Bioinformatic Tools for the Analysis and Prediction of ncRNA Interactions.

Noncoding RNAs (ncRNAs) play prominent roles in the regulation of gene expression via their interactions with other biological molecules such as proteins and nucleic acids. Although much of our knowledge about how these ncRNAs operate in different biological processes has been obtained from experimental findings, computational biology can also clearly substantially boost this knowledge by suggesting possible novel interactions of these ncRNAs with other molecules. Computational predictions are thus used as an alternative source of new insights through a process of mutual enrichment because the information obtained through experiments continuously feeds through into computational methods. The results of these predictions in turn shed light on possible interactions that are subsequently validated experimentally. This review describes the latest advances in databases, bioinformatic tools, and new in silico strategies that allow the establishment or prediction of biological interactions of ncRNAs, particularly miRNAs and lncRNAs. The ncRNA species described in this work have a special emphasis on those found in humans, but information on ncRNA of other species is also included.

NEAT1 and MALAT1 are highly expressed in saliva and nasopharyngeal swab samples of COVID-19 patients.

COVID-19, caused by the SARS-CoV-2 virus, has become a significant global public health problem, with a wide variety of clinical manifestations and disease progression outcomes. LncRNAs are key regulators of the immune response and have been associated with COVID-19 risk infection. Previous studies focused mainly on in-silico analysis of lncRNA expression in the lungs or peripheral blood cells. We evaluated the expression of lncRNAs NEAT1, MALAT1, and MIR3142 in saliva and nasopharyngeal swab from SARS-CoV-2 positive (n = 34) and negative patients (n = 46). A higher expression of the lncRNAs NEAT1 and MALAT1 (p < 0.05) were found in positive samples. NEAT1 had a higher expression mainly in saliva samples (p < 0.001), and MALAT1 was upregulated in nasopharyngeal samples (p < 0.05). Area under the ROC curve for NEAT1 in saliva was 0.8067. This study was the first to investigate the expression of lncRNAs in saliva and nasopharyngeal samples of COVID-19 patients, which gives new insights into the initial response to infection and infectivity and may provide new biomarkers for severity and targets for therapy.

Machine Learning Supports Long Noncoding RNAs as Expression Markers for Endometrial Carcinoma.

Uterine corpus endometrial carcinoma (UCEC) is the second most common type of gynecological tumor. Several research studies have recently shown the potential of different ncRNAs as biomarkers for prognostics and diagnosis in different types of cancers, including UCEC. Thus, we hypothesized that long noncoding RNAs (lncRNAs) could serve as efficient factors to discriminate solid primary (TP) and normal adjacent (NT) tissues in UCEC with high accuracy. We performed an in silico differential expression analysis comparing TP and NT from a set of samples downloaded from the Cancer Genome Atlas (TCGA) database, targeting highly differentially expressed lncRNAs that could potentially serve as gene expression markers. All analyses were performed in R software. The receiver operator characteristics (ROC) analyses and both supervised and unsupervised machine learning indicated a set of 14 lncRNAs that may serve as biomarkers for UCEC. Functions and putative pathways were assessed through a coexpression network and target enrichment analysis.

lncRNA CCAT1 promotes bladder cancer cell proliferation, migration and invasion

ABSTRACT Objective: To study the expression patterns of long noncoding RNA (lncRNA) colon cancer-associated transcript 1 (CCAT1) and the changes in cell proliferation, apoptosis, migration and invasion induced by silencing CCAT1 in bladder cancer cells. Materials and Methods: The expression levels of CCAT1 were determined using realtime quantitative polymerase chain reaction in cancerous tissues and paired normal tissues from 34 patients with bladder cancer. The relationship between clinical characteristics and CCAT1 expression was analyzed. And then we conducted cell experiments. Bladder urothelial carcinoma cell lines T24 and 5637 cells were transfected with CCAT1 small interfering RNA (siRNA) or scramble siRNA. Cell proliferation and apoptosis changes were determined using a Cell Counting Kit-8 (CCK-8) assay and a flow cytometry assay. Migration and invasion changes were measured using a wound healing assay and a trans-well assay. microRNAs (miRNAs) were predicted by Starbase 2.0, and their differential expression levels were studied. Results: CCAT1 was significantly upregulated in bladder cancer (P < 0.05). CCAT1 upregulation was positively related to tumor stage (P = 0.004), tumor grade (P = 0.001) and tumor size (P = 0.042). Cell proliferation, migration and invasion were promoted by abnormally expressed CCAT1. miRNAs miR-181b-5p, miR-152-3p, miR-24-3p, miR-148a-3p and miR-490-3p were potentially related to the aforementioned functions of CCAT1. Conclusion: CCAT1 plays an oncogenic role in urothelial carcinoma of the bladder. In addition, CCAT1 may be a potential therapeutic target in this cancer.

Expression analysis of long non-coding RNAs in a renal ischemia-reperfusion injury model.

