A peptide encoded by the circular form of the SHPRH gene induces apoptosis in neuroblastoma cells

Abstract

Background: Circular RNAs (circRNAs) and their derived peptides represent largely unchartered areas in cellular biology, with many potential roles yet to be discovered. This study aimed to elucidate the role and molecular interactions of circSHPRH and its peptide derivative SHPRH-146aa in the pathogenesis of neuroblastoma (NB).

Methods: NB samples in the GSE102285 dataset were analyzed to measure circSHPRH expression, followed by in vitro experiments for validation. The role of SHPRH-146aa in NB cell proliferation, migration, and invasion was then examined, and luciferase activity assay was performed after SHPRH-146aa and RUNX1 transfection. Finally, the regulation of NB cell apoptosis by SHPRH-146aa combined with NFKBIA was tested.

Results: The GSE102285 dataset indicated overexpression of circSHPRH in NB samples, further supported by in vitro findings. Overexpression of circ-SHPRH and SHPRH-146aa inhibited proliferation, migration, and invasion of NB cells. A significant increase in apoptosis was observed, with upregulation of Caspase-3 and downregulation of Bcl-2. Furthermore, the peptide derivative SHPRH-146aa, derived from circSHPRH, suppressed NB cell malignancy traits, suggesting its role as a therapeutic target. A direct interaction between SHPRH-146aa and the transcription factor RUNX1 was identified, subsequently leading to increased NFKBIA expression. Notably, NFKBIA knockdown inhibited the pro-apoptotic effect of SHPRH-146aa on NB cells.

Conclusion: The study demonstrates that circ-SHPRH and SHPRH-146aa play significant roles in inhibiting the malignant progression of NB. They induce apoptosis primarily by modulating key apoptotic proteins Caspase-3 and Bcl-2, a process that appears to be regulated by NFKBIA. The SHPRH-146aa-RUNX1 interaction further elucidates a novel pathway in the regulation of apoptosis in NB. These findings indicate that circ-SHPRH and its derived peptide SHPRH-146aa could be potential therapeutic targets for NB treatment.

Keywords: Apoptosis; NFKBIA; Neuroblastoma; RUNX1; circSHPRH-146aa.

Figure 1. Expression of circ-SHPRH and its associated effect on NB cell phenotype.

(A) circ-SHPRH expression in the GSE102285 dataset, with the blue box line representing normal samples and the orange box line representing tumor samples. (B) qRT-PCR analysis of circ-SHPRH mRNA levels in NB cell lines. (C) qRT-PCR detection of circ-SHPRH expression levels after overexpression transfection of SH-SY5Y and SK-N-AS cells. (D and E) CCK-8 assay examines the regulation of circ-SHPRH overexpression on the proliferation of SH-SY5Y and SK-N-AS cells. (F–H) Transwell assay to analyze the migration and invasion abilities of SH-SY5Y and SK-N-AS cells after circ-SHPRH overexpression. *P < 0.05, **P < 0.01.

Figure 2. Effect of circ-SHPRH overexpression on NB cell apoptosis.

(A and B) Flow cytometry analysis evaluating the cell apoptosis rate in neuroblastoma cell lines upon circ-SHPRH overexpression. The four quadrants represent viable cells (Q4), early apoptotic cells (Q3), late apoptotic cells (Q2), and necrotic cells (Q1), respectively. (C) Quantitative representation of flow cytometry results showing enhanced apoptosis after circ-SHPRH overexpression. (D and E) qRT-PCR analysis of Bcl-2, Caspase-3 expression changes after circ-SHPRH overexpression in SH-SY5Y and SK-N-AS cells. (F) WB analysis of changes in expression of Bcl-2 and Caspase-3 proteins after circ-SHPRH overexpression. *P < 0.05.

Figure 3. Regulation of NB cells by overexpression of circSHPRH-146aa.

(A) qRT-PCR and WB analysis of the overexpression efficiency of SHPRH-146aa in SH-SY5Y and SK-N-AS cell lines. (B and C) CCK-8 assay analysis of cell proliferation in SH-SY5Y and SK-N-AS cell lines after SHPRH-146aa overexpression. (D–F) Transwell assay assessing the effect of SHPRH-146aa overexpression on cell migration and invasion abilities in SH-SY5Y and SK-N-AS cell lines. *P < 0.05.

Figure 4. Co-IP analysis of the interaction between circSHPRH-146aa and RUNX1.

(A) qRT-PCR analysis of the overexpression efficiency of SHPRH-146aa in SH-SY5Y and SK-N-AS cell lines. *P < 0.05. (B) WB analysis of the overexpression efficiency of SHPRH-146aa in SH-SY5Y and SK-N-AS cell lines. (C and D) WB images illustrate the presence of RUNX1 in immunoprecipitates, confirming the interaction between SHPRH-146aa and RUNX1. IP: Co-immunoprecipitation, WB: Western blotting, “+” indicates that the condition is added, “−” indicates that the condition is not added, and the gray value of the band reflects the protein level after treatment with different conditions.

Figure 5. Regulation of circSHPRH-146aa and RUNX1 in promoter activity and gene transcription.

(A) qRT-PCR detection of the expression level of NFKBIA after SHPRH-146aa overexpression in SH-SY5Y and SK-N-AS cells. (B) WB detection of NFKBIA expression level after SHPRH-146aa overexpression in SH-SY5Y and SK-N-AS cells. (C) The RUNX1 binding motif and its corresponding site on the NFKBIA promoter. (D and E) Bar graphs, the left panel shows the effect of wild-type (WT) and mutant (MUT) plasmids on luciferase activity, and the right panel shows the effect of SHPRH-146aa and RUNX1 on promoter activity. The X-axis represents the respective conditions and the Y-axis represents the relative luciferase activity. *P < 0.05.

Figure 6. Effect of circSHPRH-146aa overexpression and NFKBIA knockdown on NB cell apoptosis.

(A–D) Flow cytometry analysis of apoptosis in NB cell lines under different conditions: vector, over-SHPRH-146aa, vector+NC, over-SHPRH-146aa+si-NFKBIA. The four quadrants in the flow cytometry plot represent different cell populations based on Annexin V and propidium iodide staining, indicating early apoptotic, late apoptotic, live, and dead cells, respectively. (E and F) qRT-PCR analysis detected the expression of Bcl-2 and Caspase-3 in NB lines after SHPRH-146aa overexpression and NFKBIA knockdown. (G) WB analysis to detect the expression of Bcl-2 and Caspase-3 in NB lines after SHPRH-146aa overexpression and NFKBIA knockdown. *P < 0.05, ^#P < 0.05 vs. Over-SHPRH-146aa group.