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DIFFERENTIALLY EXPRESSED MicroRNAs IN SMALL CELL LUNG CANCER

Expression of microRNAs (miRNAs) is characteristically altered in cancer, and they may play a role in cancer development and progression. The authors performed microarray and real-time quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) analyses to determine the miRNA expression profile of primary small cell lung cancer. Here we show that at least 24 miRNAs are differentially expressed between normal lung and primary small cell lung cancer (SCLC) tumors. These include miR-301, miR-183/96/182, miR-126, and miR-223, which are microRNAs deregulated in other tumor types as well; and other miRNAs, such as miR-374 and miR-210, not previously reported in association with lung cancer. The aberrant miRNA profile of SCLC may offer new insights in the biology of this aggressive tumor, and could potentially provide novel diagnostic markers.

miR-126 inhibits proliferation of small cell lung cancer cells by targeting SLC7A5

Despite intensive efforts to improve therapies, small cell lung cancer (SCLC) still has a dismal median survival of 18 months. Since miR‐126 is under‐expressed in the majority of SCLC tumors, we investigated the effect of miR‐126 overexpression on the proliferation and cell cycle distribution of H69 cells. Our results demonstrate that miR‐126 inhibits proliferation of H69 cells, by delaying the cells in the G1 phase. Short interfering RNA (siRNA) mediated suppression of SLC7A5, a predicted target of mir‐126, has the same effect on H69 cells. We also show for the first time that SLC7A5 is a direct target of miR‐126.

Reference genes for quantitative real time PCR in UVB irradiated keratinocytes.

Real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is a sensitive and highly reproducible method often used for determining mRNA levels. To enable proper comparison of gene expression genes expressed at stabile levels within the cells in the studied experimental system need to be identified and used as reference. Ultraviolet B (UVB) radiation is an exogenous carcinogenic stimulus in keratinocytes, and UVB elicited changes have extensively been studied by qRT-PCR, yet a comparison of commonly used reference genes in UVB treatment is lacking. To find the best genes for compensating slight inter-sample variations in keratinocytes in UVB experiments and to understand the potential effects of improper reference gene (RG) selection we have analyzed the mRNA expression of 10 housekeeping genes in neonatal human epidermal keratinocytes (NHEK) after UVB treatment. The biological effect of the used UVB light source was validated by trypane blue exclusion, MTT and comet assays. 20-40mJ/cm(2) dose was chosen for the experiments. The stability of the 10 RGs was assessed by the GeNorm and Normfinder software tools. Regardless of their slightly different algorithm the programs found succinate dehydrogenase complex subunit A (SDHA) to be the best individual RG and SDHA and phosphoglycerate kinase-1 (PGK1) as the most suitable combination. Analysis of the expression of tumor necrosis factor alpha (TNFalpha) and vascular endothelial growth factor (VEGF) found that while the perception of changes in TNF-alpha, a gene undergoing marked upregulation after UVB irradiation is independent of the used RG, changes seen in the more modestly upregulated VEGF are greatly effected by reference gene selection. These findings highlight the importance of reference gene selection in UVB irradiation experiments, and provide evidence that using SDHA or the combination of SDHA and PGK1 as standards could be a reliable method for normalizing qRT-PCR results in keratinocytes after UVB treatment.

Correlation among metallothionein expression, intratumoural macrophage infiltration and the risk of metastasis in human cutaneous malignant melanoma

Background  The formation of metastases and the efficacy of systemic therapies in cutaneous malignant melanoma (CMM) depend on the characteristics of the tumour cells and the host immune response. Aberrant expression of metallothionein (MT) has been observed in several types of cancers with poor prognoses.

Expression of invasion-related extracellular matrix molecules in human glioblastoma versus intracerebral lung adenocarcinoma metastasis.

