Construction and analysis of circular RNA molecular regulatory networks in liver cancer

<p>Liver cancer is the sixth most prevalent cancer, and the third most frequent cause of cancer-related deaths. Circular RNAs (circRNAs), a kind of special endogenous ncRNAs, have been coming back to the forefront of cancer genomics research. In this study, we used a <i>systems biology approach</i> to construct and analyze the circRNA molecular regulatory networks in the context of liver cancer. We detected a total of 127 differentially expressed circRNAs and 3,235 differentially expressed mRNAs. We selected the top-5 upregulated circRNAs to construct a circRNA-miRNA-mRNA network. We enriched the pathways and gene ontology items and determined their participation in cancer-related pathways such as p53 signaling pathway and pathways involved in angiogenesis and cell cycle. Quantitative real-time PCR was performed to verify the top-five circRNAs. ROC analysis showed circZFR, circFUT8, circIPO11 could significantly distinguish the cancer samples, with an AUC of 0.7069, 0.7575, and 0.7103, respectively. Our results suggest the circRNA-miRNA-mRNA network may help us further understand the molecular mechanisms of tumor progression in liver cancer, and reveal novel biomarkers and therapeutic targets.</p

ROC curve of genes as discriminators between gastric cancer and normal tissues.

<p>C&N indicates the combination of <i>COL1A1</i> and <i>NCAM1</i>.</p

Significant KEGG pathways of the hub-genes regulated by both TFs and miRNAs.

<p>Significant KEGG pathways of the hub-genes regulated by both TFs and miRNAs.</p

Transcription Factors and microRNA-Co-Regulated Genes in Gastric Cancer Invasion in <i>Ex Vivo</i>

<div><p>Aberrant miRNA expression abnormally modulates gene expression in cells and can contribute to tumorigenesis in humans. This study identified functionally relevant differentially expressed genes using the transcription factors and miRNA-co-regulated network analysis for gastric cancer. The TF-miRNA co-regulatory network was constructed based on data obtained from cDNA microarray and miRNA expression profiling of gastric cancer tissues. The network along with their co-regulated genes was analyzed using Database for Annotation, Visualization and Integrated Discovery (DAVID) and Transcriptional Regulatory Element Database (TRED). We found eighteen (17 up-regulated and 1 down-regulated) differentially expressed genes that were co-regulated by transcription factors and miRNAs. KEGG pathway analysis revealed that these genes were part of the extracellular matrix-receptor interaction and focal adhesion signaling pathways. In addition, qRT- PCR and Western blot data showed an increase in COL1A1 and decrease in NCAM1 mRNA and protein levels in gastric cancer tissues. Thus, these data provided the first evidence to illustrate that altered gene network was associated with gastric cancer invasion. Further study with a large sample size and more functional experiments is needed to confirm these data and contribute to diagnostic and treatment strategies for gastric cancer.</p></div

TF-gene regulatory network for gastric cancer.

<p>The network was constructed based on data from cDNA microarray analysis to identify differnetially expressed genes in gastric cancer. Red circles are up-regulated genes, while green circles are down-regulated genes and the yellow triangles represent transcription factors (TFs). The direction of the arrow is from the source to the target.</p

Validation of COL1A1 and NCAM1 expression in 20 pairs of gastric cancer and normal tissues.

<p>A and B, Detection of COL1A1 and NCAM1 mRNA expression in gastric cancer vs. normal tissues using PCR and qRT-PCR. Levels of COL1A1, NCAM1 mRNA were 3.10 ± 1.08 folds up-regulation and 0.37 ± 0.02 folds down-regulation in tumor tissues, respectively compared to those of the normal ones. *p<0.01. C and D, Western blot analysis of COL1A1 protein. Tumor tissues expressed higher level of COL1A1 protein compared to the normal ones (p<0.01). E and F, Western blot analysis of NCAM1 protein. Tumor tissues expressed lower level of NCAM1 protein compared to the normal ones (p<0.01). N, normal tissues; C, cancer tissues.</p

Expression of COL1A1 and NCAM1 associated with gastric cancer invasion depth.

<p>Expression of COL1A1 and NCAM1 associated with gastric cancer invasion depth.</p

Hub-genes in the TF-miRNA co-regulatory network.

<p>Circles in the central line are genes that co-regulated by TFs and miRNAs, with red for up-regulated genes and green for down-regulated genes. Yellow squares represent miRNAs and yellow triangles represent TFs.</p

miRNA-target network for gastric cancer.

<p>Red circles are up-regulated genes, while green circles are down-regulated genes and the yellow squares represent miRNAs. Lines with “T” edges in the network represent inhibitory effect of miRNAs on target genes.</p

Bi-clusters analysis of differentially expressed miRNAs in gastric cancer tissues.

<p>Each row represents a miRNA and each column represents a sample. Column “C” represents cancer tissues and column “N” represent normal tissues. The heat map with red indicates up-regulated and green for down-regulated miRNAs.</p