circRNA-TBC1D4, circRNA-NAALAD2 and circRNA-TGFBR3: Selected Key circRNAs in Neuroblastoma and Their Associations with Clinical Features

Abstract

Objective: The roles of circRNAs in neuroblastoma (NB) are unclear. We used next-generation sequencing to detect the circRNA expression profiles in NB to identify the key circRNAs and analyzed the relationships between the circRNAs and clinical features.

Methods: Five paired neuroblastoma tumor and adjacent normal fetal adrenal medulla samples were collected for high-throughput RNA sequencing. Bioinformatics analysis was performed for functional annotation of the host genes of differentially expressed circRNAs. Validation of dysregulated circRNAs was performed by real-time quantitative reverse transcription polymerase chain reaction. The relationships between the key circRNAs and clinical features were analyzed. In addition, overexpression of key circRNAs in an NB cell line, as well as cell proliferation assays, colony formation assays and cell migration assays, was conducted to investigate the biological functions of key circRNAs.

Results: A total of 4704 differentially expressed circRNAs were found, including 2462 up-regulated and 2242 down-regulated circRNAs. According to our previous studies, the predicted target circRNAs of miR-21 involved in tumorigenic signaling pathways were selected, including circRNA-TBC1D4, circRNA-NAALAD2 and circRNA-TGFBR3. These circRNAs were associated with clinical features, and the circRNA expression was significantly lower (P < 0.05) in the NB tissues than in normal adrenal tissues. Overexpression of circRNA-TBC1D4 promotes NB cell migration, but not proliferation and colony-formation in vitro.

Conclusion: We suggest that circRNA-TBC1D4, circRNA-NAALAD2 and circRNA-TGFBR3 may be cancer suppressor genes, which act by sponging miR-21 in NB. Further investigations are needed to elucidate the underlying mechanism.

Keywords: circRNA; circRNA-TBC1D4; miR-21; neuroblastoma.

Figure 1

(A) Volcano plot showing dysregulated circRNAs between neuroblastoma specimens and adjacent normal fetal adrenal medulla. The X-axis represents log₂FoldChange, and the Y-axis represents -log₁₀P value. The horizontal gray line indicates a P value of 0.05. The two vertical gray lines indicate a fold increase or decrease of 2 (left, log₂FoldChange = −1; right, log₂FoldChange = 1). The red and gray points represent significantly up-regulated (n = 2462) and down-regulated (n = 2242) circRNAs in neuroblastoma, respectively. (B) Hierarchical clustering of the dysregulated circRNAs in neuroblastoma specimens and adjacent normal fetal adrenal medulla. Each red or blue bar represents higher or lower expression of circRNA in each specimen. The circRNA expression patterns were distinguishable between the neuroblastoma group and the adjacent normal fetal adrenal medulla group, but similar within the same group. (C) Functional annotation of the host genes of differentially expressed circRNAs, conducted on the basis of gene ontology (GO) analyses. The X-axis represents the GO terms, and the Y-axis represents -log₁₀Pvalue. The green, blue and red bars represent the top ten significantly enriched GO terms in biological processes, cellular components and molecular functions. (D) Bulb map of KEGG pathway analysis for the host genes of differentially expressed circRNAs. The X-axis represents enrichment score, and the Y-axis represents the top 20 enriched pathways. The size of each bulb represents the number of enriched dysregulated circRNAs, and the P values are represented by a color scale.

Figure 2

Real-time qRT–PCR comparison of the relative expression (normalized to GAPDH) of 22 selected circRNAs in 20 paired neuroblastoma specimens and adjacent normal fetal adrenal medulla. Student t-test P values are shown in the figure. (A) Among ten selected highly up-regulated circRNAs from circRNA-seq, eight were confirmed to be significantly up-regulated in neuroblastoma, with P < 0.05. (B) Among five selected highly down-regulated circRNAs from circRNA-seq, two were confirmed to be significantly down-regulated in neuroblastoma, with P < 0.05. (C) Among seven selected miR-21 related circRNAs from circRNA-seq, three were confirmed to be significantly down-regulated in neuroblastoma, with P < 0.05.

Figure 3

Binding sites between miR-21 and (A) circRNA-TBC1D4, (B) circRNA-NAALAD2 and (C) circRNA-TGFBR3.

Figure 4

Overexpression of circRNA-TBC1D4 inhibited SH-SY5Y cell migration, but not proliferation and colony-formation. (A) SH-SY5Y cells were infected with lentivirus LV18-circTBC1D4 and LV18NC. qRT-PCR was conducted to detect circRNA-TBC1D4 levels. The bar graph showed that circRNA-TBC1D4 level of SH-SY5Y infected with LV18-circTBC1D4 was significantly higher than SH-SY5Y infected with LV18NC. (B) CCK-8 assay of SH-SY5Y with circRNA-TBC1D4 overexpression and NC on the indicated days. The growth curve showed that overexpression of circRNA-TBC1D4 did not inhibit the proliferation of SH-SY5Y cells. There is no significant difference between SH-SY5Y with circRNA-TBC1D4 overexpression and NC. (C) Colony formation assays of SH-SY5Y with circRNA-TBC1D4 overexpression and NC. The left panel visually showed that overexpression of circNRA-TBC1D4 did not inhibit the colony-forming ability of SH-SY5Y cells. And the bar graph showed that there was no significant difference between SH-SY5Y with circRNA-TBC1D4 overexpression and NC. (D) Transwell migration assay of SH-SY5Y with circRNA-TBC1D4 overexpression and NC. The left panel visually showed that overexpression of circNRA-TBC1D4 significantly inhibited the migration ability of SH-SY5Y cells. The bar graph showed that there was significant difference between SH-SY5Y with circRNA-TBC1D4 overexpression and NC. Data are shown as means ± s.d. of at least three independent experiments. ****P < 0.0001; ns, P > 0.05.