ABSTRACT
BACKGROUND: In C. elegans, germline development and early embryogenesis rely on posttranscriptional regulation of maternally transcribed mRNAs. In many cases, the 3' untranslated region (UTR) is sufficient to govern the expression patterns of these transcripts. Several RNA-binding proteins are required to regulate maternal mRNAs through the 3'UTR. Despite intensive efforts to map RNA-binding protein-mRNA interactions in vivo, the biological impact of most binding events remains unknown. Reporter studies using single copy integrated transgenes are essential to evaluate the functional consequences of interactions between RNA-binding proteins and their associated mRNAs. RESULTS: In this report, we present an efficient method of generating reporter strains with improved throughput by using a library variant of MosSCI transgenesis. Furthermore, using RNA interference, we identify the suite of RNA-binding proteins that control the expression pattern of five different maternal mRNAs. CONCLUSIONS: The results provide a generalizable and efficient strategy to assess the functional relevance of protein-RNA interactions in vivo, and reveal new regulatory connections between key RNA-binding proteins and their maternal mRNA targets. Developmental Dynamics 245:925-936, 2016. © 2016 Wiley Periodicals, Inc.
Subject(s)
Caenorhabditis elegans Proteins/metabolism , Caenorhabditis elegans/metabolism , 3' Untranslated Regions/genetics , Animals , Animals, Genetically Modified , Caenorhabditis elegans/genetics , Caenorhabditis elegans Proteins/genetics , Gene Expression Regulation, Developmental/genetics , Gene Expression Regulation, Developmental/physiology , Protein Binding/genetics , RNA Interference , RNA, Messenger, Stored/genetics , RNA-Binding Proteins/genetics , RNA-Binding Proteins/metabolismABSTRACT
Breast cancer is the most frequent cancer with second mortality rate in women worldwide. Lack of validated biomarkers for early detection of breast cancer to warranty the diagnosis and effective treatments in early stages has directed to the new therapeutic approach. Cancer/testis antigens which have restricted normal expression in testis and aberrant expression in different cancers are promising targets for generating cancer vaccines, monoclonal antibodies, or dendritic cell-based immunotherapy. In this context, we investigated the expression of two known cancer testis genes, Aurora kinase C (AURKC) and testis expressed 101 (TEX101), and one new candidate, deleted in azoospermia 1 (DAZ1), in six breast cancer cell lines including two ductal carcinomas, T47D and BT-474, and four adenocarcinomas, MDA-MB-231, MDA-MB-468, MCF7, and SKBR3 as well as 50 breast cancer tumors in comparison to normal mammary epithelial cells using quantitative real-time reverse transcription PCR (RT-PCR). Results showed significant overexpression (p = 0.000) of all three genes in BT474, DAZ1 in MDA-MB-231, and AURKC and DAZ1 in SKBR3 and significant downregulation (p = 0.000) of AURKC in MCF7 cell line relative to normal breast epithelial cells. Breast tumors showed significant overexpression of AURKC in comparison to normal breast tissues (p = 0.016). The results are noticeable especially in the case of AURKC; however, there is a little knowledge about the nature, causes, consequences, and effects of cancer/testis antigens activation in different cancers. It is suggested that AURKC has effects on cell division via its serin/threonin kinases activity and organizing microtubules in relation to centrosome/spindle function during mitosis.
Subject(s)
Aurora Kinase C/genetics , Biomarkers, Tumor/genetics , Breast Neoplasms/genetics , Breast/metabolism , Gene Expression Profiling , Membrane Proteins/genetics , RNA-Binding Proteins/genetics , Adenocarcinoma/genetics , Adenocarcinoma/pathology , Breast Neoplasms/pathology , Carcinoma, Ductal, Breast/genetics , Carcinoma, Ductal, Breast/pathology , Deleted in Azoospermia 1 Protein , Female , Humans , Neoplasm Staging , Oligonucleotide Array Sequence Analysis , Prognosis , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Tumor Cells, CulturedABSTRACT
Epigenetic toxicity, a phenomenon in which chemicals exert epigenetic effects and produce toxicity, has been attracting attention in recent years due to advances in toxicology accompanying the development of life sciences. However, it has been difficult to identify epigenetic toxicants due to the lack of a simple experimental system to evaluate epigenetic toxicity. In this study, we developed a prototype of an in vitro reporter assay system for assessing the effects of chemicals on DNA methylation using two promoters showing different degrees of DNA methylation, Agouti IAP and Daz1 promoters, and a luciferase reporter. The system successfully detected DNA demethylating activity using 5-azacytidine, a chemical having DNA demethylation activity, as a positive control chemical, and demethylation of cytosine of CpG in the promoter was confirmed by pyrosequencing analysis. Next, in order to improve the detection sensitivity of the DNA demethylating activity of this system, we tried to increase the basal level of methylation of the Daz1 promoter by pre-methylase treatment of the reporter vectors. As a result, the detection sensitivity of the system was successfully improved in cells where the basal level of methylation was indeed increased by methylase treatment. Thus, the developed assay system here is effective for the simple evaluation of chemicals that affect DNA methylation.