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. 目的: 探究富含半胱氨酸和甘氨酸蛋白2(cysteine and glycine-rich protein 2，CSRP2)在神经母细胞瘤(neuroblastoma，NB)恶性进展中的功能和作用机制。 方法: 利用R2数据库分析NB临床样本中CSRP2基因的mRNA水平与NB患儿临床预后的相关性；在NB细胞系SK-N-BE(2)和SH-SY5Y中利用靶向小干扰RNA(small interfering RNA，siRNA)干扰CSRP2的表达或利用质粒转染过表达CSRP2；通过结晶紫染色和实时无标记动态细胞分析技术观察NB细胞的增殖情况；采用克隆形成方法观察NB细胞长时间的克隆形成能力；利用免疫荧光实验检测细胞增殖标记物Ki-67的水平；利用碘化丙啶(propidium iodide，PI)染色流式细胞术分析细胞周期比例，Annexin V/7AAD染色分析细胞凋亡比例；采用划痕实验观察细胞的迁移能力；利用Western blot或实时荧光定量PCR(quantitative real-time PCR，RT-qPCR)检测NB原发肿瘤组织和细胞系中蛋白和基因的表达水平。 结果: NB临床数据库中，国际神经母细胞瘤分期(international neuroblastoma staging system，INSS)为高危险度3/4期的NB组织中CSRP2的mRNA水平显著高于低危险度的1/2期，且高表达水平组NB患儿的生存期显著低于低表达组；Western blot结果显示，CSRP2在3/4期NB组织中的蛋白水平显著高于1/2期。NB细胞中敲低CSRP2，细胞的活力减弱、增殖能力降低；NB细胞中过表达CSRP2促进细胞增殖；敲低CSRP2后，sub-G1、G0/G1和S期细胞的比例增加，Annexin V阳性细胞的比例增多；敲低CSRP2的NB细胞的划痕愈合率显著小于对照组。机制研究发现，敲低CSRP2后细胞增殖标记分子Ki-67和细胞外信号调节激酶1/2(extracellular signal-regulated kinases 1/2，ERK1/2)磷酸化水平显著低于对照组。 结论: CSRP2在高危险度3/4期NB组织中高表达，表达水平与NB患儿生存期呈负相关；CSRP2通过促进ERK1/2活化，促进NB细胞的增殖和迁移，抑制细胞凋亡，表明CSRP2通过激活ERK1/2促进NB进展，为高危NB的靶向治疗提供了潜在的靶点。
Methods
The correlation between the expression level of CSRP2 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 CSRP2 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 CSRP2 in NB primary tumor and NB cell lines were detected by Western blot and quantitative real-time PCR (RT-qPCR).
Objective
To investigate the function and underlying mechanism of cysteine and glycine-rich protein 2 (CSRP2) in neuroblastoma (NB).
Results
By analyzing the NB clinical sample databases, it was found that the expression levels of CSRP2 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 CSRP2 were shown lower overall survival rate than those with low expression levels of CSRP2. 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 CSRP2 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 CSRP2 deficiency. In the cell wound-healing assay, the healing rate of NB cells was significantly attenuated after knockdown of CSRP2. 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 CSRP2 knockdown.