Analysis of co-expression networks for circular RNAs and mRNAs reveals that circular RNAs hsa_circ_0047905, hsa_circ_0138960 and has-circRNA7690-15 are candidate oncogenes in gastric cancer

Abstract

Accumulating evidence has suggested that circular RNAs (circRNAs) play important roles in oncogenesis and tumor progression. However, our knowledge of circRNAs in gastric cancer (GC) remains limited. To investigate circRNAs involved in GC oncogenesis, we examined differentially-expressed circRNAs and mRNAs in GC tissues and paired noncancerous mucosa tissues using circRNA and mRNA microarrays. Next, we built gene co-expression networks according to the degree of correlation to predict the critical circRNAs in GC. Through bioinformatics analysis, we observed three newly identified circRNAs that are substantially upregulated in GC: hsa_circ_0047905, hsa_circ_0138960 and has-circRNA7690-15. Additionally, hsa_circ_0047905 and hsa_circ_0138960 positively correlated with their parental gene mRNA. Knockdown of hsa_circ_0047905, hsa_circ_0138960 and has-circRNA7690-15 in GC cells, resulted in downregulation of parental gene expression. Functional assays suggested that inhibition of these three circular RNAs suppresses GC cell proliferation and invasion in vitro. Those findings suggest that hsa_circ_0047905, hsa_circ_0138960 and has-circRNA7690-15 might act as tumor promoters in the pathogenesis of gastric cancer.

Keywords: Circular RNA; GDA; SERPINB5; gastric cancer; oncogenesis.

Figure 1.

Heat map of the circRNA and mRNA profiles in GC tissues versus paired normal gastric mucosa tissues. Red indicates high relative expression, and green indicates low relative expression. The expression of mRNA (A) and circRNA (B) were hierarchically clustered on the y-axis, and the tissue samples are hierarchically clustered on the x-axis (Fold change ≧ 2, p < 0.05). Expression levels are presented in red and green, indicating upregulated and downregulated RNAs, respectively. Numbers marked with T and N are from five paired gastric cancer tissues and paired adjacent normal tissues, respectively.

Figure 2.

Co-expression network in GC tissues and adjacent normal tissues. The co-expression network consists of 204 circRNAs and 169 mRNAs. (A) GC tissues; (B) Normal tissues. A round node represents a protein-coding gene, and a triangular node represents a circRNA. Solid lines between two nodes indicate positively-correlated interactions between genes, and dashed lines indicate negatively-correlated interactions. Structural cohesion levels are presented in different colors; red indicates a high cohesion level.

Figure 3.

Localization of circRNAs in AGS human GC cell line. RNA fluorescence in situ hybridization for circRNAs. Nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). Scale bar, 10 μm. (A) circRNA0047905; (B) circRNA0138960; (C) circRNA7690-15.

Figure 4.

Relative expression of critical circRNAs and parental genes mRNAs in tissues. (A) Relative expression of circRNA0047905, circRNA0138960, circRNA7690-15 in GC tissues compared with paired adjacent noncancerous tissues; (B) Relative expression of SERPINB5 mRNA and GDA mRNA in GC tissues compared with paired adjacent noncancerous tissues (T: GC tissues, N: noncancerous tissue, n = 31). (C) Correlation between expression of circRNAs and parental gene mRNAs in tissue specimens.

Figure 5.

Relative expression of circRNAs and mRNAs in AGS human GC cell lines transfected with specific siRNA targeted circRNAs. (A) Relative expression of circRNAs in AGS GC cell lines transfected with specific siRNA. *P < 0.05. (B) Relative expression of parental gene mRNAs in AGS GC cell lines transfected with specific siRNAs respectively targeted circRNAs. *P < 0.05. (C) Expression of SERPINB5 and GDA protein in AGS GC cell line transfected with specific siRNAs.

Figure 6.

Tumor-promoting effects of circRNAs in AGS human GC cell line. (A) Tumor-promoting effects of circRNA0047905, circRNA0138960, circRNA7690-15 in AGS cell lines. The proliferation curve of AGS cells following transfection with specific siRNAs by the CCK8 assay. The effects of circRNA0047905, circRNA0138960, and circRNA7690-15 knockdown on the invasive ability of AGS cells were assessed by the Transwell method. (B) si-Negative control, (C) si-circRNA0047905, (D) si-circRNA0138960, (E) circRNA7690-15), (F) Quantification was performed by light microscope (200 x), counting the stained cells that invaded the lower chamber, *P < 0.05.

Figure 7.

Receiver operating characteristic curve of specific circRNAs in distinguishing GC. AUC: area under the curve. (circRNA0047905: AUC = 0.850, P < 0.001; circRNA0138960: AUC = 0.647, P < 0.05; circRNA7690-15: AUC = 0.681, P < 0.05).