TMEM97蛋白对神经母细胞瘤侵袭迁移的影响及机制研究

Effect and potential mechanism of TMEM97 on invasion and migration of neuroblastoma cells

目的探究跨膜蛋白(TMEM97蛋白)对神经母细胞瘤(neuroblastoma,NB)转移的影响及潜在作用机制。方法从GEO和TARGET数据库中下载NB的基因测序数据,分析TMEM97与预后的关联性。免疫组织化学检测患儿NB组织和对照组织中TMEM97的蛋白表达。共表达基因富集分析预测TMEM97生物学作用,并在NB细胞株SH-SY5Y中干扰或过表达TMEM97,Transwell和细胞划痕实验检测细胞迁移和侵袭的变化,Western blot检测转移相关蛋白表达水平。分析预测TMEM97与MYCN扩增的关联性并结合荧光素酶实验检测N-myc对TMEM97转录的影响。结果TMEM97 mRNA在高危型NB中的表达量显著高于低危型,并与NB患儿总生存率、无病生存率呈负相关。TMEM97蛋白表达水平在NB组织中显著高于对照组织。TMEM97主要与细胞黏附相关,可显著促进NB细胞的侵袭和迁移能力,伴随有LMO1、GPC2、Syntenin-1和MMP9转移相关蛋白的显著上调。在MYCN扩增组织中TMEM97 mRNA表达显著高于无扩增组织,并且N-myc可以直接靶向结合启动子促进TMEM97转录,干扰TMEM97的表达可显著抑制N-myc对侵袭和迁移的促进作用。结论TMEM97是一个不良预后因素,受转录因子N-myc直接激活转录并介导NB细胞侵袭和迁移的发生。

Objective To explore the effect of transmembrane protein 97(TMEM97)on the metastasis of neuroblastoma(NB)and to elucidate its potential mechanism.Methods The gene sequencing data of NB were downloaded from the databases of GEO and TARGET and then the association was examined between TMEM97 and prognosis.The expressions of TMEM97 in NB and control tissues of children were detected by immunohistochemistry.The biological effects of TMEM97 were predicted by co-expression gene enrichment analysis.TMEM97 was interfered or overexpressed in NB cell line SH-SY5Y.The changes of cell migration and invasion were detected by Transwell and cell scratch assay and the expression of metastasis related proteins was detected by Western blot.The correlation between TMEM97 and MYCN amplification was analyzed.The effect of N-myc on TMEM97 transcription was detected by luciferase assay.Results TMEM97 mRNA expression was significantly higher in high-risk group than that in low-risk group.It was negatively correlated with overall survival(OS)and event-free survival(EFS)of NB children.The expression level of TMEM97 protein was significantly higher in NB tissues than control tissues.TMEM97 was predominantly correlated with cell adhesion and significantly promoted the invasion and migration of NB cells,accompanied by marked up-regulations of metastasis-related proteins,including LMO1,GPC2,Syntenin-1 and MMP9.The expression of TMEM97 mRNA was significantly higher in MYCN-amplified tissues than non-amplified tissues.N-myc could directly target the promoter to promote the transcription of TMEM97.Interfering TMEM97 expression significantly blocked the promoting effects of N-myc on invasion and migration of NB cells.Conclusions As a poor prognostic factor,TMEM97 is directly activated by transcription factor N-myc to enhance invasion and migration of NB cells.