PURPOSE: To investigate the long non-coding RNAs (lncRNAs) profile on renal ischemia reperfusion in a mouse model. METHODS: Microarray analysis was used to study the expression of misregulated lncRNA in a mouse model of renal ischemia reperfusion(I/R) with long ischemia time. Quantitative real-time PCR (qPCR) was used to verify the expression of selected lncRNAs and mRNAs.The potential functions of the lncRNA was analyzed by bioinformatics tools and databases. RESULTS: Kidney function was impaired in I/R group compared to the normal group. Analysis showed that a total of 2267 lncRNAs and 2341 messenger RNAs (mRNAs) were significantly expressed in I/R group (≥2.0-fold, p < 0.05).The qPCR result showed that lncRNAs and mRNAs expression were consistent with the microarray analysis. The co-expression network profile analysis based on five validated lncRNAs and 203 interacted mRNAs showed it existed a total of 208 nodes and 333 connections. The GO and KEEG pathway analysis results showed that multiple lncRNAs are involved the mechanism of I/R. CONCLUSION: Multiple lncRNAs are involved in the mechanism of I/R.These analysis results will help us to further understand the mechanism of I/R and promote the new methods targeted at lncRNA to improve I/R injury.

lncRNA CCAT1 promotes bladder cancer cell proliferation, migration and invasion.

OBJECTIVE: To study the expression patterns of long noncoding RNA (lncRNA) colon cancer-associated transcript 1 (CCAT1) and the changes in cell proliferation, apoptosis, migration and invasion induced by silencing CCAT1 in bladder cancer cells. MATERIALS AND METHODS: The expression levels of CCAT1 were determined using realtime quantitative polymerase chain reaction in cancerous tissues and paired normal tissues from 34 patients with bladder cancer. The relationship between clinical characteristics and CCAT1 expression was analyzed. And then we conducted cell experiments. Bladder urothelial carcinoma cell lines T24 and 5637 cells were transfected with CCAT1 small interfering RNA (siRNA) or scramble siRNA. Cell proliferation and apoptosis changes were determined using a Cell Counting Kit-8 (CCK-8) assay and a fl ow cytometry assay. Migration and invasion changes were measured using a wound healing assay and a trans-well assay. microRNAs (miRNAs) were predicted by Starbase 2.0, and their differential expression levels were studied. RESULTS: CCAT1 was signifi cantly upregulated in bladder cancer (P < 0.05). CCAT1 upregulation was positively related to tumor stage (P = 0.004), tumor grade (P = 0.001) and tumor size (P = 0.042). Cell proliferation, migration and invasion were promoted by abnormally expressed CCAT1. miRNAs miR-181b-5p, miR-152-3p, miR-24-3p, miR-148a-3p and miR-490-3p were potentially related to the aforementioned functions of CCAT1. CONCLUSION: CCAT1 plays an oncogenic role in urothelial carcinoma of the bladder. In addition, CCAT1 may be a potential therapeutic target in this cancer.

Expression analysis of long non-coding RNAs in a renal ischemia-reperfusion injury model

Purpose: To investigate the long non-coding RNAs (lncRNAs) profile on renal ischemia reperfusion in a mouse model. Methods: Microarray analysis was used to study the expression of misregulated lncRNA in a mouse model of renal ischemia reperfusion (I/R) with long ischemia time. Quantitative real-time PCR (qPCR) was used to verify the expression of selected lncRNAs and mRNAs. The potential functions of the lncRNA was analyzed by bioinformatics tools and databases. Results: Kidney function was impaired in I/R group compared to the normal group. Analysis showed that a total of 2267 lncRNAs and 2341 messenger RNAs (mRNAs) were significantly expressed in I/R group (≥2.0-fold, p < 0.05). The qPCR result showed that lncRNAs and mRNAs expression were consistent with the microarray analysis. The co-expression network profile analysis based on five validated lncRNAs and 203 interacted mRNAs showed it existed a total of 208 nodes and 333 connections. The GO and KEEG pathway analysis results showed that multiple lncRNAs are involved the mechanism of I/R. Conclusion: Multiple lncRNAs are involved in the mechanism of I/R. These analysis results will help us to further understand the mechanism of I/R and promote the new methods targeted at lncRNA to improve I/R injury.(AU)

Effect of lncRNA HULC knockdown on rat secreting pituitary adenoma GH3 cells.

Pituitary adenoma is one of the most common tumors in the neuroendocrine system. This study investigated the effects of long non-coding RNAs (lncRNAs) highly up-regulated in liver cancer (HULC) on rat secreting pituitary adenoma GH3 cell viability, migration, invasion, apoptosis, and hormone secretion, as well as the underlying potential mechanisms. Cell transfection and qRT-PCR were used to change and measure the expression levels of HULC, miR-130b, and FOXM1. Cell viability, migration, invasion, and apoptosis were assessed using trypan blue staining assay, MTT assay, two-chamber transwell assay, Guava Nexin assay, and western blotting. The concentrations of prolactin (PRL) and growth hormone (GH) in culture supernatant of GH3 cells were assessed using ELISA. The targeting relationship between miR-130b and FOXM1 was verified using dual luciferase activity. Finally, the expression levels of key factors involved in PI3K/AKT/mTOR and JAK1/STAT3 pathways were evaluated using western blotting. We found that HULC was highly expressed in GH3 cells. Overexpression of HULC promoted GH3 cell viability, migration, invasion, PRL and GH secretion, as well as activated PI3K/AKT/mTOR and JAK1/STAT3 pathways. Knockdown of HULC had opposite effects and induced cell apoptosis. HULC negatively regulated the expression of miR-130b, and miR-130b participated in the effects of HULC on GH3 cells. FOXM1 was a target gene of miR-130b, which was involved in the regulation of GH3 cell viability, migration, invasion, and apoptosis, as well as PI3K/AKT/mTOR and JAK1/STAT3 pathways. In conclusion, HULC tumor-promoting roles in secreting pituitary adenoma might be via down-regulating miR-130b, up-regulating FOXM1, and activating PI3K/AKT/mTOR and JAK1/STAT3 pathways.

