Interactions of RNA with DNA are principles of gene expression control that have recently gained considerable attention. Among RNA-DNA interactions are R-loops and RNA-DNA hybrid G-quadruplexes, as well as RNA-DNA triplexes. It is proposed that RNA-DNA triplexes guide RNA-associated regulatory proteins to specific genomic locations, influencing transcription and epigenetic decision making. Although triplex formation initially was considered solely an in vitro event, recent progress in computational, biochemical, and biophysical methods support in vivo functionality with relevance for gene expression control. Here, we review the central methodology and biology of triplexes, outline paradigms required for triplex function, and provide examples of physiologically important triplex-forming long non-coding RNAs.

Emerging evidence suggests that long noncoding RNAs (lncRNAs) play crucial roles in various cancers. In the present study, we aim to investigate the function and molecular mechanism of an up-regulated and survival-associated lncRNA, LINC00525, in lung adenocarcinoma (LUAD).
The expression level of LINC00525 in tissues was determined by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and in situ hybridization (ISH). The functional role of LINC00525 in LUAD was investigated using gain-and loss-of-function approaches, both in vivo and in vitro. RNA pull-down, RNA immunoprecipitation (RIP), chromatin immunoprecipitation (ChIP), triplex-capture assay, dual-luciferase assay, gene expression microarray, and bioinformatics analysis were used to investigate the potential underlying mechanisms involved.
LINC00525 is highly expressed in LUAD cells and tissues. Survival analysis indicated that upregulation of LINC00525 was associated with poor prognosis in patients with LUAD patients. Knockdown of LINC00525 inhibited cell proliferation and cell cycle progression in vitro. In xenograft models, LINC00525 knockdown suppressed tumor growth and tumorigenesis of tumor-bearing mice. Mechanistically, LINC00525 epigenetically suppressed p21 transcription by guiding Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2) to the p21 promoter through an formation of RNA-DNA triplex with the p21 promoter, leading to increased trimethylation of lysine 27 on histone 3 (H3K27me3) of the p21 promoter. In addition, LINC00525 repressed p21 expression post-transcriptionally by enhancing p21 mRNA decay. LINC00525 promoted p21 mRNA decay by competitively binding to RNA Binding Motif Single Stranded Interacting Protein 2 (RBMS2).
Our findings demonstrate that LINC00525 promotes the progression of LUAD by reducing the transcription and stability of p21 mRNA in concert with EZH2 and RBMS2, thus suggesting that LINC00525 may be a potential therapeutic target for clinical intervention in LUAD.

Endometrial cancer features abnormal growth of cells of the inner lining of the uterus with the potential to invade to other organs. Accumulating evidence suggests that aberrant expression of long non-coding RNA (lncRNA) may facilitate cancer progression. The aim of the present study was to identify the molecular mechanisms of the lncRNA known as DLX6 antisense RNA 1 (DLX6-AS1) in endometrial cancer. Microarray-based analysis was utilized to predict expression profile and possible function pattern of DLX6-AS1 and DLX6 in endometrial cancer, and their expression was quantified in 78 clinically obtained endometrial cancer tissues and also in cell lines. We next assessed the effects of DLX6-AS1 and DLX6 on proliferation, invasion and apoptosis of endometrial cancer cells. A mouse xenograft model was established to confirm DLX6-AS1 functions and explore its underlying regulatory mechanisms in vivo. DLX6-AS1 and DLX6 were highly expressed in endometrial cancer tissues and cells, and their silencing weakened the proliferative and invasive abilities of endometrial cancer cells and tumours, while promoting apoptosis. Mechanistic investigations indicated that DLX6-AS1 formed a triplex structure with DLX6 via interaction with p300/E2F1 acetyltransferase. Thus, we find that functional up-regulation of DLX6-AS1 can promote endometrial cancer progression via a novel triplex mechanism that may prove to be great clinical significance for future treatments of endometrial cancer.

Atherosclerosis involves a slow process of plaque formation on the walls of arteries, and comprises a leading cause of cardiovascular disease. Long non-coding RNAs (lncRNAs) have been implicated in the pathogenesis of atherosclerosis. In this study, we aim to explore the possible involvement of lncRNA \'cyclin-dependent kinase inhibitor 2B antisense noncoding RNA\' (CDKN2B-AS1) and CDKN2B in the progression of atherosclerosis.
Initially, we quantified the expression of CDKN2B-AS1 in atherosclerotic plaque tissues and, in THP-1 macrophage-derived, and human primary macrophage (HPM)-derived foam cells. Next, we established a mouse model of atherosclerosis using apolipoprotein E knockout (ApoE-/-) mice, where lipid uptake, lipid accumulation, and macrophage reverse cholesterol transport (mRCT) were assessed, in order to explore the contributory role of CDKN2B-AS1 to the progression of atherosclerosis. RIP and ChIP assays were used to identify interactions between CDKN2B-AS1, CCCTC-binding factor (CTCF), enhancer of zeste homologue 2 (EZH2), and CDKN2B. Triplex formation was determined by RNA-DNA pull-down and capture assay as well as EMSA experiment.
CDKN2B-AS1 showed high expression levels in atherosclerosis, whereas CDKN2B showed low expression levels. CDKN2B-AS1 accelerated lipid uptake and intracellular lipid accumulation whilst attenuating mRCT in THP-1 macrophage-derived foam cells, HPM-derived foam cells, and in the mouse model. EZH2 and CTCF were found to bind to the CDKN2B promoter region. An RNA-DNA triplex formed by CDKN2B-AS1 and CDKN2B promoter was found to recruit EZH2 and CTCF in the CDKN2B promoter region and consequently inhibit CDKN2B transcription by accelerating histone methylation.
The results demonstrated that CDKN2B-AS1 promotes atherosclerotic plaque formation and inhibits mRCT in atherosclerosis by regulating CDKN2B promoter, and thereby could be a potential therapeutic target for atherosclerosis.

Long noncoding RNAs (lncRNAs) play important roles in regulating various biological processes including growth and stress responses in plants. RNA-seq data sets provide a good resource to exploring the noncoding transcriptome and studying their comprehensive interactions with the coding transcriptome. Here, we describe computational procedures for studying plant lncRNAs including long intergenic noncoding RNAs (lincRNAs) and long noncoding natural antisense transcripts (lncNATs). Bioinformatics tools for transcriptome assembly, lncRNA identification, and functional interpretations are included. Finally, we also introduce PLncDB, a user-friendly database that provides comprehensive information of plant lncRNAs for researchers to compare their own data sets to those in public database.