Although long noncoding RNAs (lncRNAs) have been recognized in recent years to constitute a significant portion of mammalian transcriptome, and the functional impact of several lncRNAs has been characterized by a few studies, yet it is still difficult to large-scale ascertain their functions from lab experiment or structure prediction. To address this deficit, we have developed a computational pipeline to large-scale annotate the functions of lncRNA based on coding-noncoding gene co-expression network. In this network, a node (circle) represents the protein-coding gene or lncRNA, and an edge (connecting line between nodes) indicates that the two genes are co-expressed (the correlation coefficients of connected genes reached the defined cutoff). In this chapter, we show how to use an lncRNA functional annotation pipeline to construct a co-expression network based on gene expression profiles in prostate cancer and how to further predict lncRNA functions using model-based and hub-based sub-networks.
Each day, more and more transcripts are being discovered along the genome (especially in poorly annotated species) thanks to the rapid progress of high-throughput technology such as RNA sequencing. However, this situation unravels the challenge of how to classify the newly identified transcripts into protein coding or noncoding. Here, we describe a de novo approach named coding-noncoding index (CNCI), a powerful signature tool by profiling adjoining nucleotide triplets (ANT) to effectively distinguish between protein-coding and noncoding sequences independently of known annotations. The main advantage of CNCI is its ability to accurately classify transcripts assembled from whole-transcriptome sequencing data in a cross-species manner, which allowed it to be used for all vertebrates and invertebrates based on the training data of well-annotated species (such as human and Arabidopsis). In this chapter, we illustrate the CNCI method in detail through an example of RNA-sequencing data generated from six biological replicates of six mouse tissues. CNCI software is available at http://www.bioinfo.org/software/cnci.
The differentiation of distinct multifocal hepatocellular carcinoma (HCC): multicentric disease vs. intrahepatic metastases, in which the management and prognosis varies substantively, remains problematic. We aim to stratify multifocal HCC and identify novel diagnostic and prognostic biomarkers by performing whole genome and transcriptome sequencing, as part of a multi-omics strategy.
Nicotine, the addictive component of cigarettes, promotes lung cancer proliferation via the ?7-nicotinic acetylcholine receptor (?7-nAChR) subtype. The present manuscript explores the effect of nicotine exposure on ?7-nAChR levels in squamous cell carcinoma of the lung (SCC-L) in vitro and in vivo. Nicotine (at concentrations present in the plasma of average smokers) increased ?7-nAChR levels in human SCC-L cell lines. Nicotine-induced up-regulation of ?7-nAChR was confirmed in vivo by chicken chorioallantoic membrane models. We also observed that the levels of ?7-nAChR in human SCC-L tumors (isolated from patients who are active smokers) correlated with their smoking history. Nicotine increased the levels of ?7-nAChR mRNA and ?7-nAChR transcription in human SCC-L cell lines and SCC-L tumors. Nicotine-induced up-regulation of ?7-nAChR required GATA4 and GATA6. ChIP assays showed that nicotine induced the binding of GATA4 or GATA6 to Sp1 on the ?7-nAChR promoter, thereby inducing its transcription and increasing its levels in human SCC-L. Our data are clinically relevant because SCC-L patients smoked for decades before being diagnosed with cancer. It may be envisaged that continuous exposure to nicotine (in such SCC-L patients) causes up-regulation of ?7-nAChRs, which facilitates tumor growth and progression. Our results will also be relevant to many SCC-L patients exposed to nicotine via second-hand smoke, electronic cigarettes, and patches or gums to quit smoking.
Mammals and other complex organisms can transcribe an abundance of long non-coding RNAs (lncRNAs) that fulfill a wide variety of regulatory roles in many biological processes. These roles, including as scaffolds and as guides for protein-coding genes, mainly depend on the structure and expression level of lncRNAs. In this review, we focus on the current methods for analyzing lncRNA structure and expression, which is basic but necessary information for in-depth, large-scale analysis of lncRNA functions.