Expression analysis of long non-coding RNAs in a renal ischemia-reperfusion injury model

Abstract Purpose: To investigate the long non-coding RNAs (lncRNAs) profile on renal ischemia reperfusion in a mouse model. Methods: Microarray analysis was used to study the expression of misregulated lncRNA in a mouse model of renal ischemia reperfusion(I/R) with long ischemia time. Quantitative real-time PCR (qPCR) was used to verify the expression of selected lncRNAs and mRNAs.The potential functions of the lncRNA was analyzed by bioinformatics tools and databases. Results: Kidney function was impaired in I/R group compared to the normal group. Analysis showed that a total of 2267 lncRNAs and 2341 messenger RNAs (mRNAs) were significantly expressed in I/R group (≥2.0-fold, p < 0.05).The qPCR result showed that lncRNAs and mRNAs expression were consistent with the microarray analysis. The co-expression network profile analysis based on five validated lncRNAs and 203 interacted mRNAs showed it existed a total of 208 nodes and 333 connections. The GO and KEEG pathway analysis results showed that multiple lncRNAs are involved the mechanism of I/R. Conclusion: Multiple lncRNAs are involved in the mechanism of I/R.These analysis results will help us to further understand the mechanism of I/R and promote the new methods targeted at lncRNA to improve I/R injury.

Identificação e anotação funcional de RNAs longos não codificadores associados à subtipos moleculares em tumores pancreáticos / Identification of long non coding RNAs associated to molecular subtypes in pancreatic tumors

O Adenocarcinoma Pancreático Ductal (Pancreatic Ductal Adenocarcinoma - PDAC) é a sé- tima causa de mortes por câncer no mundo, com uma taxa de sobrevida de apenas 6%. Embora alguns genes estejam recorrentemente mutados em grande parte dos tumores e sejam críticos para a oncogênese, a heterogeneidade das alterações moleculares tanto no tumor quanto em componentes do microambiente tumoral se reflete em diferentes características fenotípicas com comportamentos clínicos distintos e que têm sido associados a diferentes subtipos moleculares através da análise computacional de dados de alterações somáticas e transcricionais no PDAC. RNAs não codificadores longos (lncRNAs) têm sido reconhecidos como importantes reguladores da expressão gênica em doenças proliferativas mas sua associação com subtipos em PDAC e sua contribuição para o estabelecimento de diferentes fenótipos moleculares e clínicos da doença não foi explorada até o momento. Neste trabalho, foi implementada uma abordagem computacional com o objetivo de identificar e anotar funcionalmente lncRNAs associados a subtipos moleculares de PDAC. Inicialmente, a classificação não supervisionada por Fatoração Matricial Não Negativa (Non-Negative Matrix Factorization - NMF) de dados de expressão gê- nica global de amostras clínicas disponíveis publicamente (The Cancer Genome Atlas - TCGA) resultou na identificação de quatro subgrupos distintos de PDAC, que recapitulam os fenótipos Exócrino/Endócrino, Imunogênico, Escamoso e Progenitor descritos na literatura. Uma análise de expressão diferencial permitiu a identificação de assinaturas de expressão gênica características que incluem lncRNAs associados a cada subgrupo. Através da construção de redes de coexpressão de mRNAs e lncRNAs e a identificação de módulos da rede significativamente enriquecidos em genes que participam em vias moleculares conhecidas foi possível inferir possíveis funções biológicas à lncRNAs associados aos diferentes subtipos moleculares, tais como funções exócrinas/neuroendócrinas, imunogênicas, reparo de DNA/progressão do ciclo celular e progenitoras/morfogênicas. Entre ele, o subgrupo 3, enriquecido para fenótipo Escamoso e associado a hiper-expressão do supressor tumoral TP63, possui dois lncRNAS hiper-expressos neste subgrupo em relação aos outros subgrupos, sendo que o lncRNA antissenso FAM83A-AS1 tem a predição de interagir com as proteínas FGFR2, AXIN1, PTEN, BRAF, SMAD4, TGFBR2, TP53 e CDKN2A, que exercem funções importantes na transdução de sinal e supressão tumoral no câncer incluindo o de pâncreas. Entre os lncRNAs hipo-regulados no subgrupo 3 em relação ao outros subgrupos, alguns, como FLJ42875, LOC338651, C20orf56 e LOC38838 tem predição de interação com alta afinidade à proteína BRCA2, que está envolvida no reparo de DNA e participa de processos de resistência à quimioterápicos. As informações trazidas por este estudo permitem gerar hipóteses sobre a contribuição de lncRNAs para a definição de subtipos moleculares de PDAC e priorizar candidatos e experimentos para estudos funcionais de modo a contribuir para um melhor entendimento sobre os mecanismos de ação de lncRNAs na tumorigênese e agressividade do câncer de pâncreas

