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Transcription profiling by array of human malignant melanoma cell line, A375, following interferon-gamma-induced gene transcription

ABSTRACT: MicroRNAs are major regulators of post-transcriptional gene regulation. Even small changes in miRNA levels may have profound consequences for the expression levels of target genes. Hence, miRNAs themselves need to be tightly, albeit dynamically, regulated. Here, we investigated the dynamic behaviour of miRNAs over a wide time range following transcriptional activation of melanoma cells by interferon-? (IFN-?), which activates the transcription factor STAT1. By applying several software packages for analyses, visualisation and identification of differentially expressed miRNAs derived from time-series microarray experiments, 8.9% (98) of 1102 miRNAs appeared to be directly or indirectly regulated by STAT1. Focussing on distinct dynamic expression patterns, we found that the majority of differentially expressed miRNAs were up-or down-regulated in the intermediate time range (24 h - 48 h), one (miR-27a*) was up-regulated early (already at 18 h), while none reacted late (after 72 h – 96 h). Expression of individual miRNAs was altered gradually over time or it abruptly increased between two time points. Furthermore, we have observed co-ordinated dynamic transcription of several clustered miRNAs. However, we also detected that some of those can be regulated independently of their genetic cluster. Most interestingly, several “star” or passenger strand sequences were specifically regulated over time while their “guide” strands were not.

ORGANISM(S): Homo sapiens

SUBMITTER: Reinsbach S

PROVIDER: S-ECPF-MEXP-3544 | biostudies-other | 2012 Jul

REPOSITORIES: biostudies-other

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Project description:Skin cancer is one of the most common cancers worldwide. Melanoma accounts for ~5% of skin cancers but causes the large majority of skin cancer‑related deaths. Recent discoveries have shown that the mitogen‑activated protein kinase (MAPK) signaling pathway is critical for melanoma development and progression. Many oncogenic pathways that cause melanoma tumorigenesis have been identified, most of which are due to RAF/MEK/ERK (MAPK) pathway activation. However, the precise role of p38 remains unclear. Using specific short hairpin (sh) RNA to silence p38α and p38β, the present findings demonstrated that p38α was a crucial factor in regulating cell migration in the A375 melanoma cell line. Silencing p38α downregulated the expression of epithelial‑mesenchymal transition markers, such as matrix metallopeptidase (MMP) 2, MMP9, twist family bHLH transcription factor 1, snail family transcriptional repressor 1 and vimentin, while mesenchymal‑epithelial transition markers, such as E‑cadherin, were upregulated. Of note, the results also demonstrated that p38α silencing impaired vascular endothelial growth factor expression, which regulates tumor angiogenesis. Furthermore, p38α knockdown inhibited cell proliferation in melanoma cells. In addition, silencing p38α induced senescence‑like features, but not cell cycle arrest. Expression of the senescence markers p16, p21, p53 and β‑galactosidase was upregulated, and an increase in the number of senescence‑associated β‑galactosidase‑positive cells was observed in a p38α knockdown stable clone. However, no significant difference was found between control and p38β stable knockdown cells. Taken together, the present results suggested that p38α knockdown impaired migration and proliferation, and increased senescence, in A375 melanoma cells. However, p38β may not be involved in melanoma tumorigenesis. Therefore, targeting p38α may be a valuable approach towards inhibiting tumor growth and metastasis in patients with melanoma.

Project description:The innate response of melanocytes to exogenous or endogenous stress stimuli like extreme pH and temperature, metabolite and oxygen deficiency or a high UV dose initiates a cellular stress response. This process activates adaptive processes to minimize the negative impact of the stressor on the pigment cell. Under physiological conditions, a non-cancer cell is directed to apoptosis if the stressor persists. However, malignant melanoma cells will survive persistent stress thanks to distinct "cancerous" signaling pathways (e.g. MEK) and transcription factors that regulate the expression of so-called "survival genes" (e.g. HIF, MITF). In this survival response of cancer cells, MEK pathway directs melanoma cells to deregulate mitochondrial metabolism, to accumulate reduced species (NADH), and to centralize metabolism in the cytosol. The aim of this work was to study the effect of gene silencing in malignant melanoma A375 cells on metabolic processes in cytosol and mitochondria. Gene silencing of HIF-1α, and miR-210 in normoxia and pseudohypoxia, and analysis of its effect on MITF-M, and PDHA1 expression. Detection of cytosolic NADH by Peredox-mCherry Assay. Detection of OCR, and ECAR using Seahorse XF96. Measurement of produced O2•- with MitoTracker Red CMXRos. 1H NMR analysis of metabolites present in cell suspension, and medium. By gene silencing of HIF-1α and miR-210 the expression of PDHA1 was upregulated while that of MITF-M was downregulated, yielding acceleration of mitochondrial respiratory activity and thus elimination of ROS. Hence, we detected a significantly reduced A375 cell viability, an increase in alanine, inositol, nucleotides, and other metabolites that together define apoptosis. Based on the results of measurements of mitochondrial resipiratory activity, ROS production, and changes in the metabolites obtained in cells under the observed conditions, we concluded that silencing of HIF-1α and miR-210 yields apoptosis and, ultimately, apoptotic cell death in A375 melanoma cells.

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Transcription profiling by array of human malignant melanoma cell line, A375, following interferon-gamma-induced gene transcription

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