Tumor cell invasion into the surrounding brain tissue is mainly responsible for the failure of radical surgical resection, with tumor recurrence in the form of microdisseminated disease. Extracellular matrix (ECM)-related molecules and their receptors predominantly participate in the invasion process, including cell adhesion to the surrounding microenvironment and cell migration. The extent of infiltration of the healthy brain by malignant tumors strongly depends on the tumor cell type. Malignant gliomas show much more intensive peritumoral invasion than do metastatic tumors. In this study, the mRNA expression of 30 invasion-related molecules (twenty-one ECM components, two related receptors, and seven ECM-related enzymes) was investigated by quantitative reverse transcriptase-polymerase chain reaction. Fresh frozen human tissue samples from glioblastoma (GBM), intracerebral lung adenocarcinoma metastasis, and normal brain were evaluated. Significant differences were established for 24 of the 30 molecules. To confirm our results at the protein level, immunohistochemical analysis of seven molecules was performed (agrin, neurocan, syndecan, versican, matrix metalloproteinase 2 [MMP-2], MMP-9, and hyaluronan). Determining the differences in the levels of invasion-related molecules for tumors of different origins can help to identify the exact molecular mechanisms that facilitate peritumoral infiltration by glioblastoma cells. These results should allow the selection of target molecules for potential chemotherapeutic agents directed against highly invasive malignant gliomas.

Transfection of pseudouridine-modified mRNA encoding CPD-photolyase leads to repair of DNA damage in human keratinocytes: a new approach with future therapeutic potential

UVB irradiation induces harmful photochemical reactions, including formation of Cyclobutane Pyrimidine Dimers (CPDs) in DNA. Accumulation of unrepaired CPD lesions causes inflammation, premature ageing and skin cancer. Photolyases are DNA repair enzymes that can rapidly restore DNA integrity in a light-dependent process called photoreactivation, but these enzymes are absent in humans. Here, we present a novel mRNA-based gene therapy method that directs synthesis of a marsupial, Potorous tridactylus, CPD-photolyase in cultured human keratinocytes. Pseudouridine was incorporated during in vitro transcription to make the mRNA non-immunogenic and highly translatable. Keratinocytes transfected with lipofectamine-complexed mRNA expressed photolyase in the nuclei for at least 2days. Exposing photolyase mRNA-transfected cells to UVB irradiation resulted in significantly less CPD in those cells that were also treated with photoreactivating light, which is required for photolyase activity. The functional photolyase also diminished other UVB-mediated effects, including induction of IL-6 and inhibition of cell proliferation. These results demonstrate that pseudouridine-containing photolyase mRNA is a powerful tool to repair UVB-induced DNA lesions. The pseudouridine-modified mRNA approach has a strong potential to discern cellular effects of CPD in UV-related cell biological studies. The mRNA-based transient expression of proteins offers a number of opportunities for future application in medicine.

Metabolic roles of poly(ADP-ribose) polymerases

Poly(ADP-ribosyl)ation (PARylation) is an evolutionarily conserved reaction that had been associated with numerous cellular processes such as DNA repair, protein turnover, inflammatory regulation, aging or metabolic regulation. The metabolic regulatory tasks of poly(ADP-ribose) polymerases (PARPs) are complex, it is based on the regulation of metabolic transcription factors (e.g. SIRT1, nuclear receptors, SREBPs) and certain cellular energy sensors. PARP over-activation can cause damage to mitochondrial terminal oxidation, while the inhibition of PARP-1 or PARP-2 can induce mitochondrial oxidation by enhancing the mitotropic tone of gene transcription and signal transduction. These PARP-mediated processes impact on higher order metabolic regulation that modulates lipid metabolism, circadian oscillations and insulin secretion and signaling. PARP-1, PARP-2 and PARP-7 are related to metabolic diseases such as diabetes, alcoholic and non-alcoholic fatty liver disease (AFLD, NAFLD), or on a broader perspective to Warburg metabolism in cancer or the metabolic diseases accompanying aging.

