. 2015

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“…miRNA validation miRNA microarray results were validated by employing the stem-loop quantitative real-time polymerase chain reaction technique published by Mhammadi-Yeganeh, which is a modified form of the technique from Life Technologies (Mohammadi-Yeganeh et al, 2013). Briefly, in a reverse transcription step, a so-called stem-loop primer binds with 6-8 bp of its 3¢-end to the respective miRNA.…”

Section: Microrna Screeningmentioning

confidence: 99%

Atypical Cell Populations Associated with Acquired Resistance to Cytostatics and Cancer Stem Cell Features: The Role of Mitochondria in Nuclear Encapsulation

Díaz-Carballo

Gustmann

Jastrow

et al. 2014

DNA and Cell Biology

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Until recently, acquired resistance to cytostatics had mostly been attributed to biochemical mechanisms such as decreased intake and/or increased efflux of therapeutics, enhanced DNA repair, and altered activity or deregulation of target proteins. Although these mechanisms have been widely investigated, little is known about membrane barriers responsible for the chemical imperviousness of cell compartments and cellular segregation in cytostatic-treated tumors. In highly heterogeneous cross-resistant and radiorefractory cell populations selected by exposure to anticancer agents, we found a number of atypical recurrent cell types in (1) tumor cell cultures of different embryonic origins, (2) mouse xenografts, and (3) paraffin sections from patient tumors. Alongside morphologic peculiarities, these populations presented cancer stem cell markers, aberrant signaling pathways, and a set of deregulated miRNAs known to confer both stem-cell phenotypes and highly aggressive tumor behavior. The first type, named spiral cells, is marked by a spiral arrangement of nuclei. The second type, monastery cells, is characterized by prominent walls inside which daughter cells can be seen maturing amid a rich mitochondrial environment. The third type, called pregnant cells, is a giant cell with a syncytium-like morphology, a main nucleus, and many endoreplicative functional progeny cells. A rare fourth cell type identified in leukemia was christened shepherd cells, as it was always associated with clusters of smaller cells. Furthermore, a portion of resistant tumor cells displayed nuclear encapsulation via mitochondrial aggregation in the nuclear perimeter in response to cytostatic insults, probably conferring imperviousness to drugs and long periods of dormancy until nuclear eclosion takes place. This phenomenon was correlated with an increase in both intracellular and intercellular mitochondrial traffic as well as with the uptake of free extracellular mitochondria. All these cellular disorders could, in fact, be found in untreated tumor cells but were more pronounced in resistant entities, suggesting a natural mechanism of cell survival triggered by chemical injury, or a primitive strategy to ensure stemming, self-renewal, and differentiation under adverse conditions, a fact that may play a significant role in chemotherapy outcomes.

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“…Samples were assessed in triplicate along with pertaining controls, including positive, negative, no-template and RT-minus controls. A previously approved and non-treated RNA, SNORD 47, was used as an internal control (21). The Ct is the fractional cycle number at which the fluorescence passes the fixed threshold.…”

Section: Methodsmentioning

confidence: 99%

Regulation of connexin 43 and microRNA expression via β2-adrenoceptor signaling in 1321N1 astrocytoma cells

Khaksarian

Mostafavi

Soleimani

et al. 2015

Molecular Medicine Reports

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Abstract. Connexin 43 (Cx43) is the main gap junction protein in astrocytes and exerts the same effects on growth inhibition in astrocytoma and glioma as microRNA-146a (miR-146a) in glioma. β2-adrenergic receptor (AR) signaling modulates Cx43 expression in myocytes via components downstream of protein kinase A (PKA) and exchange protein directly activated by cAMP (Epac). However, it remains to be elucidated how expression of Cx43 is modulated in astrocytes. In the present study, 1321N1 astrocytoma cells were treated with β2-AR signaling agents in order to evaluate the expression of Cx43 and miRNAs. RNA and protein were extracted from the cells for use in reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. The results revealed that clenbuterol increased miR-146a level and upregulated Cx43 expression via cAMP/PKA at the mRNA and protein level. Pre-inhibition of adenyl cyclase decreased expression of Cx43 and miR-146a. PKA activation and overexpression of miR-146a in A-1321N1 cells increased the expression of Cx43. β2-AR stimulation and 6Bnz, a PKA activator, suppressed oncomiRs miR-155 and miR-27a, while 8-(4-chlorophenylthio)-2'-O-methyl adenosine-3',5'-cyclic monophosphate, an Epac activator, increased their levels. The current findings demonstrated that β2-AR signaling has growth inhibitory effects via modulation of the cAMP/PKA pathway in A-1321N1 cells through increasing the expression level of Cx43 and miR-146a as well as decreasing miR-155 and miR-27a levels. Thus, stimulation of the β2-AR and PKA signaling pathway may be a useful approach for astrocytoma therapy.

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Development of a robust, low cost stem-loop real-time quantification PCR technique for miRNA expression analysis

Cited by 83 publications

References 28 publications

miR-141 as potential suppressor of β-catenin in breast cancer

miR-141 as potential suppressor of β-catenin in breast cancer

Atypical Cell Populations Associated with Acquired Resistance to Cytostatics and Cancer Stem Cell Features: The Role of Mitochondria in Nuclear Encapsulation

Regulation of connexin 43 and microRNA expression via β2-adrenoceptor signaling in 1321N1 astrocytoma cells

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