It is a challenge to classify protein-coding or non-coding transcripts, especially those re-constructed from high-throughput sequencing data of poorly annotated species. This study developed and evaluated a powerful signature tool, Coding-Non-Coding Index (CNCI), by profiling adjoining nucleotide triplets to effectively distinguish protein-coding and non-coding sequences independent of known annotations. CNCI is effective for classifying incomplete transcripts and sense-antisense pairs. The implementation of CNCI offered highly accurate classification of transcripts assembled from whole-transcriptome sequencing data in a cross-species manner, that demonstrated gene evolutionary divergence between vertebrates, and invertebrates, or between plants, and provided a long non-coding RNA catalog of orangutan. CNCI software is available at http://www.bioinfo.org/software/cnci.
Ovarian cancer is the fifth leading cause of cancer deaths for women in America. With no known carcinogens or manageable risk factors, targeted prevention is currently unavailable. Angioprevention is a nonspecific strategy to limit the growth of solid tumors and is especially suitable for ovarian cancers. In search of angiopreventive agents, we examined chaetoglobosin K (ChK), a natural cytochalasan compound from the fungus Diplodia macrospora. We found that ChK significantly inhibits cell viability at concentrations as low as 0.5 ?mol/l for A2780/CP70 ovarian cancer cells and 1.0 ?mol/l for OVCAR-3 cells. ChK also significantly inhibits the secretion of key angiogenesis mediators, including Akt (which is also known as protein kinase B), hypoxia-inducible factor 1? (HIF-1?), and vascular epithelial growth factor (VEGF) by ovarian cancer cells. More importantly, ChK inhibits in-vitro and in-vivo angiogenesis induced by ovarian cancer cells and reduces the migratory capability of human umbilical vein endothelial cells. Through transfection of HIF-1? plasmids in luciferase assays, we found that ChK executes its VEGF inhibition by mediating the downregulation of HIF-1?. Furthermore, chromatin immunoprecipitation assays using the HIF-1? antibody revealed that ChK inhibits the interaction of HIF-1? with the VEGF promoter. Through transfection of Akt plasmids, we found that inhibition of HIF-1? by ChK occurs through downregulation of Akt. To our knowledge, this is the first report about the potential angioprevention of ChK. Our data suggest that this natural fungal bioactive compound effectively inhibits angiogenesis through downregulation of VEGF-binding HIF-1? and could be an effective agent for cancer treatment.
The functional impact of several long intergenic non-coding RNAs (lincRNAs) has been characterized in previous studies. However, it is difficult to identify lincRNAs on a large-scale and to ascertain their functions or predict their structures in laboratory experiments because of the diversity, lack of knowledge and specificity of expression of lincRNAs. Furthermore, although there are a few well-characterized examples of lincRNAs associated with cancers, these are just the tip of the iceberg owing to the complexity of cancer. Here, by combining RNA-Seq data from several kinds of human cell lines with chromatin-state maps and human expressed sequence tags, we successfully identified more than 3000 human lincRNAs, most of which were new ones. Subsequently, we predicted the functions of 105 lincRNAs based on a coding-non-coding gene co-expression network. Finally, we propose a genetic mediator and key regulator model to unveil the subtle relationships between lincRNAs and lung cancer. Twelve lincRNAs may be principal players in lung tumorigenesis. The present study combines large-scale identification and functional prediction of human lincRNAs, and is a pioneering work in characterizing cancer-associated lincRNAs by bioinformatics.
Ovarian cancer is one of the primary causes of death for women all through the Western world. Baicalin and baicalein are naturally occurring flavonoids that are found in the roots and leaves of some Chinese medicinal plants and are thought to have antioxidant activity and possible anti-angiogenic, anti-cancer, anxiolytic, anti-inflammatory and neuroprotective activities. Two kinds of ovarian cancer (OVCAR-3 and CP-70) cell lines and a normal ovarian cell line (IOSE-364) were selected to be investigated in the inhibitory effect of baicalin and baicalein on cancer cells. Largely, baicalin and baicalein inhibited ovarian cancer cell viability in both ovarian cancer cell lines with LD50 values in the range of 45-55 µM for baicalin and 25-40 µM for baicalein. On the other hand, both compounds had fewer inhibitory effects on normal ovarian cells viability with LD50 values of 177 µM for baicalin and 68 µM for baicalein. Baicalin decreased expression of VEGF (20 µM), cMyc (80 µM), and NFkB (20 µM); baicalein decreased expression of VEGF (10 µM), HIF-1? (20 µM), cMyc (20 µM), and NFkB (40 µM). Therefore baicalein is more effective in inhibiting cancer cell viability and expression of VEGF, HIF-1?, cMyc, and NF?B in both ovarian cancer cell lines. It seems that baicalein inhibited cancer cell viability through the inhibition of cancer promoting genes expression including VEGF, HIF-1?, cMyc, and NF?B. Overall, this study showed that baicalein and baicalin significantly inhibited the viability of ovarian cancer cells, while generally exerting less of an effect on normal cells. They have potential for chemoprevention and treatment of ovarian cancers.
