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Objective To investigate the regulatory effects of transmembrane protein with EGF-like and two follistatin-like domain 1(TMEFF1/tomoregulin 1) on the proliferation and migration of neuroblastoma (NB) cell line SN-K-BE(2). Methods TMEFF1 mRNA in neuroblastoma cell lines SN-K-BE(2), IMR32, SK-N-SH, SK-N-AS and normal cell lines MCF10A and hTERT RPE-1 was detected by RT-qPCR. Small interfering RNA (siRNA) was used to construct transient knockdown TMEFF1 normal and SN-K-BE (2) NB cell lines. The effect of transient knockdown of TMEFF1 mRNA was examined by RT-qPCR. After transfection of SN-K-BE (2) cells with siRNA, cell proliferation was detected by colony-forming unit assay and real-time cell analysis (RTCA). The positive rate of Ki-67 cells was determined by immunofluorescence staining. CellTiter-Glo (CTG) was used to detect cell activity. Stable knockdown of TMEFF1 was performed in SK-N-BE(2) cells by short hairpin RNA (shRNA) lentiviral infection, the stable outcomes of TMEFF1 was also detected by RT-qPCR. Cell proliferation was detected by coloning formation, RTCA and CTG. In addition, the positive rate of Ki-67 cells was detected by immunofluorescence,the cell mobility was measured by cell scratch assay. Results Compared with normal cell lines, the expression of TMEFF1 in NB cell lines was significantly higher (P＜0.001). Transient or stable knockdown of TMEFF1 had no significant effect on the proliferation of normal cell lines. However, by knocking down TMEFF1 decreased cell proliferation of SN-K-BE (2) cells as showen RTCA and colony-forming unit assay (P＜0.05). Immunofluorescence results showed that the rate of Ki-67 positive cells was reduced (P＜0.001). CTG results showed that the cell activity was decreased(P＜0.01). Cell scratch assay also showed that the cell migration ratio in the knockdown group was significantly lower than that of control group (P＜0.01). Conclusions TMEFF1 is a protein specifically highly expressed in neuroblastoma. TMEFF1 knockdown has no effect on normal cell lines, but significantly inhibited the proliferation and migration of NB cells, suggesting that TMEFF1 may play an important role in the occurrence and development of neuroblastoma, and so may be a potential target for NB targeting therapy.

Objective To explore the effect of open reading frame 66 (C12ORF66) located at chromosome 12 on the viability of MYCN amplified NB cell lines. Methods DDatasets GSE16476 and GSE49710 in R2 database were analyzed for expression level of C12ORF66 in MYCN amplified and MYCN non-amplified NB cells and its potential correlation with the prognosis of pediatric patients. C12ORF66 mRNA expression level in normal tissue immortalized cell lines, MYCN amplified and MYCN non-amplified cell lines were detected by RT-qRCR. Transient or stable knockdown of C12ORF66 cell lines were constructed to compare the difference in real time cellular analysis (RTCA), colony formation, Ki67 positive cells between the control group and the C12ORF66 knockdown group. Results By analyzing R2 datasets, C12ORF66 level in MYCN amplified samples was significantly higher than that in MYCN non-amplified samples, and the expression of C12ORF66 was negatively correlated with the prognosis of pediatric patients(P＜0.05). C12ORF66 highly expressed in MYCN-amplified BE(2)-C and SK-N-BE(2) cell lines than in MYCN non-amplified CHLA-255 and SH-SY5Y cell lines (P＜0.001). Transient or stable knockdown of C12ORF66 resulted in significant slow down of proliferation of MYCN amplified NB cells (P＜0.001), the colony formation ability was significantly reduced (P＜0.001), and the proportion of Ki67 positive cells was significantly decreased (P＜0.05). Conclusions C12ORF66 was highly expressed in MYCN amplified clinical NB samples and cell lines which is believed to be correlated with poor prognosis of pediatric patients. C12ORF66 knockdown significantly inhibits cell viability of NB cells.

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 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)., 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., 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.

Immunoglobulin (Ig) is considered a part of the innate immune system and cooperates with the complementary system as the first line of defense. In this study, a monoclonal antibody (MAb) direct against the light chain of goose Ig (GoIgCL) was generated, characterized and identified in various immunoassays to detect goose Ig. An immunoaffinity chromatography column prepared with this MAb was used to separate the goose Ig from sera. After being conjugated with horseradish peroxidase (HRP), this MAb was used as the secondary antibody to evaluate the goose-specific antibody. In addition, this MAb distinguished and localized the SIg+ lymphocytes from peripheral blood lymphocytes. MAb against GoIgCL may be good candidate to detect or purify goose Ig under various conditions and as a powerful tool for humoral immunity research on goose.