Naturally occurring circular RNAs efficiently impair miRNA functions. Synthetic circular RNAs may thus serve as potent agents for miRNA inhibition. Their therapeutic effect critically relies on (i) the identification of optimal miRNA targets, (ii) the optimization of decoy structures and (iii) the development of efficient formulations for their use as drugs. In this study, we extensively explored the functional relevance of miR-21-5p in cancer cells. Analyses of cancer transcriptomes reveal that miR-21-5p is the by far most abundant miRNA in human cancers. Deletion of the MIR21 locus in cancer-derived cells identifies several direct and indirect miR-21-5p targets, including major tumor suppressors with prognostic value across cancers. To impair miR-21-5p activities, we evaluate synthetic, circular RNA decoys containing four repetitive binding elements. In cancer cells, these decoys efficiently elevate tumor suppressor expression and impair tumor cell vitality. For their in vivo delivery, we for the first time evaluate the formulation of decoys in polyethylenimine (PEI)-based nanoparticles. We demonstrate that PEI/decoy nanoparticles lead to a significant inhibition of tumor growth in a lung adenocarcinoma xenograft mouse model via the upregulation of tumor suppressor expression. These findings introduce nanoparticle-delivered circular miRNA decoys as a powerful potential therapeutic strategy in cancer treatment.

BackgroundLung cancer is the leading cause of cancer-related deaths with 1.8 million new cases each year and poor 5-year prognosis. Promoter hypermethylation of tumour suppressors leads to their inactivation and thereby can promote cancer development and progression.ResultsIn this study, we analysed ZAR1 (zygote arrest 1), which has been said to be a maternal-effect gene and its expression mostly limited to certain reproductive tissues. Our study shows that ZAR1 is expressed in normal lung but inactivated by promoter methylation in lung cancer. ZAR1 is hypermethylated in primary lung cancer samples (22% small cell lung carcinoma (SCLC) and 76% non-small cell lung carcinoma (NSCLC), p < 0.001) vs. normal control lung tissue (11%). In lung cancer cell lines, ZAR1 was significantly methylated in 75% of SCLC and 83% of NSCLC vs. normal tissue (p < 0.005/0.05). In matching tumours and control tissues, we observed that NSCLC primary tumour samples exhibited a tumour-specific promoter methylation of ZAR1 in comparison to the normal control lung tissue. Demethylation treatment of various lung cancer cell lines reversed ZAR1 promoter hypermethylation and subsequently re-established ZAR1 expression. In addition, we could show the growth inhibitory potential of ZAR1 in lung cancer cell lines and cancer cell lines. Exogenous expression of ZAR1 not only inhibited colony formation but also blocked cell cycle progression of cancer cell lines.ConclusionsOur study shows for the first time the lung tumour-specific epigenetic inactivation of ZAR1 due to DNA methylation of its CpG island promoter. Furthermore, ZAR1 was characterised by the ability to block tumour growth through the inhibition of cell cycle progression in cancer cell lines. We propose that ZAR1 could serve as an epigenetically inactivated biomarker in lung cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s13148-017-0360-4) contains supplementary material, which is available to authorized users.

Characterized by their covalently closed structure and thus an elevated stability compared to linear RNA molecules, circular RNAs (circRNAs) form a novel class of mainly non-coding RNAs. Although the biological functions of naturally occurring circRNAs are largely unknown, they were reported to act as molecular sponges, sequestering microRNAs (miRNAs), resulting in a de-repression of target mRNAs. Taking these characteristics of naturally occurring circRNAs into account, artificial circRNAs could be a potential tool in molecular biology and medicine. Using the Hepatitis C virus (HCV) as a model system, this application of artificial circular RNAs was demonstrated. The virus requires cellular miRNA miR-122 for its life cycle, and circRNAs specifically engineered to efficiently sequester this miRNA impacted viral propagation. Since in this context the production of engineered circRNA remains the limiting factor, we present a method to produce and efficiently purify artificial circRNA sponges (ciRS) in vitro. In this protocol we provide insights into a small-scale and large-scale production technique of artificial circular RNA sponges relying on in vitro transcription and RNA ligation.

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BackgroundCancer still is one of the leading causes of death and its death toll is predicted to rise further. We identified earlier the potential tumour suppressor zygote arrest 1 (ZAR1) to play a role in lung carcinogenesis through its epigenetic inactivation.ResultsWe are the first to report that ZAR1 is epigenetically inactivated not only in lung cancer but also across cancer types, and ZAR1 methylation occurs across its complete CpG island. ZAR1 hypermethylation significantly correlates with its expression reduction in cancers. We are also the first to report that ZAR1 methylation and expression reduction are of clinical importance as a prognostic marker for lung cancer and kidney cancer. We further established that the carboxy (C)-terminally present zinc-finger of ZAR1 is relevant for its tumour suppression function and its protein partner binding associated with the mRNA/ribosomal network. Global gene expression profiling supported ZAR1's role in cell cycle arrest and p53 signalling pathway, and we could show that ZAR1 growth suppression was in part p53 dependent. Using the CRISPR-dCas9 tools, we were able to prove that epigenetic editing and reactivation of ZAR1 is possible in cancer cell lines.ConclusionZAR1 is a novel cancer biomarker for lung and kidney, which is epigenetically silenced in various cancers by DNA hypermethylation. ZAR1 exerts its tumour suppressive function in part through p53 and through its zinc-finger domain. Epigenetic therapy can reactivate the ZAR1 tumour suppressor in cancer.

Transcriptome-wide analysis of RNA-binding partners is commonly achieved using UV crosslinking and immunoprecipitation (CLIP). Individual-nucleotide-resolution CLIP (iCLIP)enables identification of the specific position of the protein–RNA interaction. In addition to RNA-binding proteins (RBPs), microRNA (miRNA)–mRNA interactions also play a crucial role in the regulation of gene expression. Argonaute-2 (Ago2) mediates miRNA binding to a multitude of mRNA target sites, enabling the identification of miRNA–mRNA interactions by employing modified Ago2-CLIP protocols. Here, we describe an Ago2-specific CLIP protocol optimized for the use of small quantities of cell material, targeting endogenous Ago2 while avoiding possible methodological biases such as metabolic labeling or Ago2 overexpression and applying the latest advances in CLIP library preparation, the iCLIP2 protocol. In particular, we focus on the optimization of lysis conditions and improved radioactive labeling of the 5′ end of the miRNA.

The sodium-dependent organic anion transporter (SOAT, gene symbol SLC10A6) specifically transports 3′- and 17′-monosulfated steroid hormones, such as estrone sulfate and dehydroepiandrosterone sulfate, into specific target cells. These biologically inactive sulfo-conjugated steroids occur in high concentrations in the blood circulation and serve as precursors for the intracrine formation of active estrogens and androgens that contribute to the overall regulation of steroids in many peripheral tissues. Although SOAT expression has been detected in several hormone-responsive peripheral tissues, its quantitative contribution to steroid sulfate uptake in different organs is still not completely clear. Given this fact, the present review provides a comprehensive overview of the current knowledge about the SOAT by summarizing all experimental findings obtained since its first cloning in 2004 and by processing SOAT/SLC10A6-related data from genome-wide protein and mRNA expression databases. In conclusion, despite a significantly increased understanding of the function and physiological significance of the SOAT over the past 20 years, further studies are needed to finally establish it as a potential drug target for endocrine-based therapy of steroid-responsive diseases such as hormone-dependent breast cancer.

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