Eukaryotic mRNAs consist of two forms of transcripts: poly(A)+ and poly(A)-, based on the presence or absence of poly(A) tails at the 3 end. Poly(A)+ mRNAs are mainly protein coding mRNAs, whereas the functions of poly(A)- mRNA are largely unknown. Previous studies have shown that a significant proportion of gene transcripts are poly(A)- or bimorphic (containing both poly(A)+ and poly(A)- transcripts). We compared the expression levels of poly(A)- and poly(A)+ RNA mRNAs in normal and cancer cell lines. We also investigated the potential functions of these RNA transcripts using an integrative workflow to explore poly(A)+ and poly(A)- transcriptome sequences between a normal human mammary gland cell line (HMEC) and a breast cancer cell line (MCF-7), as well as between a normal human lung cell line (NHLF) and a lung cancer cell line (A549). The data showed that normal and cancer cell lines differentially express these two forms of mRNA. Gene ontology (GO) annotation analyses hinted at the functions of these two groups of transcripts and grouped the differentially expressed genes according to the form of their transcript. The data showed that cell cycle-, apoptosis-, and cell death-related functions corresponded to most of the differentially expressed genes in these two forms of transcripts, which were also associated with the cancers. Furthermore, translational elongation and translation functions were also found for the poly(A)- protein-coding genes in cancer cell lines. We demonstrate that poly(A)- transcripts play an important role in cancer development.
Large intergenic noncoding RNAs (lincRNAs) have been recognized in recent years to constitute a significant portion of the mammalian transcriptome, yet their biological functions remain largely elusive. This is partly due to an incomplete annotation of tissue-specific lincRNAs in essential model organisms, particularly in mice, which has hindered the genetic annotation and functional characterization of these novel transcripts. In this report, we performed ab initio assembly of 1.9 billion tissue-specific RNA-sequencing reads across six tissue types, and identified 3,965 novel expressed lincRNAs in mice. Combining these with 6,606 documented lincRNAs, we established a comprehensive catalog of 10,571 transcribed lincRNAs. We then systemically analyzed all mouse lincRNAs to reveal that some of them are evolutionally conserved and that they exhibit striking tissue-specific expression patterns. We also discovered that mouse lincRNAs carry unique genomic signatures, and that their expression level is correlated with that of neighboring protein-coding transcripts. Finally, we predicted that a large portion of tissue-specific lincRNAs are functionally associated with essential biological processes including the cell cycle and cell development, and that they could play a key role in regulating tissue development and functionality. Our analyses provide a framework for continued discovery and annotation of tissue-specific lincRNAs in model organisms, and our transcribed mouse lincRNA catalog will serve as a roadmap for functional analyses of lincRNAs in genetic mouse models.
Facilitated by the rapid progress of high-throughput sequencing technology, a large number of long noncoding RNAs (lncRNAs) have been identified in mammalian transcriptomes over the past few years. LncRNAs have been shown to play key roles in various biological processes such as imprinting control, circuitry controlling pluripotency and differentiation, immune responses and chromosome dynamics. Notably, a growing number of lncRNAs have been implicated in disease etiology. With the increasing number of published lncRNA studies, the experimental data on lncRNAs (e.g. expression profiles, molecular features and biological functions) have accumulated rapidly. In order to enable a systematic compilation and integration of this information, we have updated the NONCODE database (http://www.noncode.org) to version 3.0 to include the first integrated collection of expression and functional lncRNA data obtained from re-annotated microarray studies in a single database. NONCODE has a user-friendly interface with a variety of search or browse options, a local Genome Browser for visualization and a BLAST server for sequence-alignment search. In addition, NONCODE provides a platform for the ongoing collation of ncRNAs reported in the literature. All data in NONCODE are open to users, and can be downloaded through the website or obtained through the SOAP API and DAS services.