Pancreatic Ductal Adenocarcinoma (PDAC) is the seventh cause of worldwide cancer related deaths, with an overall survival rate of only 6%. Some genes might be recurrently mutated in a large number of tumors, and be critical for oncogenesis, molecular alteration heterogeneity both in the tumor as all as in the tumor microenvironment is reflected in diverse phenotypic features with distinct clinical outcomes, and this distinction in multiple molecular subtypes has been drawn through transcriptional and somatic alteration computational analysis within PDAC. Long Non Coding RNAs (lncRNAs) have been recognized as important gene expression regulators in proliferative diseases, but its association to molecular subtypes in PDAC and its contribution in the establishment of diverse molecular and clinical phenotypes hasnt been explored at length until the present. This work focused on the implementation of a computational approach with the objective of lncRNA identification and functional annotation associated to distinct molecular subtypes in PDAC. Initially, Non-negative Matrix Factorization (NMF), an unsupervised classification method, applied to global gene expression data from publicly available clinical samples (The Cancer Genome Atlas - TCGA) resulted in the identification of four distinct PDAC molecular subgroups reminiscent of Exocrine/Endocrine, Immunogenic, Squamous and Progenitor phenotypes. Differential expression analysis allowed a characteristic gene expression signature identification, including distinct molecular subtype associated lncRNAs. mRNA and lncRNA containing gene co-expression modules significantly enriched annotated pathways containing the molecular subtype associated lncRNAs allowed to designate possible molecular functions of the distinct molecular subtype associated lncRNAs, such as exocrine/neuroendocrine, immunogenic, DNA repair/cell cycle progression and progenitor/morphogenic functions. Subgroup 3, enriched with a Squamous phenotype and associated to TP63 over-expression contains two lncRNAs over-expressed compared to other subgroups; furthermore, the antisense lncRNA FAM83A-AS1 yielded a predicted lncRNA-protein interaction to FGFR2, AXIN1, PTEN, BRAF, SMAD4, TGFBR2, TP53 and CDKN2A, proteins that play important signal transduction and tumor suppressor roles in several cancer types, including pancreas. Among under-expressed lncRNAs in subgroup 3 compared to the other subgroups, some, such as FLJ42875, LOC338651, C20orf56 and LOC38838 yilded a high protein interaction prediction score with BRCA2, a protein involved in DNA repair and processes resuling in chemotherapy resistance. The information brought by this study allowed to generate hypothesis on lncRNA contribution to define PDAC molecular subtypes, helping prioritize candidates and experiments for functional studies, thus contributing to a better understanding on lncRNA mechanisms related to tumor progression and aggressiveness in pancreatic cancer

Long non-coding RNA LINC00261 sensitizes human colon cancer cells to cisplatin therapy.

Colon cancer is one of the most common digestive tumors. The present study aimed to explore the functional role, as well as the underlying mechanism of long non-coding RNA LINC00261 in colon cancer. Expression of LINC00261 was analyzed in colon cancer cell lines and human normal cell lines. Acquired resistance cell lines were then built and the acquired resistance efficiency was detected by evaluating cell viability. Thereafter, the effects of LINC00261 overexpression on cisplatin-resistant colon cancer cells were measured, as well as cell apoptosis, viability, migration, and invasion. Subsequently, we investigated the interaction of LINC00261 and ß-catenin. The results showed that the LINC00261 gene was down-regulated in colon cancer cell lines and tissues, and in cisplatin-resistant cells. LINC00261 overexpression might relieve cisplatin resistance of colon cancer cells via promoting cell apoptosis, and inhibiting cell viability, migration, and invasion. Moreover, LINC00261 might down-regulate nuclear ß-catenin through restraining ß-catenin from cytoplasm into nuclei or it could also promote ß-catenin degradation and inhibit activation of Wnt pathway. Finally, LINC00261 reduced cisplatin resistance of colon cancer in vivo and enhanced the anti-colon cancer effect of cisplatin through reducing tumor volume and weight.

Long non-coding RNA CHCHD4P4 promotes epithelial-mesenchymal transition and inhibits cell proliferation in calcium oxalate-induced kidney damage.

Kidney stone disease is a major cause of chronic renal insufficiency. The role of long non-coding RNAs (lncRNAs) in calcium oxalate-induced kidney damage is unclear. Therefore, we aimed to explore the roles of lncRNAs in glyoxylate-exposed and healthy mouse kidneys using microarray technology and bioinformatics analyses. A total 376 mouse lncRNAs were differentially expressed between the two groups. Using BLAST, 15 lncRNA homologs, including AU015836 and CHCHD4P4, were identified in mice and humans. The AU015836 expression in mice exposed to glyoxylate and the CHCHD4P4 expression in human proximal tubular epithelial (HK-2) cells exposed to calcium oxalate monohydrate were analyzed, and both lncRNAs were found to be upregulated in response to calcium oxalate. To further evaluate the effects of CHCHD4P4 on the cell behavior, we constructed stable CHCHD4P4-overexpressing and CHCHD4P4-knockdown HK-2 cells. The results showed that CHCHD4P4 inhibited cell proliferation and promoted the epithelial-mesenchymal transition in kidney damage and fibrosis caused by calcium oxalate crystallization and deposition. The silencing of CHCHD4P4 reduced the kidney damage and fibrosis and may thus be a potential molecular target for the treatment of kidney stones.

