Jichu yixue yu linchuang (May 2024)
Regulation of TMEFF1 on proliferation of neuroblastoma cell lines
Abstract
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.
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