Small cell lung cancer (SCLC) demonstrates a strong etiological association with smoking. Although cigarette smoke is a mixture of about 4,000 compounds, nicotine is the addictive component of cigarette smoke. Several convergent studies have shown that nicotine promotes angiogenesis in lung cancers via the ?7-nicotinic acetylcholine receptor (?7-nAChR) on endothelial cells. Therefore, we conjectured that ?7-nAChR antagonists may attenuate nicotine-induced angiogenesis and be useful for the treatment of human SCLC. For the first time, our study explores the anti-angiogenic activity of MG624, a small-molecule ?7-nAChR antagonist, in several experimental models of angiogenesis. We observed that MG624 potently suppressed the proliferation of primary human microvascular endothelial cells of the lung (HMEC-Ls). Furthermore, MG624 displayed robust anti-angiogenic activity in the Matrigel, rat aortic ring and rat retinal explant assays. The anti-angiogenic activity of MG624 was assessed by two in vivo models, namely the chicken chorioallantoic membrane model and the nude mice model. In both of these experimental models, MG624 inhibited angiogenesis of human SCLC tumors. Most importantly, the administration of MG624 was not associated with any toxic side effects, lethargy or discomfort in the mice. The anti-angiogenic activity of MG624 was mediated via the suppression of nicotine-induced FGF2 levels in HMEC-Ls. MG624 decreased nicotine-induced early growth response gene 1 (Egr-1) levels in HMEC-Ls, and reduced the levels of Egr-1 on the FGF2 promoter. Consequently, this process decreased FGF2 levels and angiogenesis. Our findings suggest that the anti-angiogenic effects of MG624 could be useful in anti-angiogenic therapy of human SCLCs.
Recent interest in the non-coding transcriptome has resulted in the identification of large numbers of long non-coding RNAs (lncRNAs) in mammalian genomes, most of which have not been functionally characterized. Computational exploration of the potential functions of these lncRNAs will therefore facilitate further work in this field of research. We have developed a practical and user-friendly web interface called ncFANs (non-coding RNA Function ANnotation server), which is the first web service for functional annotation of human and mouse lncRNAs. On the basis of the re-annotated Affymetrix microarray data, ncFANs provides two alternative strategies for lncRNA functional annotation: one utilizing three aspects of a coding-non-coding gene co-expression (CNC) network, the other identifying condition-related differentially expressed lncRNAs. ncFANs introduces a highly efficient way of re-using the abundant pre-existing microarray data. The present version of ncFANs includes re-annotated CDF files for 10 human and mouse Affymetrix microarrays, and the server will be continuously updated with more re-annotated microarray platforms and lncRNA data. ncFANs is freely accessible at http://www.ebiomed.org/ncFANs/ or http://www.noncode.org/ncFANs/.
Ovarian cancer is a significant malignancy for women in the western world, and its death rate has remained unchanged over the past 50 years, leaving room for proper chemoprevention. Kaempferol is a natural flavonoid widely distributed in fruits and vegetables, and epidemiological studies have found a negative correlation between kaempferol consumption and ovarian cancer risk. To understand the mechanism behind this negative correlation, we investigated kaempferols ability to induce apoptosis in A2780/CP70, A2780/wt, and OVCAR-3 ovarian cancer cell lines. Kaempferol inhibited cell proliferation but did not cause necrosis in all 3 cell lines. For the apoptosis, caspase 3/7 levels were induced in a concentration-dependent manner by kaempferol treatment, with A2780/wt cells being the most responsive. This induction can be diminished by pre-treatment with a caspase-9 inhibitor, indicating an intrinsic apoptosis pathway. Western blot analysis revealed that protein levels of Bcl-x(L) were decreased in ovarian cancer cells, while p53, Bad, and Bax proteins were up-regulated by kaempferol treatment. Our data indicate that kaempferol induces apoptosis in ovarian cancer cells through regulating pro-apoptotic and anti-apoptotic protein expressions in the intrinsic apoptosis pathways, and is a good candidate for the chemoprevention of ovarian cancers in humans. Further studies in animal models and clinical trials are therefore warranted.
