Dysregulated circular RNAs in medulloblastoma regulate proliferation and growth of tumor cells via host genes

Abstract

Circular RNAs (circRNAs) have been demonstrated to be involved in various biological processes. Nevertheless, the function of circRNAs in medulloblastoma (MB) is still unknown. The present study aimed to investigate the expression profiles of circRNAs and related mechanisms for regulating the proliferation and growth of tumor cells in MB. The expression profiles of circRNAs were screened from four normal cerebellum and four MB samples using a HiSeq Sequencer. Bioinformatic analysis was employed to predict the interaction between circRNAs and mRNAs in MB. Subsequently, the expression levels of eight differential circRNAs [circ-SKA3 (hsa_circ_0029696), circ-DTL (hsa_circ_0000179), circ-CRTAM, circ-MAP3K5 (hsa_circ_0006856), circ-RIMS1-1 (hsa_circ_0132250), circ-RIMS1-2 (hsa_circ_0076967), circ-FLT3-1 (hsa_circ_0100165), and circ-FLT3-2 (hsa_circ_0100168)] were validated using quantitative reverse transcription-polymerase chain reaction. Moreover, circ-SKA3 and circ-DTL were silenced using small interfering RNAs and their host genes were overexpressed to investigate their role in the pathogenesis of MB. A total of 33 circRNAs were found to be differentially expressed in MB tissues (fold change ≥ 2.0, FDR <0.05), of which three were upregulated and 30 were downregulated; six circRNAs were experimentally validated successfully. Upregulated circ-SKA3 and circ-DTL promoted the proliferation migration and invasion in vitro by regulating the expression of host genes. This novel study exploited the profiling of circRNAs in MB and demonstrated that circ-SKA3 and circ-DTL were crucial in the tumorigenesis and development of MB and might be considered as novel and potential biomarkers for the diagnosis and new targets for the intervention of MB.

Keywords: circular RNAs; invasion; medulloblastoma; migration; proliferation.

Figure 1

Profiling of circRNAs in human medulloblastoma (MB) and normal cerebellum tissues. A, The number of circRNAs and back‐spliced reads. B, The length distribution for exonic circRNAs. C, The number of circRNAs produced from one host gene. D, The differentially expressed mRNAs between MB and normal cerebellum tissues by hierarchical clustering analysis. E, The differentially expressed circRNAs between MB and normal cerebellum tissues by detected using hierarchical clustering analysis. Each column represents the expression profile of a sample, whereas each row represents circRNA

Figure 2

The coexpression network of gene and circRNAs. A, The coexpression network of circ‐SKA3 and circ‐DTL and related mRNAs as drawn via k‐core analysis. The circ‐SKA3 and circ‐DTL were the two circRNAs that gained the greatest k‐core difference in the network. B, The k‐core difference was calculated to identify the core gene and circRNAs in the medulloblastoma group

Figure 3

Differentially expressed circRNAs in medulloblastoma (MB). A, Volcano plot shows differentially expressed circRNAs with statistical significance. The vertical dotted lines represent 2.0‐fold change up and down. The horizontal dotted line corresponds to an FDR value of 0.05. The red points in the plot represent upregulated circRNAs, the blue points represent the downregulated circRNAs, and the gray points represent the normal circRNAs. B, The relative expression of circRNAs in MB and normal cerebellum tissues detected using qRT‐PCR. C, RT‐PCR gels show that circ‐SKA3 and circ‐DTL are enriched in MB. *P < .05, **P < .01

Figure 4

Effect of circ‐SKA3 and circ‐DTL on the proliferation of DAOY cell line in vitro. A, The effect of Si‐circ‐SKA3 plasmids on the expression of circ‐SKA3. B, The effect of Si‐circ‐DTL plasmids on the expression of circ‐DTL. C, The effect of circ‐SKA3 silencing and overexpression of linear transcript of SKA3 on the expression of circ‐SKA3 and linear transcript of SKA3. D, The effect of circ‐DTL silencing and overexpression of linear transcript of DTL on the expression of circ‐SKA3 and linear transcript of DTL. E, The effect of circ‐SKA3 silencing and overexpression of mRNA of SKA3 on the proliferation potential of the medulloblastoma (MB) DAOY cell line. F, The effect of circ‐DTL silencing and overexpression of mRNA of DTL on the proliferation potential of the MB DAOY cell line. The data are expressed as means ± standard deviation; *P < .05

Figure 5

Effect of circ‐SKA3 and circ‐DTL on the migration and invasion capacity of DAOY cell line in vitro. A, Circ‐SKA3 silencing reduced the migration and invasion capacity of DAOY cells in Transwell migration assays, while overexpression of mRNA of SKA3 recovered the migration and invasion ability. B, Circ‐DTL silencing and overexpression of mRNA of DTL showed the same trend. C, The quantification number of migration and invasion cells with different expression levels of circRNAs and mRNAs. Scale bars represent 50 μm. Magnification 100×; data expressed as means ± standard deviation; *P < .05