ABSTRACT
CircRNAs are a subclass of lncRNAs that have been found to be abundantly present in a wide range of species, including humans. CircRNAs are generally produced by a noncanonical splicing event called backsplicing that is dependent on the canonical splicing machinery, giving rise to circRNAs classified into three main categories: exonic circRNA, circular intronic RNA, and exon-intron circular RNA. Notably, circRNAs possess functional importance and display their functions through different mechanisms of action including sponging miRNAs, or even being translated into functional proteins. In addition, circRNAs also have great potential as biomarkers, particularly in cancer, thanks to their high stability, tissue type and developmental stage specificity, and their presence in biological fluids, which make them promising candidates as noninvasive biomarkers. In this chapter, we describe the most commonly used techniques for the study of circRNAs as cancer biomarkers, including high-throughput techniques such as RNA-Seq and microarrays, and other methods to analyze the presence of specific circRNAs in patient samples.
Subject(s)
Biomarkers, Tumor , Neoplasms/diagnosis , Neoplasms/genetics , RNA, Circular/genetics , Animals , Gene Expression Regulation , Gene Expression Regulation, Neoplastic , High-Throughput Nucleotide Sequencing , Humans , Liquid Biopsy/methods , Neoplasms/metabolism , RNA Splicing , RNA Stability , RNA-Binding Proteins/metabolism , Real-Time Polymerase Chain Reaction , Sequence Analysis, RNAABSTRACT
The Golgi organelle duplicates its protein and lipid content to segregate evenly between two daughter cells after mitosis. However, how Golgi biogenesis is regulated during interphase remains largely unknown. Here we show that messenger RNA (mRNA) expression of GOLPH3 and GOLGA2, two genes encoding Golgi proteins, is induced specifically in G1 phase, suggesting a link between cell cycle regulation and Golgi growth. We have examined the role of E2F transcription factors, critical regulators of G1 to S progression of the cell cycle, in the expression of Golgi proteins during interphase. We show that promoter activity for GOLPH3, a Golgi protein that is also oncogenic, is induced by E2F1-3 and repressed by E2F7. Mutation of the E2F motifs present in the GOLPH3 promoter region abrogates E2F1-mediated induction of a GOLPH3 luciferase reporter construct. Furthermore, we identify a critical CREB/ATF element in the GOLPH3 promoter that is required for its steady state and ATF2-induced expression. Interestingly, depletion of GOLPH3 with small interfering RNA (siRNA) delays the G1 to S transition in synchronized U2OS cells. Taken together, our results reveal a link between cell cycle regulation and Golgi function, and suggest that E2F-mediated regulation of Golgi genes is required for the timely progression of the cell cycle.
Subject(s)
Activating Transcription Factor 2/metabolism , E2F Transcription Factors/metabolism , Membrane Proteins/chemistry , Membrane Proteins/genetics , Animals , Binding Sites , Cell Cycle , Cell Line, Tumor , Gene Expression Regulation , Golgi Apparatus/genetics , Golgi Apparatus/metabolism , Humans , Membrane Proteins/metabolism , Mice , Mutation , NIH 3T3 Cells , Phosphoproteins/genetics , Promoter Regions, GeneticABSTRACT
Huntington's disease (HD) is a polyglutamine disorder caused by a CAG expansion in the Huntingtin (HTT) gene exon 1. This expansion encodes a mutant protein whose abnormal function is traditionally associated with HD pathogenesis; however, recent evidence has also linked HD pathogenesis to RNA stable hairpins formed by the mutant HTT expansion. Here, we have shown that a locked nucleic acid-modified antisense oligonucleotide complementary to the CAG repeat (LNA-CTG) preferentially binds to mutant HTT without affecting HTT mRNA or protein levels. LNA-CTGs produced rapid and sustained improvement of motor deficits in an R6/2 mouse HD model that was paralleled by persistent binding of LNA-CTG to the expanded HTT exon 1 transgene. Motor improvement was accompanied by a pronounced recovery in the levels of several striatal neuronal markers severely impaired in R6/2 mice. Furthermore, in R6/2 mice, LNA-CTG blocked several pathogenic mechanisms caused by expanded CAG RNA, including small RNA toxicity and decreased Rn45s expression levels. These results suggest that LNA-CTGs promote neuroprotection by blocking the detrimental activity of CAG repeats within HTT mRNA. The present data emphasize the relevance of expanded CAG RNA to HD pathogenesis, indicate that inhibition of HTT expression is not required to reverse motor deficits, and further suggest a therapeutic potential for LNA-CTG in polyglutamine disorders.