Western blotting is a basic technique for protein detection. For proteins of less abundance or antibodies of poorer quality, an increased sensitivity is often desired. Although it is commonly known that higher concentrations of antibodies and prolonged film exposure times will help improve sensitivity in western blots, both measures come with their own risks, and it is often unclear to which extent these measures should be applied.
Although accumulating evidence has provided insight into the various functions of long-non-coding RNAs (lncRNAs), the exact functions of the majority of such transcripts are still unknown. Here, we report the first computational annotation of lncRNA functions based on public microarray expression profiles. A coding-non-coding gene co-expression (CNC) network was constructed from re-annotated Affymetrix Mouse Genome Array data. Probable functions for altogether 340 lncRNAs were predicted based on topological or other network characteristics, such as module sharing, association with network hubs and combinations of co-expression and genomic adjacency. The functions annotated to the lncRNAs mainly involve organ or tissue development (e.g. neuron, eye and muscle development), cellular transport (e.g. neuronal transport and sodium ion, acid or lipid transport) or metabolic processes (e.g. involving macromolecules, phosphocreatine and tyrosine).
Ovarian cancer is one of the most significant malignancies in the western world. Studies showed that Ovarian cancers tend to grow resistance to cisplatin treatment. Therefore, new approaches are needed in ovarian cancer treatment. Kaempferol is a dietary flavonoid that is widely distributed in fruits and vegetables, and epidemiology studies have revealed a protective effect of kaempferol against ovarian cancer risk. Our early studies also found that kaempferol is effective in reducing vascular endothelial growth factor (VEGF) expression in ovarian cancer cells. In this study, we investigated kaempferols effects on sensitizing ovarian cancer cell growth in response to cisplatin treatment.
Small cell lung cancer (SCLC) is characterized by rapid progression and low survival rates. Therefore, novel therapeutic agents are urgently needed for this disease. Capsaicin, the active ingredient of chilli peppers, displays anti-proliferative activity in prostate and epidermoid cancer in vitro. However, the anti-proliferative activity of capsaicin has not been studied in human SCLCs. The present manuscript fills this void of knowledge and explores the anti-proliferative effect of capsaicin in SCLC in vitro and in vivo.
Ovarian cancer is 1 of the most significant malignancies in the Western world, and the antiangiogenesis strategy has been postulated for prevention and treatment of ovarian cancers. Kaempferol is a natural flavonoid present in many fruits and vegetables. The antiangiogenesis potential of kaempferol and its underlying mechanisms were investigated in two ovarian cancer cell lines, OVCAR-3 and A2780/CP70. Kaempferol mildly inhibits cell viability but significantly reduces VEGF gene expression at mRNA and protein levels in both ovarian cancer cell lines. In chorioallantoic membranes of chicken embryos, kaempferol significantly inhibits OVCAR-3-induced angiogenesis and tumor growth. HIF-1alpha, a regulator of VEGF, is downregulated by kaempferol treatment in both ovarian cancer cell lines. Kaempferol also represses AKT phosphorylation dose dependently at 5 to 20 muM concentrations. ESRRA is a HIF-independent VEGF regulator, and it is also downregulated by kaempferol in a dose-dependent manner. Overall, this study demonstrated that kaempferol is low in cytotoxicity but inhibits angiogenesis and VEGF expression in human ovarian cancer cells through both HIF-dependent (Akt/HIF) and HIF-independent (ESRRA) pathways and deserves further studies for possible application in angio prevention and treatment of ovarian cancers.
A snapshot imaging polarimeter utilizing Savart plates is integrated into a fundus camera for retinal imaging. Acquired retinal images can be processed to reconstruct Stokes vector images, giving insight into the polarization properties of the retina. Results for images from a normal healthy retina and retinas with pathology are examined and compared.
