Expression and regulatory roles of lncRNAs in G-CIMP-low vs G-CIMP-high Glioma: an in-silico analysis

BACKGROUND: Clinically relevant glioma subtypes, such as the glioma-CpG island methylator phenotype (G-CIMP), have been defined by epigenetics. In this study, the role of long non-coding RNAs in association with the poor-prognosis G-CMIP-low phenotype and the good-prognosis G-CMIP-high phenotype was investigated. Functional associations of lncRNAs with mRNAs and miRNAs were examined to hypothesize influencing factors of the aggressive phenotype. METHODS: RNA-seq data on 250 samples from TCGA\u27s Pan-Glioma study, quantified for lncRNA and mRNAs (GENCODE v28), were analyzed for differential expression between G-CIMP-low and G-CIMP-high phenotypes. Functional interpretation of the differential lncRNAs was performed by Ingenuity Pathway Analysis. Spearman rank order correlation estimates between lncRNA, miRNA, and mRNA nominated differential lncRNA with a likely miRNA sponge function. RESULTS: We identified 4371 differentially expressed features (mRNA = 3705; lncRNA = 666; FDR ≤ 5%). From these, the protein-coding gene TP53 was identified as an upstream regulator of differential lncRNAs PANDAR and PVT1 (p = 0.0237) and enrichment was detected in the development of carcinoma (p = 0.0176). Two lncRNAs (HCG11, PART1) were positively correlated with 342 mRNAs, and their correlation estimates diminish after adjusting for either of the target miRNAs: hsa-miR-490-3p, hsa-miR-129-5p. This suggests a likely sponge function for HCG11 and PART1. CONCLUSIONS: These findings identify differential lncRNAs with oncogenic features that are associated with G-CIMP phenotypes. Further investigation with controlled experiments is needed to confirm the molecular relationships

Noninvasive approaches to detect methylation-based markers to monitor gliomas

In this review, we summarize the current approaches used to detect glioma tissue-derived DNA methylation markers in liquid biopsy specimens with the aim to diagnose, prognosticate and potentially track treatment response and evolution of patients with gliomas

An early requirement for maternal FoxH1 during zebrafish gastrulation

AbstractThe Forkhead Box H1 (FoxH1) protein is a co-transcription factor recruited by phosphorylated Smad2 downstream of several TGFβs, including Nodal-related proteins. We have reassessed the function of zebrafish FoxH1 using antisense morpholino oligonucleotides (MOs). MOs targeting translation of foxH1 disrupt embryonic epiboly movements during gastrulation and cause death on the first day of development. The FoxH1 morphant phenotype is much more severe than that of zebrafish carrying foxh1/schmalspur (sur) DNA-binding domain mutations, FoxH1 splice-blocking morphants or other Nodal pathway mutants, and it cannot be altered by concomitant perturbations in Nodal signaling. Apart from disrupting epiboly, FoxH1 MO treatment disrupts convergence and internalization movements. Late gastrula-stage FoxH1 morphants exhibit delayed mesoderm and endoderm marker gene expression and failed patterning of the central nervous system. Probing FoxH1 morphant RNA by microarray, we identified a cohort of five keratin genes – cyt1, cyt2, krt4, krt8 and krt18 – that are normally transcribed in the embryo's enveloping layer (EVL) and which have significantly reduced expression in FoxH1-depleted embryos. Simultaneously disrupting these keratins with a mixture of MOs reproduces the FoxH1 morphant phenotype. Our studies thus point to an essential role for maternal FoxH1 and downstream keratins during gastrulation that is epistatic to Nodal signaling

