The human genome encodes thousands of long noncoding RNA (lncRNA) genes; the function of majority of them is poorly understood. Aberrant expression of a significant number of lncRNAs is observed in various diseases, including cancer. To gain insights into the role of lncRNAs in breast cancer progression, we performed genome-wide transcriptome analyses in an isogenic, triple negative breast cancer (TNBC/basal-like) progression cell lines using a 3D cell culture model. We identified significantly altered expression of 1853 lncRNAs, including ~500 natural antisense transcript (NATs) lncRNAs. A significant number of breast cancer-deregulated NATs displayed co-regulated expression with oncogenic and tumor suppressor protein-coding genes in cis. Further studies on one such NAT, PDCD4-AS1 lncRNA reveal that it positively regulates the expression and activity of the tumor suppressor PDCD4 in mammary epithelial cells. Both PDCD4-AS1 and PDCD4 show reduced expression in TNBC cell lines and in patients, and depletion of PDCD4-AS1 compromised the cellular levels and activity of PDCD4. Further, tumorigenic properties of PDCD4-AS1-depleted TNBC cells were rescued by exogenous expression of PDCD4, implying that PDCD4-AS1 acts upstream of PDCD4. Mechanistically, PDCD4-AS1 stabilizes PDCD4 RNA by forming RNA duplex and controls the interaction between PDCD4 RNA and RNA decay promoting factors such as HuR. Our studies demonstrate crucial roles played by NAT lncRNAs in regulating post-transcriptional gene expression of key oncogenic or tumor suppressor genes, thereby contributing to TNBC progression.

RNA editing by adenosine deaminases that act on RNA (ADARs) diversifies the transcriptome by changing adenosines to inosines. In mammals, editing levels vary in different tissues, during development, and also in pathogenic conditions. From a screen for repressors of editing we have isolated three proteins that repress ADAR2-mediated RNA editing. The three proteins RPS14, SFRS9 and DDX15 interact with RNA. Overexpression or depletion of these proteins can decrease or increase editing levels by 15%, thus allowing a modulation of RNA editing up to 30%. Interestingly, the three proteins alter RNA editing in a substrate-specific manner that correlates with their RNA binding preferences. In mammalian cells, SFRS9 significantly affects editing of the two substrates CFLAR and cyFIP2, while the ribosomal protein RPS14 mostly inhibits editing of cyFIP2 messenger RNA. The helicase DDX15, in turn, has a strong effect on editing in Caenorhabditis elegans.Expression of the three factors decreases during mouse brain development. Moreover, expression levels of SFRS9 and DDX15 respond strongly to neuronal stimulation or repression, showing an inverse correlation with editing levels.Colocalization and immunoprecipitation studies demonstrate a direct interaction of SFRS9 and RPS14 with ADAR2, while DDX15 associates with other helicases and splicing factors. Our data show that different editing sites can be specifically altered in their editing pattern by changing the local RNP landscape.

RNA editing by adenosine deaminases that act on RNA converts adenosines to inosines in coding and non-coding regions of mRNAs. Inosines are interpreted as guanosines and hence, this type of editing can change codons, alter splice patterns, or influence the fate of an RNA. A to I editing is most abundant in the central nervous system (CNS). Here, targets for this type of nucleotide modification frequently encode receptors and channels. In many cases, the editing-induced amino acid exchanges alter the properties of the receptors and channels. Consistently, changes in editing patterns are frequently found associated with diseases of the CNS. In this review we describe the mechanisms of RNA editing and focus on target mRNAs of editing that are functionally relevant to normal and aberrant CNS activity.

Adenosine to inosine deamination of RNA is widespread in metazoa. Inosines are recognized as guanosines and, therefore, this RNA-editing can influence the coding potential, localization and stability of RNAs. Therefore, RNA editing contributes to the diversification of the transcriptome in a flexible manner. The editing reaction is performed by adenosine deaminases that act on RNA (ADARs), which are essential for normal life and development in many organisms. Changes in editing levels are observed during development but also in neurological pathologies like schizophrenia, depression or tumors. Frequently, changes in editing levels are not reflected by changes in ADAR levels suggesting a regulation of enzyme activity. Until now, only a few factors are known that influence the activity of ADARs. Here we present a two-stage in vivo editing screen aimed to isolate enhancers of editing. A primary, high-throughput yeast-screen is combined with a more accurate secondary screen in mammalian cells that uses a fluorescent read-out to detect minor differences in RNA-editing. The screen was successfully employed to identify DSS1/SHFM1, the RNA binding protein hnRNP A2/B1 and a 3′ UTR as enhancers of editing. By varying intracellular DSS1/SHFM1 levels, we can modulate A to I editing by up to 30%. Proteomic analysis indicates an interaction of DSS1/SHFM1 and hnRNP A2/B1 suggesting that both factors may act by altering the cellular RNP landscape. An extension of this screen to cDNAs from different tissues or developmental stages may prove useful for the identification of additional enhancers of RNA-editing.