Recent case-controlled clinical studies show that bronchioalveolar carcinomas (BAC) are correlated with smoking. Nicotine, the addictive component of cigarettes, accelerates cell proliferation through nicotinic acetylcholine receptors (nAChR). In this study, we show that human BACs produce acetylcholine (ACh) and contain several cholinergic factors including acetylcholinesterase (AChE), choline acetyltransferase (ChAT), choline transporter 1 (CHT1, SLC5A7), vesicular acetylcholine transporter (VAChT, SLC18A3), and nACh receptors (AChRs, CHRNAs). Nicotine increased the production of ACh in human BACs, and ACh acts as a growth factor for these cells. Nicotine-induced ACh production was mediated by ?7-, ?3?2-, and ?3-nAChRs, ChAT and VAChT pathways. We observed that nicotine upregulated ChAT and VAChT. Therefore, we conjectured that VAChT antagonists, such as vesamicol, may suppress the growth of human BACs. Vesamicol induced potent apoptosis of human BACs in cell culture and nude mice models. Vesamicol did not have any effect on EGF or insulin-like growth factor-II-induced growth of human BACs. siRNA-mediated attenuation of VAChT reversed the apoptotic activity of vesamicol. We also observed that vesamicol inhibited Akt phosphorylation during cell death and that overexpression of constitutively active Akt reversed the apoptotic activity of vesamicol. Taken together, our results suggested that disruption of nicotine-induced cholinergic signaling by agents such as vesamicol may have applications in BAC therapy.
More and more evidences demonstrate that the long non-coding RNAs (lncRNAs) play many key roles in diverse biological processes. There is a critical need to annotate the functions of increasing available lncRNAs. In this article, we try to apply a global network-based strategy to tackle this issue for the first time. We develop a bi-colored network based global function predictor, long non-coding RNA global function predictor (lnc-GFP), to predict probable functions for lncRNAs at large scale by integrating gene expression data and protein interaction data. The performance of lnc-GFP is evaluated on protein-coding and lncRNA genes. Cross-validation tests on protein-coding genes with known function annotations indicate that our method can achieve a precision up to 95%, with a suitable parameter setting. Among the 1713 lncRNAs in the bi-colored network, the 1625 (94.9%) lncRNAs in the maximum connected component are all functionally characterized. For the lncRNAs expressed in mouse embryo stem cells and neuronal cells, the inferred putative functions by our method highly match those in the known literature.
Ovarian cancer is one of the leading causes of cancer death for women throughout the Western world. Kaempferol, a natural flavonoid, has shown promise in the chemoprevention of ovarian cancer. A common concern about using dietary supplements for chemoprevention is their bioavailability. Nanoparticles have shown promise in increasing the bioavailability of some chemicals. Here we developed five different types of nanoparticles incorporating kaempferol and tested their efficacy in the inhibition of viability of cancerous and normal ovarian cells. We found that positively charged nanoparticle formulations did not lead to a significant reduction in cancer cell viability, whereas nonionic polymeric nanoparticles resulted in enhanced reduction of cancer cell viability. Among the nonionic polymeric nanoparticles, poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) (PEO-PPO-PEO) nanoparticles incorporating kaempferol led to significant reduction in cell viability of both cancerous and normal cells. Poly(DL-lactic acid-co-glycolic acid) (PLGA) nanoparticles incorporating kaempferol resulted in enhanced reduction of cancer cell viability together with no significant reduction in cell viability of normal cells compared with kaempferol alone. Therefore, both PEO-PPO-PEO and PLGA nanoparticle formulations were effective in reducing cancer cell viability, while PLGA nanoparticles incorporating kaempferol had selective toxicity against cancer cells and normal cells. A PLGA nanoparticle formulation could be advantageous in the prevention and treatment of ovarian cancers. On the other hand, PEO-PPO-PEO nanoparticles incorporating kaempferol were more effective inhibitors of cancer cells, but they also significantly reduced the viability of normal cells. PEO-PPO-PEO nanoparticles incorporating kaempferol may be suitable as a cancer-targeting strategy, which could limit the effects of the nanoparticles on normal cells while retaining their potency against cancer cells. We have identified two nanoparticle formulations incorporating kaempferol that may lead to breakthroughs in cancer treatment. Both PEO-PPO-PEO and PLGA nanoparticle formulations had superior effects compared with kaempferol alone in reducing cancer cell viability.
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