TMEFF1对神经母细胞瘤细胞系增殖的调控

doi:10.16352/j.issn.1001-6325.2024.05.0637

基础医学与临床 ›› 2024, Vol. 44 ›› Issue (5): 637-644.doi: 10.16352/j.issn.1001-6325.2024.05.0637

TMEFF1对神经母细胞瘤细胞系增殖的调控

贾安娜^#, 郭金鑫^#, 张璇, 战世佳, 于永波, 郭永丽, 常艳^*

国家儿童医学中心首都医科大学附属北京儿童医院儿科重大疾病研究教育部重点实验室北京市儿科研究所儿童耳鼻咽喉头颈外科疾病北京市重点实验室,北京 100045

收稿日期:2024-01-08 修回日期:2024-03-21 出版日期:2024-05-05 发布日期:2024-04-23
通讯作者: ^*changyan809@ccmu.edu.cn
^#对本文有相同贡献
基金资助:
国家自然科学基金(82141118,82293660/82293665,82172849);北京市教育委员会科技/社科计划项目(KM202210025010)

Regulation of TMEFF1 on proliferation of neuroblastoma cell lines

JIA Anna^#, GUO Jinxin^#, ZHANG Xuan, ZHAN Shijia, YU Yongbo, GUO Yongli, CHANG Yan^*

Beijing Key Laboratory for Pediatric Diseases of Otolaryngology, Head and Neck Surgery, Key Laboratory of Major Diseases in Children, Ministry of Education, Beijing Pediatric Research Institute, Beijing Children′s Hospital, Capital Medical University, National Center for Children′s Health, Beijing 100045, China

Received:2024-01-08 Revised:2024-03-21 Online:2024-05-05 Published:2024-04-23
Contact: ^*changyan809@ccmu.edu.cn

摘要/Abstract

摘要： 目的探究具有表皮生长因子结构域和两个卵泡抑素样结构域的跨膜蛋白1(TMEFF1/tomoregulin1)对神经母细胞瘤(NB)细胞系SN-K-BE(2)增殖和迁移的调控效应。方法 RT-qPCR检测神经母细胞瘤细胞系SN-K-BE(2)、IMR32、SK-N-SH、SK-N-AS和正常细胞系MCF 10A、hTERT RPE-1中TMEFF1 mRNA表达量。利用小分子干扰RNA(siRNA)瞬时敲低MCF 10A、hTERT RPE-1细胞和SN-K-BE(2) NB细胞中TMEFF1表达,RT-qPCR检测TMEFF1 mRNA瞬时敲低效果后,结晶紫染色、实时无标记细胞分析(RTCA)检测细胞增殖;CellTiter-Glo(CTG)检测细胞活性。利用短发夹RNA(shRNA)稳定敲低正常细胞系和NB细胞系中TMEFF1表达,进一步通过集落形成实验、RTCA、CTG检测细胞增殖和活性,此外,通过免疫荧光检测Ki-67细胞阳性率,细胞划痕实验检测细胞迁移率。结果与正常细胞系相比,NB细胞系中TMEFF1 mRNA表达量显著较高(P＜0.001)。敲低TMEFF1对正常细胞系增殖影响无统计学意义,但在SN-K-BE(2)细胞中敲低TMEFF1,RTCA和集落形成实验结果显示细胞增殖能力减弱(P＜0.05),免疫荧光结果显示Ki-67阳性细胞率减少(P＜0.001),CTG结果显示细胞活性降低(P＜0.01),细胞划痕实验结果显示敲低组细胞迁移率显著低于对照组(P＜0.01)。结论 TMEFF1是一种特异在NB细胞中高表达的膜蛋白,TMEFF1敲低后不影响正常细胞系的增殖,但显著抑制NB细胞系的增殖、迁移,提示TMEFF1可能在NB发生发展中发挥重要作用,可能成为NB靶向治疗的潜在新靶点。

关键词: 神经母细胞瘤, 表皮生长因子结构域和两个卵泡抑素样结构域的跨膜蛋白1(TMEFF1/tomoregulin1), 增殖, 迁移

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.

Key words: neuroblastoma, transmembrane protein with EGF-like and two follistatin-like domain 1(TMEFF1/tomoregulin 1), cell proliferation, cell migration

中图分类号:

R739.43

贾安娜, 郭金鑫, 张璇, 战世佳, 于永波, 郭永丽, 常艳. TMEFF1对神经母细胞瘤细胞系增殖的调控[J]. 基础医学与临床, 2024, 44(5): 637-644.

JIA Anna, GUO Jinxin, ZHANG Xuan, ZHAN Shijia, YU Yongbo, GUO Yongli, CHANG Yan. Regulation of TMEFF1 on proliferation of neuroblastoma cell lines[J]. Basic & Clinical Medicine, 2024, 44(5): 637-644.

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TMEFF1对神经母细胞瘤细胞系增殖的调控

Regulation of TMEFF1 on proliferation of neuroblastoma cell lines

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