Adenosine (A) to inosine (I) RNA editing is a hydrolytic deamination reaction catalyzed by the adenosine deaminase (ADAR) enzyme acting on double-stranded RNA. This posttranscriptional process diversifies a plethora of transcripts, including coding and noncoding RNAs. Interestingly, few studies have been carried out to determine the role of RNA editing in vascular disease. The aim of this study was to determine the potential role of ADARs in congenital heart disease. Strong downregulation of ADAR2 and increase in ADAR1 expression was observed in blood samples from congenital heart disease (CHD) patients. The decrease in expression of ADAR2 was in line with its downregulation in ventricular tissues of dilated cardiomyopathy patients. To further decipher the plausible regulatory pathway of ADAR2 with respect to heart physiology, miRNA profiling of ADAR2 was performed on tissues from ADAR2-/- mouse hearts. Downregulation of miRNAs (miR-29b, miR-405, and miR-19) associated with cardiomyopathy and cardiac fibrosis was observed. Moreover, the upregulation of miR-29b targets COL1A2 and IGF1, indicated that ADAR2 might be involved in cardiac myopathy. The ADAR2 target vascular development associated protein-coding gene filamin B (FLNB) was selected. The editing levels of FLNB were dramatically reduced in ADAR2 -/- mice; however, no observable changes in FLNB expression were noted in ADAR2 -/- mice compared to wild-type mice. This study proposes that sufficient ADAR2 enzyme activity might play a vital role in preventing cardiovascular defects.

Point-of-care (POC) or near-patient testing allows clinicians to accurately achieve real-time diagnostic results performed at or near to the patient site. The outlook of POC devices is to provide quicker analyses that can lead to well-informed clinical decisions and hence improve the health of patients at the point-of-need. Microfluidics plays an important role in the development of POC devices. However, requirements of handling expertise, pumping systems and complex fluidic controls make the technology unaffordable to the current healthcare systems in the world. In recent years, capillary-driven flow microfluidics has emerged as an attractive microfluidic-based technology to overcome these limitations by offering robust, cost-effective and simple-to-operate devices. The internal wall of the microchannels can be pre-coated with reagents, and by merely dipping the device into the patient sample, the sample can be loaded into the microchannel driven by capillary forces and can be detected via handheld or smartphone-based detectors. The capabilities of capillary-driven flow devices have not been fully exploited in developing POC diagnostics, especially for antimicrobial resistance studies in clinical settings. The purpose of this review is to open up this field of microfluidics to the ever-expanding microfluidic-based scientific community.

Caveolae-mediated endocytosis regulates cell adhesion and growth in an anchorage-dependent manner. Studies of the endocytic function of caveolae have suggested a wide-ranging list of cargoes, including a number of receptors and extracellular proteins, ligands and nutrients from the extracellular matrix. Disruption of the processes of caveolae-mediated endocytosis mediated by signaling proteins is critical to cellular integrity. Caveolin-1 and dynamin-2 are the 2 major proteins associated with endocytotic function. Mechanistically, dynamin-2 has a co-equal role with caveolin-1 in terms of caveolae-derived endosome formation. Recent studies have revealed the pathological outcomes associated with the dysregulation of caveolin-1 and dynamin-2 expression. Increased expression levels of the gene for caveolin, Cav-1, resulting in augmented cellular metastasis and invasion, have been demonstrated in various types of cancer, and overexpression of the gene for dynamin-2, DNM2, has been associated with tumorigenesis in cervical, pancreatic and lung cancer. An increased expression of Cav-1 and DNM2 is known to be associated with the invasive behavior of cancer cells, and with cancer progression. Furthermore, it has been previously demonstrated that, in caveolar assembly and caveolae mediated endocytosis, Cav-1 interacts directly with DNM2 during the processes. Altered expression of the 2 genes is critical for the normal function of the cell. The expression patterns of Cav-1 and DNM2 have been previously examined in bladder cancer cell lines, and were each demonstrated to be overexpressed. In the present study, the expression levels of these 2 genes in bladder cancer samples were quantified. The gene expression levels of Cav-1 and DNM2 were identified to be increased 8.88-and 8.62-fold, respectively, in tumors compared with the normal controls. Furthermore, high-grade tumors exhibited significantly increased expression levels of Cav-1 and DNM2 (both P<0.0001) compared with the low-grade tumors. In addition, compared with normal control samples, the expression of the 2 genes in tumor samples was observed to be highly significant (P<0.0001), with a marked positive correlation identified for the tumors (Pearson's correlation coefficient, r=0.80 for the tumor samples vs. r=0.32 in the normal control samples). Taken together, the results of the present study demonstrated that the overexpression of Cav-1 and DNM2 genes, and a determination of their correlation coefficients, may be a potential risk factor for bladder cancer, in addition to other clinical factors.

Triple-negative breast cancer (TNBC) is one of the most aggressive breast cancer (BC) subtypes with a poor prognosis and high recurrence rate. Recent studies have identified vital roles played by several lncRNAs (long noncoding RNAs) in BC pathobiology. Cell type-specific expression of lncRNAs and their potential role in regulating the expression of oncogenic and tumor suppressor genes have made them promising cancer drug targets. By performing a transcriptome screen in an isogenic TNBC/basal subtype BC progression cell line model, we recently reported ∼1800 lncRNAs that display aberrant expression during breast cancer progression. Mechanistic studies on one such nuclear-retained lncRNA, linc02095, reveal that it promotes breast cancer proliferation by facilitating the expression of oncogenic transcription factor, SOX9. Both linc02095 and SOX9 display coregulated expression in BC patients as well in basal subtype BC cell lines. Knockdown of linc02095 results in decreased BC cell proliferation, whereas its overexpression promotes cells proliferation. Linc02095-depleted cells display reduced expression of SOX9 concomitant with reduced RNA polymerase II occupancy at the SOX9 gene body as well as defective SOX9 mRNA export, implying that linc02095 positively regulates SOX9 transcription and mRNA export. Finally, we identify a positive feedback loop in BC cells that controls the expression of both linc02095 and SOX9. Thus, our results unearth tumor-promoting activities of a nuclear lncRNA linc02095 by facilitating the expression of key oncogenic transcription factor in BC.