[Role and mechanism of cysteine and glycine-rich protein 2 in the malignant progression of neuroblastoma].

OBJECTIVE

To investigate the function and underlying mechanism of cysteine and glycine-rich protein 2 (CSRP2) in neuroblastoma (NB).

METHODS

The correlation between the expression level of mRNA and the prognosis of NB children in NB clinical samples was analyzed in R2 Genomics Analysis and Visualization Platform. The small interfering RNA (siRNA) targeting or CSRP2 plasmid were transfected to NB cell lines SK-N-BE(2) and SH-SY5Y. Cell proliferation was observed by crystal violet staining and real-time cellular analysis. The ability of colony formation of NB cells was observed by colony-forming unit assay. Immunofluorescence assay was used to detect the expression of the proliferation marker Ki-67. Flow cytometry analysis for cell cycle proportion was used with cells stained by propidium iodide (PI). Annexin V/7AAD was used to stain cells and analyze the percentage of cell apoptosis. The ability of cell migration was determined by cell wound-healing assay. The level of protein and mRNA expression of in NB primary tumor and NB cell lines were detected by Western blot and quantitative real-time PCR (RT-qPCR).

RESULTS

By analyzing the NB clinical sample databases, it was found that the expression levels of in high-risk NB with 3/4 stages in international neuroblastoma staging system (INSS) were significantly higher than that in low-risk NB with 1/2 INSS stages. The NB patients with high expression levels of were shown lower overall survival rate than those with low expression levels of . We detected the protein levels of CSRP2 in the NB samples by Western blot, and found that the protein level of CSRP2 in 3/4 INSS stages was significantly higher than that in 1/2 INSS stages. Knockdown of inhibited cell viability and proliferation of NB cells. Overexpression of CSRP2 increased the proliferation of NB cells. Flow cytometry showed that the proportion of sub-G1, G0/G1 and S phase cells and Annexin V positive cells were increased after deficiency. In the cell wound-healing assay, the healing rate of NB cells was significantly attenuated after knockdown of . Further mechanism studies showed that the proportion of the proliferation marker Ki-67 and the phosphorylation levels of extracellular signal-regulated kinases 1/2 (ERK1/2) were significantly decreased after knockdown.

CONCLUSION

CSRP2 is highly expressed in high-risk NB with 3/4 INSS stages, and the expression levels of CSRP2 are negatively correlated with the overall survival of NB patients. CSRP2 significantly increased the proliferation and cell migration of NB cells and inhibited cell apoptosis via the activation of ERK1/2. All these results indicate that CSRP2 promotes the progression of NB by activating ERK1/2, and this study will provide a potential target for high-risk NB therapy.