Long non-coding RNA AFAP1-AS1 facilitates tumor growth and promotes metastasis in colorectal cancer.

BACKGROUND AND OBJECTIVE: Long non-coding RNAs can regulate tumorigenesis of various cancers. Dys-regulation of lncRNA-AFAP1-AS1 has not been studied in colorectal carcinoma (CRC). This study was to examine the function involvement of AFAP1-AS1 in tumor growth and metastasis of CRC. METHODS: Relative expression of AFAP1-AS1 in CRC tissues and CRC cells lines was determined using quantitative real-time PCR (qRT-PCR). Functional involvement of AFAP1-AS1 in tumor proliferation and metastasis was evaluated in AFAP1-AS1-specific siRNA-treated CRC cells and in CRC cell xenograft. Expression of epithelial-mesenchymal transition (EMT)-related gene expression was determined using western blot. RESULTS: Relative expression of AFAP1-AS1 was significantly elevated in CRC tissues and CRC HCT116 and SW480 cell lines. AFAP1-AS1 knock-down suppressed SW480 cell proliferation, colony formation, migration and invasion. Also AFAP1-AS1 knock-down inhibited tumor metastasis-associated genes expression in terms of EMT. This carcinostatic action by AFAP1-AS1 knock-down was further confirmed by suppression of tumor formation and hepatic metastasis of CRC cells in nude mice. CONCLUSION: lncRNA-AFAP1-AS1 knock-down exhibits antitumor effect on colorectal carcinoma in respects of suppression of cell proliferation and metastasis of cancer cells.

Long noncoding RNAs (lncRNAs) dynamics evidence immunomodulation during ISAV-Infected Atlantic salmon (Salmo salar).

Despite evidence for participation in the host response to infection, the roles of many long non-coding RNAs (lncRNAs) remain unknown. Therefore, the aims of this study were to identify lncRNAs in Atlantic salmon (Salmo salar) and evaluate their transcriptomic regulation during ISA virus (ISAV) infection, an Orthomyxoviridae virus associated with high mortalities in salmonid aquaculture. Using next-generation sequencing, whole-transcriptome analysis of the Salmo salar response to ISAV infection was performed, identifying 5,636 putative lncRNAs with a mean length of 695 base pairs. The transcriptional modulation evidenced a similar number of differentially expressed lncRNAs in the gills (3,294), head-kidney (3,275), and liver (3,325) over the course of the infection. Moreover, analysis of a subset of these lncRNAs showed the following: (i) Most were similarly regulated in response to ISA virus infection; (ii) The transcript subsets were uniquely modulated in each tissue (gills, liver, and head-kidney); and (iii) A subset of lncRNAs were upregulated for each tissue and time analysed, indicating potential markers for ISAV infection. These findings represent the first discovery of widespread differential expression of lncRNAs in response to virus infection in non-model species, suggesting that lncRNAs could be involved in regulating the host response during ISAV infection.

Characterization of HIPSTR highlights the heterogeneous expression pattern of lncRNAs in human embryos and stable cell lines / Caracterização do HIPSTR destaca o padrão de expressão heterogênea de IncRNAs em embriões humanos e linhagens estáveis de células

There is a growing appreciation that eukaryotic genomes are transcribed into numerous, previously undetected - and thus uncharacterized regulatory long non-coding RNAs (lncRNAs). Recent studies are primarily focused on lncRNAs transcribed from intergenic regions and enhancers, leaving antisense lncRNAs the least studied group of lncRNAs. At the same time, antisense transcription occurs in up to 74 % of human gene loci, frequently - from the opposite strand of genes encoding proteins involved in regulation of transcription. Here, we identified HIPSTAR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), a novel conserved lncRNA that is transcribed antisense to the TFAP2A gene. Unlike previously reported antisense lncRNAs, HIPSTR expression does not correlate with the expression of its antisense counterpart. Although HIPSTAR and TFAP2A are co-expressed in in vitro derived neural crest and trophoblast cells, only HIPSTAR and not TFAP2A is specifically expressed in a subset of cells within 8-cell- and morula-stage human embryos. We show that, similar to HIPSTAR, in the individual cells of developing human embryos or of stable cell lines the expression of lncRNAs is more highly heterogeneous than the expression of mRNAs. Finally, we demonstrate that HIPSTAR depletion in HEK293 and H1BP, a human embryonic stem cell line, predominantly affects the expression levels of genes involved in early organismal development and cell differentiation. Together, we show that expression of HIPSTAR and hundreds other lncRNAs is highly heterogeneous in human embryos and cell lines. We use HIPSTAR to exemplify the functional relevance of lncRNAs with heterogeneous and developmental stage-specific expression patterns