TCGAbiolinks: an R/Bioconductor package for integrative analysis of TCGA data

The Cancer Genome Atlas (TCGA) research network has made public a large collection of clinical and molecular phenotypes of more than 10 000 tumor patients across 33 different tumor types. Using this cohort, TCGA has published over 20 marker papers detailing the genomic and epigenomic alterations associated with these tumor types. Although many important discoveries have been made by TCGA’s research network, opportunities still exist to imple- ment novel methods, thereby elucidating new bio- logical pathways and diagnostic markers. However, mining the TCGA data presents several bioinformat- ics challenges, such as data retrieval and integra- tion with clinical data and other molecular data types (e.g. RNA and DNA methylation). We developed an R/Bioconductor package called TCGAbiolinks to ad- dress these challenges and offer bioinformatics so- lutions by using a guided workflow to allow users to query, download and perform integrative analyses of TCGA data. We combined methods from computer science and statistics into the pipeline and incor- porated methodologies developed in previous TCGA marker studies and in our own group. Using four dif- ferent TCGA tumor types (Kidney, Brain, Breast and Colon) as examples, we provide case studies to illus- trate examples of reproducibility, integrative analysis and utilization of different Bioconductor packages to advance and accelerate novel discoveries

RGBM: regularized gradient boosting machines for identification of the transcriptional regulators of discrete glioma subtypes

We propose a generic framework for gene regulatory network (GRN) inference approached as a feature selection problem. GRNs obtained using Machine Learning techniques are often dense, whereas real GRNs are rather sparse. We use a Tikonov regularization inspired optimal L-curve criterion that utilizes the edge weight distribution for a given target gene to determine the optimal set of TFs associated with it. Our proposed framework allows to incorporate a mechanistic active biding network based on cis-regulatory motif analysis. We evaluate our regularization framework in conjunction with two non-linear ML techniques, namely gradient boosting machines (GBM) and random-forests (GENIE), resulting in a regularized feature selection based method specifically called RGBM and RGENIE respectively. RGBM has been used to identify the main transcription factors that are causally involved as master regulators of the gene expression signature activated in the FGFR3-TACC3-positive glioblastoma. Here, we illustrate that RGBM identifies the main regulators of the molecular subtypes of brain tumors. Our analysis reveals the identity and corresponding biological activities of the master regulators characterizing the difference between G-CIMP-high and G-CIMP-low subtypes and between PA-like and LGm6-GBM, thus providing a clue to the yet undetermined nature of the transcriptional events among these subtypes

Optimizing exosomal RNA isolation for RNA-Seq analyses of archival sera specimens

Exosomes are endosome-derived membrane vesicles that contain proteins, lipids, and nucleic acids. The exosomal transcriptome mediates intercellular communication, and represents an understudied reservoir of novel biomarkers for human diseases. Next-generation sequencing enables complex quantitative characterization of exosomal RNAs from diverse sources. However, detailed protocols describing exosome purification for preparation of exosomal RNA-sequence (RNA-Seq) libraries are lacking. Here we compared methods for isolation of exosomes and extraction of exosomal RNA from human cell-free serum, as well as strategies for attaining equal representation of samples within pooled RNA-Seq libraries. We compared commercial precipitation with ultracentrifugation for exosome purification and confirmed the presence of exosomes via both transmission electron microscopy and immunoblotting. Exosomal RNA extraction was compared using four different RNA purification methods. We determined the minimal starting volume of serum required for exosome preparation and showed that high quality exosomal RNA can be isolated from sera stored for over a decade. Finally, RNA-Seq libraries were successfully prepared with exosomal RNAs extracted from human cell-free serum, cataloguing both coding and non-coding exosomal transcripts. This method provides researchers with strategic options to prepare RNA-Seq libraries and compare RNA-Seq data quantitatively from minimal volumes of fresh and archival human cell-free serum for disease biomarker discovery

