Supplementary Material for: Interference with NTSR1 Expression Exerts an Anti-Invasion Effect via the Jun/miR-494/SOCS6 Axis of Glioblastoma Cells

<b><i>Background/Aims:</i></b> Glioblastoma is the most common and aggressive brain tumor and carries a poor prognosis. Previously, we found that neurotensin receptor 1 (NTSR1) contributes to glioma progression, but the underlying mechanisms of NTSR1 in glioblastoma invasion remain to be clarified. The aim of this study was to investigate the molecular mechanisms of NTSR1 in glioblastoma invasion. <b><i>Methods:</i></b> Cell migration and invasion were evaluated using wound-healing and transwell assays. Cell proliferation was detected using CCK-8. The expression of NTSR1, Jun, and suppressor of cytokine signaling 6 (SOCS6) was detected using western blotting. The expression of miR-494 was detected by Quantitative real-time PCR. Chromatin immunoprecipitation assay was performed to examine the interaction between Jun and miR-494 promoter. Dual-luciferase reporter assay and western blotting were performed to identify the direct regulation of SOCS6 by miR-494. An orthotopic xenograft mouse model was conducted to assess tumor growth and invasion. <b><i>Results:</i></b> NTSR1 knockdown attenuated the invasion of glioblastoma cells. Jun was positively regulated by NTSR1, which promoted miR-494 expression through binding to miR-494 promoter. SOCS6 was confirmed as a direct target of miR-494, thus, NTSR1-induced miR-494 upregulation resulted in SOCS6 downregulation. Both miR-494 and SOCS6 were involved in the NTSR1-induced invasion of glioblastoma cells. <i>In vivo</i>, tumor invasion and growth were inhibited by NTSR1 knockdown, but were restored with miR-494 overexpression. <b><i>Conclusion:</i></b> NTSR1 knockdown inhibited glioblastoma invasion via the Jun/miR-494/SOCS6 axis.

**<i>背景与研究目的：</i>** 胶质母细胞瘤（Glioblastoma）是最常见且恶性程度最高的脑肿瘤，预后极差。本团队既往研究表明，神经降压素受体1（neurotensin receptor 1, NTSR1）可促进胶质瘤进展，但NTSR1在胶质母细胞瘤侵袭中的具体分子机制仍有待阐明。本研究旨在探讨NTSR1调控胶质母细胞瘤侵袭的分子机制。
**<i>方法：</i>** 采用划痕愈合实验（wound-healing assay）与Transwell实验（Transwell assay）检测细胞迁移与侵袭能力；采用CCK-8法检测细胞增殖活性；采用蛋白质印迹法（Western Blotting）检测NTSR1、Jun及细胞因子信号抑制因子6（suppressor of cytokine signaling 6, SOCS6）的蛋白表达水平；采用定量实时PCR（Quantitative real-time PCR）检测miR-494的表达水平；采用染色质免疫沉淀实验（Chromatin immunoprecipitation assay）分析Jun与miR-494启动子的相互作用；采用双荧光素酶报告基因实验（Dual-luciferase reporter assay）结合蛋白质印迹法验证miR-494对SOCS6的直接调控作用；构建原位异种移植小鼠模型（orthotopic xenograft mouse model）评估肿瘤生长与侵袭情况。
**<i>结果：</i>** 敲低NTSR1可显著减弱胶质母细胞瘤细胞的侵袭能力。NTSR1可正向调控Jun的表达，Jun通过结合miR-494启动子区域促进其转录。实验证实SOCS6是miR-494的直接靶基因，因此NTSR1介导的miR-494上调可导致SOCS6表达下调。miR-494与SOCS6均参与了NTSR1诱导的胶质母细胞瘤细胞侵袭过程。*体内*实验显示，敲低NTSR1可抑制肿瘤生长与侵袭，但该效应可被miR-494过表达所逆转。
**<i>结论：</i>** 敲低NTSR1可通过调控Jun/miR-494/SOCS6信号轴抑制胶质母细胞瘤的侵袭。