Screening differential circular RNA expression profiles reveals hsa_circ_0004018 is associated with hepatocellular carcinoma

Abstract

Circular RNAs (circRNAs) have been emerged as an indispensable part of endogenous RNA network. However, the expression significance of circRNAs in hepatocellular carcinoma (HCC) is rarely revealed. The aim of this study was to determine the circRNA expression profile in HCC, and to investigate their clinical significances and relevant mechanisms for cancer progression. The global circRNA expression profile in HCC was measured by circRNA microarray. Levels of one representative circRNAs, hsa_circ_0004018, were confirmed by real-time reverse transcription-polymerase chain reaction. The expression levels of hsa_circ_0004018 in HCC were significantly lower compared with para-tumorous tissue (P<0.001). Our data further showed that lower expression of hsa_circ_0004018 was correlated with serum alpha-fetoprotein (AFP) level, tumor diameters, differentiation, Barcelona Clinic Liver Cancer stage and Tumor-node-metastasis stage. More importantly, we detected liver tissues from chronic hepatitis, cirrhosis and HCC patients; and found that hsa_circ_0004018 harbored HCC-stage-specific expression features in diverse chronic liver diseases (P<0.001). The area under receiver operating characteristic curve was up to 0.848 (95% CI=0.803-0.894, P<0.001). The sensitivity and specificity were 0.716 and 0.815, respectively. Finally, hsa_circ_0004018 might be involved in cancer-related pathways via interactions with miRNAs.

Keywords: biomarker; circular RNA; hepatocellular carcinom; hsa_circ_0004018; microRNA.

Figure 1. Overview of the microarray signatures

(A) The boxplot view is used to compare the distributions of expression values for the samples in an experiment after standardization. After standardization, the distributions of log2 ratios among ten samples are nearly the same (CA: HCC; PARA: para-tumorous tissue). (B) Volcano plot of the differentially expressed circRNAs. The vertical lines correspond to 2.0-fold up and down, respectively, and the horizontal line represents a p-value of 0.05. The red point in the plot represents the differentially expressed circRNAs with statistical significance. (C) The result of hierarchical clustering shows a distinguishable circRNA expression profiling among samples. Each column represents the expression profile of a tissue sample, and each row corresponds to a circRNA. ‘‘Red’’ indicates higher expression level, and ‘‘green’’ indicates lower expression level.

Figure 2. Hsa_circ_0004018 expression features

(A) Electrophoresis of qRT-PCR products. Lane M is DL600 Marker, lanes 1-3 are qRT-PCR products. (B) Validation of hsa_circ_0004018 splicing junction site through sequencing of its qRT-PCR product. (C) Decreased expression of hsa_circ_0004018 in HCC tissues. Real-time RT-PCR was used to determine the expression level. The ΔCt value was determined by subtracting the Ct value of GAPDH from the Ct value of circRNA. Larger ΔCt value indicates lower expression. (n=102, P<0.001). (D) Hsa_circ_0004018 expression level among liver tissues from HCC (n=102), cirrhosis (n=63), and chronic hepatitis (n=66). (E) Expression of hsa_circ_4018 in HCC cell lines and normal hepatic cell line L02. Data are means± SD.

Figure 3. The ROC curve of using hsa_circ_0004018 as a biomarker

P<0.05.

Figure 4. Prediction for hsa_circ_0004018/miRNA interactions

Figure 5. Prediction for hsa_circ_0004018/miRNA-mediated pathways

(A) The heatmap reveals statistically significant correlations between hsa-miR-30e-5p, hsa-miR-647, hsa-miR-92a-1-5p, hsa-miR-660-3p and hsa-miR-626 and their mediated pathways by P-value (log scaled). Red represents high significance. (B) A network comprising has_circ_0004018 (red diamond), hsa-miR-30e-5p, hsa-miR-626(green hexagon), their target miRNAs (pink nodes) and MYC (yellow node) is presented. (C) Venn diagram revealed the gene showed MYC are the common target gene of hsa-miR-30e-5p and hsa-miR-626.