SIRT1 regulates Mxd1 during malignant melanoma progression

In a murine melanoma model, malignant transformation promoted by a sustained stress condition was causally related to increased levels of reactive oxygen species resulting in DNA damage and massive epigenetic alterations. Since the chromatin modifier Sirtuin-1 (SIRT1) is a protein attracted to double-stranded DNA break (DSB) sites and can recruit other components of the epigenetic machinery, we aimed to define the role of SIRT1 in melanomagenesis through our melanoma model. The DNA damage marker, gamma H2AX was found increased in melanocytes after 24 hours of deadhesion, accompanied by increased SIRT1 expression and decreased levels of its target, H4K16ac. Moreover, SIRT1 started to be associated to DNMT3B during the stress condition, and this complex was maintained along malignant progression. Mxd1 was identified by ChIP-seq among the DNA sequences differentially associated with SIRT1 during deadhesion and was shown to be a common target of both, SIRT1 and DNMT3B. In addition, Mxd1 was found downregulated from pre-malignant melanocytes to metastatic melanoma cells. Treatment with DNMT inhibitor 5AzaCdR reversed the Mxd1 expression. Sirt1 stable silencing increased Mxd1 mRNA expression and led to down-regulation of MYC targets, such as Cdkn1a, Bcl2 and Psen2, whose upregulation is associated with human melanoma aggressiveness and poor prognosis. We demonstrated a novel role of the stress responsive protein SIRT1 in malignant transformation of melanocytes associated with deadhesion. Mxd1 was identified as a new SIRT1 target gene. SIRT1 promoted Mxd1 silencing, which led to increased activity of MYC oncogene contributing to melanoma progression.FAPESP [2011/0166-38, 2011/12306-1, 2014/13663-0, 2015/07925-5, 2016/06488-3]DAAD [PKZ A/12/79134]FAPESP/BAYLAT [2012/51300-7]Univ Fed Sao Paulo UNIFESP, Dept Pharmacol, Ontogeny & Epigenet Lab, Sao Paulo, SP, BrazilUniv Sao Paulo, Ribeirao Preto Med Sch, Dept Genet, Ribeirao Preto, SP, BrazilFriedrich Alexander Univ Erlangen Nurnberg FAU, Inst Pathol, Expt Tumorpathol, Erlangen, GermanyFriedrich Alexander Univ Erlangen Nurnberg FAU, Dept Pediat & Adolescent Med, Erlangen, GermanyUniv Fed Sao Paulo UNIFESP, Dept Pharmacol, Ontogeny & Epigenet Lab, Sao Paulo, SP, BrazilFAPESP [2011/0166-38, 2011/12306-1, 2014/13663-0, 2015/07925-5, 2016/06488-3]DAAD [PKZ A/12/79134]FAPESP/BAYLAT [2012/51300-7]Web of Scienc

Genome-wide Runx2 occupancy in prostate cancer cells suggests a role in regulating secretion

Runx2 is a metastatic transcription factor (TF) increasingly expressed during prostate cancer (PCa) progression. Using PCa cells conditionally expressing Runx2, we previously identified Runx2-regulated genes with known roles in epithelial–mesenchymal transition, invasiveness, angiogenesis, extracellular matrix proteolysis and osteolysis. To map Runx2-occupied regions (R2ORs) in PCa cells, we first analyzed regions predicted to bind Runx2 based on the expression data, and found that recruitment to sites upstream of the KLK2 and CSF2 genes was cyclical over time. Genome-wide ChIP-seq analysis at a time of maximum occupancy at these sites revealed 1603 high-confidence R2ORs, enriched with cognate motifs for RUNX, GATA and ETS TFs. The R2ORs were distributed with little regard to annotated transcription start sites (TSSs), mainly in introns and intergenic regions. Runx2-upregulated genes, however, displayed enrichment for R2ORs within 40 kb of their TSSs. The main annotated functions enriched in 98 Runx2-upregulated genes with nearby R2ORs were related to invasiveness and membrane trafficking/secretion. Indeed, using SDS–PAGE, mass spectrometry and western analyses, we show that Runx2 enhances secretion of several proteins, including fatty acid synthase and metastasis-associated laminins. Thus, combined analysis of Runx2's transcriptome and genomic occupancy in PCa cells lead to defining its novel role in regulating protein secretion