Tem sido cada vez mais reconhecido que a transcrição dos genomas eucarióticos produz múltiplos transcritos novos, anteriormente não detectados e ainda não caracterizados, sendo que a maioria é constituida de RNAs não-codificantes longos (lncRNAs) regulatórios. Estudos recentes estão focados principalmente nos lncRNAs transcritos de regiões intergênicas e enhancers; assim, o grupo dos lncRNAs antisenso permanece o menos estudado de todos. Ao mesmo tempo, a transcrição antisenso ocorre em até 74% dos loci de genes humanos, frequentemente - a partir da fita oposta de genes que codificam proteínas envolvidas na regulação da transcrição. No presente trabalho, nós identificamos HIPSTR (Heterogeneously expressed from the Intronic Plus Strand of the TFAP2A-locus RNA), um lncRNA novo conservado que é transcrito a partir da fita antisenso do gene TFAP2A. Ao contrário do anteriormente relatado para os lncRNAs antisenso, a expressão de HIPSTR não está correlacionada com a expressão do gene da fita oposta. HIPSTR e TFAP2A são co-expressos em células da crista neural e em trofoblastos derivadas in vitro, mas somente HIPSTR e não TFAP2A está especificamente expresso num subconjunto de células de embriões humanos nos estágios de 8-células e mórula. Mostramos que, semelhante a HIPSTR, a expressão de lncRNAs é mais altamente heterogênea que a expressão de mRNAs em células individuais de embriões humanos em desenvolvimento ou em linhagens estáveis de células. Finalmente, nós demonstramos que a depleção de HIPSTAR em células HEK293 e H1BP, uma linhagem de células tronco embrionárias humanas, afeta predominantemente os níveis de genes envolvidos no início do desenvolvimento do organismo e na diferenciação de células. No conjunto, nós mostramos que a expressão de HIPSTR e de centenas de outros lncRNAs é altamente heterogênea em embriões humanos e linhagens celulares. Usamos HIPSTR para exemplificar a relevância funcional de lncRNAs com padrões de expressão heterogêneos e estágio-de-desenvolvimento específicos

Long non-coding RNA AFAP1-AS1 facilitates tumor growth and promotes metastasis in colorectal cancer

BACKGROUND AND OBJECTIVE: Long non-coding RNAs can regulate tumorigenesis of various cancers. Dys-regulation of lncRNA-AFAP1-AS1 has not been studied in colorectal carcinoma (CRC). This study was to examine the function involvement of AFAP1-AS1 in tumor growth and metastasis of CRC. METHODS: Relative expression of AFAP1-AS1 in CRC tissues and CRC cells lines was determined using quantitative real-time PCR (qRT-PCR). Functional involvement of AFAP1-AS1 in tumor proliferation and metastasis was evaluated in AFAP1-AS1-specific siRNA-treated CRC cells and in CRC cell xenograft. Expression of epithelial-mesenchymal transition (EMT)-related gene expression was determined using western blot. RESULTS: Relative expression of AFAP1-AS1 was significantly elevated in CRC tissues and CRC HCT116 and SW480 cell lines. AFAP1-AS1 knock-down suppressed SW480 cell proliferation, colony formation, migration and invasion. Also AFAP1-AS1 knock-down inhibited tumor metastasis-associated genes expression in terms of EMT. This carcinostatic action by AFAP1-AS1 knock-down was further confirmed by suppression of tumor formation and hepatic metastasis of CRC cells in nude mice. CONCLUSION: lncRNA-AFAP1-AS1 knock-down exhibits antitumor effect on colorectal carcinoma in respects of suppression of cell proliferation and metastasis of cancer cells.

INXS, um longo RNA não codificador de proteínas mediador da apoptose / INXS, a long noncoding RNA that mediates apoptosis

O splicing alternativo do pré-mRNA de BCL-X produz duas isoformas de mRNAs com funções antagônicas, a pró-apoptótica BCL-XS e a anti-apoptótica BCL-XL, cujo balanço regula a homeostasia celular. Entretanto, o mecanismo que regula esse processamento ainda é desconhecido. Nesse trabalho, nós identificamos e caracterizamos um longo RNA não codificador de proteínas (lncRNA) nomeado INXS, que é transcrito a partir da fita oposta do locus genômico de BCL-X, sendo menos abundante em linhagens celulares tumorais e tecidos tumorais de pacientes quando comparados com os respectivos pares não tumorais. INXS é um RNA unspliced de 1903 nts, é transcrito pela RNA Polimerase II, possui cap 5', está enriquecido na fração nuclear das células e se liga à proteína Sam68 do complexo modulador de splicing. O tratamento de células tumorais 786-O com cada um de três agentes indutores de apoptose aumentou a expressão endógena do INXS, levando ao aumento expressivo da proporção entre os mRNAs de BCL-XS / BCL-XL, e ativação das caspases 3, 7 e 9. Estes efeitos foram anulados na presença do knockdown do INXS. Da mesma forma, a superexpressão ectópica do INXS causou uma mudança no splicing favorecendo a isoforma BCL-XS e ativação das caspases, aumentando os níveis da proteína BCL-XS e conduzindo as células à apoptose. Utilizando um modelo in vivo, cinco injeções intra-tumorais do INXS durante 15 dias causaram uma regressão acentuada no volume dos xenotumores. Portanto, INXS é um lncRNA que induz a apoptose, sugerindo que essa molécula seja um possível alvo a ser explorado na terapia contra o câncer

