Identification and Initial Functional Characterization of a Human Vascular Cell-Enriched Long Noncoding RNA

OBJECTIVE: Long noncoding RNAs (lncRNAs) represent a rapidly growing class of RNA genes with functions related primarily to transcriptional and post-transcriptional control of gene expression. There is a paucity of information about lncRNA expression and function in human vascular cells. Thus, we set out to identify novel lncRNA genes in human vascular smooth muscle cells and to gain insight into their role in the control of smooth muscle cell phenotypes. APPROACH AND RESULTS: RNA sequencing of human coronary artery smooth muscle cells revealed 31 unannotated lncRNAs, including a vascular cell-enriched lncRNA (smooth muscle and endothelial cell-enriched migration/differentiation-associated long noncoding RNA [SENCR]). Strand-specific reverse transcription polymerase chain reaction (PCR) and rapid amplification of cDNA ends indicate that SENCR is transcribed antisense from the 5' end of the FLI1 gene and exists as 2 splice variants. RNA fluorescence in situ hybridization and biochemical fractionation studies demonstrate SENCR is a cytoplasmic lncRNA. Consistent with this observation, knockdown studies reveal little to no cis-acting effect of SENCR on FLI1 or neighboring gene expression. RNA-sequencing experiments in smooth muscle cells after SENCR knockdown disclose decreased expression of Myocardin and numerous smooth muscle contractile genes, whereas several promigratory genes are increased. Reverse transcription PCR and Western blotting experiments validate several differentially expressed genes after SENCR knockdown. Loss-of-function studies in scratch wound and Boyden chamber assays support SENCR as an inhibitor of smooth muscle cell migration. CONCLUSIONS: SENCR is a new vascular cell-enriched, cytoplasmic lncRNA that seems to stabilize the smooth muscle cell contractile phenotype

Fluorescent Labeling, Co-Tracking, and Quantification of RNA In Cellulo.

RNA plays a fundamental, pervasive role in cellular physiology, through the maintenance and controlled readout of all genetic information, a functional landscape we are only beginning to understand. In particular, the cellular mechanisms for the spatiotemporal control of the plethora of RNAs are still poorly understood. Intracellular single-molecule fluorescence microscopy provides a powerful emerging tool for probing the pertinent biophysical and biochemical parameters that govern cellular RNA functions, including those of protein-encoding mRNAs. Yet progress has been hampered by the scarcity of high-yield, efficient methods to fluorescently label RNA molecules without the need to drastically increase their molecular weight through artificial appendages that may result in altered behavior. Herein, we employ a series of in vitro enzymatic techniques to efficiently, extensively and in high-yield, incorporate chemically modified nucleoside triphosphates into a transcribed messenger RNA body, between its body and tail (BBT), or randomly throughout the poly(A) tail (tail). Of these, BBT and tail modified strategies proved the most promising methods to functionally label messenger RNA and single-particle track their behaviors using our in-house single-molecule assay: intracellular single-molecule high resolution localization and counting (iSHiRLoC). From this research also was spawned a novel method to anchor an RNA to the actin cytoskeleton for the study of long-term interactions within a cellular context, termed: Gene-Actin Tethered Intracellular Co-tracking Assay (GATICA). Here, biotinylated RNA is tethered to the actin surface, either through complexation with a streptavidin coupled to a biotinylated phalloidin molecule or actin protein. Taken together, this body of work represents strategies for the labeling and visualizing, both freely diffusing and actin tethered, long-RNAs and their interactome in real-time.PHDChemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/135832/1/tcuster_1.pd

An evaluation of cancer biomarkers in normal ovarian epithelial cells and ovarian cancer cell lines

Philosophiae Doctor - PhDIntroduction: Globally, there are over 190,000 new reported cases of ovarian cancers per annum. This comprises 3% to 4% of all cancers in women. Ovarian cancer is one of the leading causes of deaths in women. Ovarian cancer is the second most diagnosed gynaecological malignancy and over all the fifth cause leading to death among all types of cancer in the UK in 2004. More than 70% of epithelial ovarian cancers are diagnosed at an advanced stage. Consequently, the prognosis is poor and the mortality rate high. Thus, the survival rate is affected by how far the disease has progressed or spread. A dire need exists to identify ovarian cancer biomarkers, which could be used as good indicators of expression in ovarian cancer cells in vitro Aim: The aim of this study was to analyse selected cancer biomarkers, which are currently under intense investigation for their suitability to diagnose epithelial ovarian cancer at an early stage. These biomarkers were analysed in terms of their in vitro expression in normal epithelial cells and ovarian cancer cell lines, which allows for their genomic and proteomic classification. The expression analysis of each biomarker is related to the malignancy of a tumour and, therefore, advocates its use for potential future improvement of sensitive tumour markers. Methods: The primary human ovarian surface epithelial cell line (HOSEpiC), SKOV-3 cells and the OAW42 human epithelial ovarian tumour cell lines were used to evaluate the selected cancer biomarkers. Cells were cultured using appropriate media and supplements, and real-time quantitative polymerase chain reaction (RT-PCR) utilized to validate expression levels of the following genes: HDAC1, HDAC2, HDCA3, HDAC5, HDAC6, HDAC7, HDAC8, LPAR1, LPAR2, MUC16 and FOSL1, against normal housekeeping genes GAPDH and HPRT. In addition, immunocytochemistry was also used in the validation process of the aforementioned genes. Significance: ovarian cancer cells express gene signatures, which pose significant challenges for cancer drug development, therapeutics, prevention and management. The present study is an effort to explore ovarian cancer biomarkers to provide a better diagnostic method that may offer translational therapeutic possibilities to increase five- year survival rate. Results: HDAC5, HDAC6, LPAR1, LPAR2 and MUC16 expressed distinctively in ovarian cancers matched to other tissues or cancer types have already been identified by RT-QPCR and confirmed by immunocytochemistry and efforts to generate monoclonal antibodies to the other six genes (HDAC1, HDAC2, HDAC3, HDAC7, HDAC8 and FOSL1) encoded proteins are underway. Conclusions: here we provide strong evidence suggesting that HDAC5, HDAC6, LPAR1, LPAR2, except MUC16 are up regulated in ovarian cancer. These data were confirmed by examining Human Protein Atlas (HPA) databases, in addition to protein expression of HDAC5, HDAC6, LPAR1, LPAR2 and MUC16 in cells cytoplasm. For future prospective, using other techniques that assess the variant expression that could explain the release of these gene candidates into the circulation with serum tumour markers, and protein expression will be strengthened