Translational aspects of the microbiome—to be exploited

The scope of Cell Biology and Toxicology had been opened towards translational research as was discussed in the editorial of the Xiandong Wang (Gu and Wang 2016). Translational research supports the close collaboration of different specialties ranging from basic science research to clinical studies. That approach may shorten the time it takes for developing new treatment possibilities or schemes. To better understand the complexity of such methodology, hereby we review an exciting field, the microbiome—from a translational point of view (Fig. 1). The human body harbors symbiotic, commensal and pathogenic bacteria in enormous numbers. These microbes live in the cavities (e.g., gut, genitals, or airways) or on the surface of the human body, the skin. The ensemble of the microbes in an organism is referred as the normal flora. Changes in the composition of the normal flora, the invasion or over-proliferation of pathogenic bacteria had long been associated with diseases (e.g., Helicobacter pylori infection of the stomach) and had been translated already to the everyday clinical practice. Recent developments in research technology have vastly increased our knowledge on the Bnormal flora,^Next generation highthroughput sequencing experiments have demonstrated that there are more bacterial species in the gut than it was known from classical microbiological cultures. These studies have identified numerous new bacterial species, among them several obligatory anaerobic strains that are impossible to culture. The ensemble of microbes in a compartment (e.g., gut or airways), identified by sequencing, is referred as the microbiome or microbiota. Due to the abundance and variance of microbes, the overall size of the genomes of these organisms exceeds that of the human genome, extending vastly the variability of genes in a compartment. Therefore, several authors consider the microbiome as an additional organ and recently proposed to consider the ensemble of the human and microbial genomes present in one human being the Bmetagenome.^ There is an intricate bidirectional interaction between the host and the microbiome. The composition of the microbiome is governed by the behavior (e.g., hygiene), feeding, metabolism, and immunological characteristics of the host. While the microbiome influences host metabolism, immune reactions, and behavior through (1) releasing its own metabolites (e.g., short chain fatty acids), (2) modifying themetabolites of the host (e.g., secondary bile acids, primary amines, metabolites of aromatic amino acids (e.g., Trp), lactate, pyruvate, redox-modified sex steroids or polyphenols), (3) metabolizing nutrients (e.g., dietary fibers), or (4) synthesizing vitamins and Cell Biol Toxicol (2016) 32:153–156 DOI 10.1007/s10565-016-9320-6

Identification of Cyclobutane Pyrimidine Dimer-Responsive Genes Using UVB-Irradiated Human Keratinocytes Transfected with In Vitro-Synthesized Photolyase mRNA.

Major biological effects of UVB are attributed to cyclobutane pyrimidine dimers (CPDs), the most common photolesions formed on DNA. To investigate the contribution of CPDs to UVB-induced changes of gene expression, a model system was established by transfecting keratinocytes with pseudouridine-modified mRNA (Ψ-mRNA) encoding CPD-photolyase. Microarray analyses of this model system demonstrated that more than 50% of the gene expression altered by UVB was mediated by CPD photolesions. Functional classification of the gene targets revealed strong effects of CPDs on the regulation of the cell cycle and transcriptional machineries. To confirm the microarray data, cell cycle-regulatory genes, CCNE1 and CDKN2B that were induced exclusively by CPDs were selected for further investigation. Following UVB irradiation, expression of these genes increased significantly at both mRNA and protein levels, but not in cells transfected with CPD-photolyase Ψ-mRNA and exposed to photoreactivating light. Treatment of cells with inhibitors of c-Jun N-terminal kinase (JNK) blocked the UVB-dependent upregulation of both genes suggesting a role for JNK in relaying the signal of UVB-induced CPDs into transcriptional responses. Thus, photolyase mRNA-based experimental platform demonstrates CPD-dependent and -independent events of UVB-induced cellular responses, and, as such, has the potential to identify novel molecular targets for treatment of UVB-mediated skin diseases.

Lithocholic acid, a bacterial metabolite reduces breast cancer cell proliferation and aggressiveness

Our study aimed at finding a mechanistic relationship between the gut microbiome and breast cancer. Breast cancer cells are not in direct contact with these microbes, but disease could be influenced by bacterial metabolites including secondary bile acids that are exclusively synthesized by the microbiome and known to enter the human circulation. In murine and bench experiments, a secondary bile acid, lithocholic acid (LCA) in concentrations corresponding to its tissue reference concentrations (< 1 μM), reduced cancer cell proliferation (by 10-20%) and VEGF production (by 37%), aggressiveness and metastatic potential of primary tumors through inducing mesenchymal-to-epithelial transition, increased antitumor immune response, OXPHOS and the TCA cycle. Part of these effects was due to activation of TGR5 by LCA. Early stage breast cancer patients, versus control women, had reduced serum LCA levels, reduced chenodeoxycholic acid to LCA ratio, and reduced abundance of the baiH (7α/β-hydroxysteroid dehydroxylase, the key enzyme in LCA generation) gene in fecal DNA, all suggesting reduced microbial generation of LCA in early breast cancer.