BCL-X mRNA alternative splicing generates pro-apoptotic BCL-XS or anti-apoptotic BCL-XL, whose balance regulates cell homeostasis. However, the mechanism that regulates the splice shifting is incompletely understood. Here, we identified and characterized a long noncoding RNA (lncRNA) named INXS, transcribed from the opposite genomic strand of BCL-X, that was less abundant in tumor cell lines and patient tumor tissues compared with non-tumors. INXS is an unspliced 1903 nt-long RNA, is transcribed by RNA Polymerase II, 5'-capped, nuclear enriched and binds Sam68 splicing-modulator. The treatment of tumor cell line 786-O with each of three apoptosis-inducing agents increased endogenous INXS lncRNA, increased BCL-XS / BCL-XL mRNA ratio, and activated caspases 3, 7 and 9. These effects were abrogated in the presence of INXS knockdown. Similarly, ectopic INXS overexpression caused a shift in splicing towards BCL-XS and activation of caspases, increasing the levels of BCL-XS protein and then leading the cells to apoptosis. In a mouse xenograft model, five intra-tumor injections of INXS along 15 days caused a marked regression in tumor volume. INXS is an lncRNA that induces apoptosis, suggesting that INXS is a possible target to be explored in cancer therapies

Long non-coding RNA INXS is a critical mediator of BCL-XS induced apoptosis.

BCL-X mRNA alternative splicing generates pro-apoptotic BCL-XS or anti-apoptotic BCL-XL gene products and the mechanism that regulates splice shifting is incompletely understood. We identified and characterized a long non-coding RNA (lncRNA) named INXS, transcribed from the opposite genomic strand of BCL-X, that was 5- to 9-fold less abundant in tumor cell lines from kidney, liver, breast and prostate and in kidney tumor tissues compared with non-tumors. INXS is an unspliced 1903 nt-long RNA, is transcribed by RNA polymerase II, 5'-capped, nuclear enriched and binds Sam68 splicing-modulator. Three apoptosis-inducing agents increased INXS lncRNA endogenous expression in the 786-O kidney tumor cell line, increased BCL-XS/BCL-XL mRNA ratio and activated caspases 3, 7 and 9. These effects were abrogated in the presence of INXS knockdown. Similarly, ectopic INXS overexpression caused a shift in splicing toward BCL-XS and activation of caspases, thus leading to apoptosis. BCL-XS protein accumulation was detected upon INXS overexpression. In a mouse xenograft model, intra-tumor injections of an INXS-expressing plasmid caused a marked reduction in tumor weight, and an increase in BCL-XS isoform, as determined in the excised tumors. We revealed an endogenous lncRNA that induces apoptosis, suggesting that INXS is a possible target to be explored in cancer therapies.

Identificação de RNAs longos não-codificadores de proteínas regulados por micro-RNAs / Identification of long non-protein coding RNAs regulated by micro-RNAs

Estudos recentes têm revelado que a maior parte dos transcritos gerados em células humanas é composta por RNAs não-codificadores de proteínas (ncRNAs). Uma parte desses ncRNAs compreende a classe de RNAs curtos, que possuem menos que 200 nucleotídeos. Os micro-RNAs (miRNAs) fazem parte dessa classe e têm sido alvo de grande interesse, pois são preditos como possíveis reguladores de mais de 60% dos RNAs mensageiros (mRNAs) humanos. Outra classe dos ncRNAs é composta por ncRNAs longos (lncRNAs, com mais de 200 nucleotídeos), que são transcritos a partir de regiões intergênicas e intrônicas do genoma humano e possuem várias funções, muitas delas relacionadas ao controle da expressão de mRNAs. Recentemente, os lncRNAs têm sido caracterizados quanto à sua estrutura e função. No entanto, muito pouco se sabe sobre os mecanismos pelos quais os lncRNAs são regulados. Este trabalho teve como objetivo avaliar se lncRNAs são regulados por miRNAs em células humanas. Para tanto, identificamos lncRNAs ligados ao complexo de silenciamento induzido por RNA (RISC) em células da linhagem HeLa, utilizando um método aqui desenvolvido de geração de bibliotecas de cDNA direcionadas para sequenciamento em larga escala na plataforma 454/Roche. Em paralelo, sequenciamos os miRNAs ligados ao RISC nestas mesmas células. Os resultados obtidos mostram que centenas de lncRNAs de diversas classes se ligam ao RISC em células HeLa, juntamente com milhares de mRNAs e várias centenas de miRNAs. Entre os miRNAs, encontramos 37 que são preditos como alvejando os lncRNAs detectados. Estes miRNAs constituem possíveis reguladores dos lncRNAs e, portanto, nosso trabalho estabelece um mapa experimental de interações diretas entre lncRNAs e miRNAs. Dentre os lncRNAs identificados ligados ao RISC neste trabalho, destaca-se o TUG1, lincRNA sabidamente envolvido na regulação de genes relacionados à apoptose e ao ciclo celular. Mostramos por ensaio de super-expressão de miRNAs e qPCR que TUG1 é regulado pelo miRNA-148b, um dos miRNAs por nós detectados que possui um sítio alvo altamente conservado em mamíferos localizado na extremidade 3' de TUG1. Em conjunto, este trabalho contribui para o entendimento da regulação dos níveis de expressão de lncRNAs em células humanas e abre perspectivas para a modulação de miRNAs como estratégia de regulação dos níveis e das funções de lncRNAs

Recent studies have revealed that the largest fraction of the transcripts generated in human cells is composed of non-protein coding RNAs (ncRNAs). A portion of these RNAs encompasses the class of short RNAs, which are less than 200 nucleotides in length. Micro-RNAs (miRNAs) are part of this class and are of great interest, as they are predicted to target over 60% of the human messenger RNAs (mRNAs). Another class of ncRNAs is composed of long ncRNAs (lncRNAs, longer than 200 nucleotides), which are transcribed from intergenic and intronic regions of the human genome and have several functions, many of them related to the control of the mRNA expression. Recently, the structure and function of lncRNAs have been characterized. However, little is known about the mechanisms involved in lncRNA regulation. This work aimed to evaluate whether lncRNAs are regulated by miRNAs in human cells. For this purpose, we identified lncRNAs bound to the RNA-induced silencing complex (RISC) in HeLa cells using a method developed here for the generation of strand-specific cDNA libraries for large scale RNA-sequencing in the 454/Roche plataform. In parallel, we sequenced the miRNAs bound to RISC in these cells. Our results show that hundreds of lncRNAs from diverse classes are bound to RISC in HeLa cells, along with thousands of mRNAs and several hundred miRNAs. Among the miRNAs we identified 37 that are predicted to target the detected lncRNAs. These miRNAs are possible regulators of the lncRNAs, and therefore our work establishes an experimental map of direct interactions between lncRNAs and miRNAs. The lncRNA TUG1, a lincRNA involved in the regulation of genes related to apoptosis and cell cycle, was identified among the lncRNAs bound to RISC. We showed by miRNA over-expression and qPCR that TUG-1 is regulated by the miRNA-148b, which is one of the miRNAs detected in our sequencings and has a binding site highly conserved in mammals located at the TUG1 3` end. Taken together, our results contribute to the understanding of the regulation of the lncRNA expression levels in human cells and open perspectives for the modulation of miRNAs as a strategy to regulate the levels and functions of lncRNAs

Análise da expressão de RNAs não codificadores longos em adenocarcinoma de pâncreas / Expression analysis of long noncoding RNAs in pancreatic adecarcinoma

RNAs não codificadores longos (lncRNAs) compõem uma fração significativa do transcriptoma. Alterações na expressão de lncRNAs já foram observadas em vários cânceres humanos, mas ainda não foram exploradas no adenocarcinoma pancreático ductal (PDAC), uma doença devastadora e agressiva para a qual faltam métodos para diagnóstico precoce e tratamentos efetivos. Utilizando uma plataforma de microarranjo de cDNA com sondas para 984 lncRNAs e 2371 mRNAs, o presente estudo identificou conjuntos de lncRNAs expressos em 38 amostras clínicas pancreáticas. O enriquecimento de (i) elementos regulatórios associados às regiões promotoras (H3K4me3); (ii) possíveis inícios de transcrição (CAGE-tags); (iii) presença de elementos conservados sugere que ao menos uma fração desses RNAs seja originada a partir de unidades transcricionais independentes, reguladas e possivelmente funcionais. Foram identificadas assinaturas de expressão gênica compostas por mRNA e lncRNAs associadas ao tumor primário e à metástase pancreática. A assinatura gIenica associada à metástase apresentou enriquecimento RNAs intrônicos de loci gênicos associados à via MAPK quinase. O aumento de expressão dos transcritos intrônicos dos loci PPP3CB, MAP3K14 e DAPK1 foi confirmado por qPCR em metástases. Em conjunto, este trabalho aponta para a importância de lncRNAs intrônicos no PDAC e para a necessidade de estudos mais aprofundados para uma melhor compreensão do papel dessa classe de transcritos na biologia da doença

Long noncoding RNAs (lncRNAs) compose a significant fraction of transcriptome. Altered expression of lncRNAs has been observed in diverse human cancers, but has not being investigated in pancreatic ductal adenocarcinoma (PDAC), a devastating and aggressive disease that lack early diagnosis methods and effective treatments. Using a cDNA microarray platform with probes interrogating 984 lncRNAs and 2371 mRNA, the present study identified subsets of lncRNAs expressed in 38 pancreatic clinical samples. Enrichment of (i) regulatory elements associated to promoter region (H3K4me3); (ii) putative transcription start site (CAGEtags) and (iii) conserved elements, suggest that at least a fraction of these RNAs could be independent transcriptional unit, regulated, an possibly functional. Gene expression signatures comprised of mRNAs and lncRNAs and associated to primary or metastatic tumors were found. A gene signature associated to metastasis was enriched in intronic ncRNAs mapping to gene loci associated to the MAPK pathway. Over expression of intronic RNAs from PPP3CB, MAP3K14 and DAPK1 was confirmed by qPCR in metastatic samples. Taken together, this study points to the importance of intronic lncRNAs in PDAC and for the need to study this class of ncRNAs in greater detail to better understand its role in the biology of PDAC