背景:由于肿瘤的异质性,鉴定与胃癌(GC)诊断和治疗相关的生物标志物尚未取得重大进展。组织学分类和遗传相关性研究中涉及的基因对于开发适当的GC治疗至关重要。
方法:进行体外和体内慢病毒shRNA文库筛选。免疫组化证实GC患者肿瘤组织中Synaptotagmin(SYT11)的表达,并分析其表达水平与患者生存率的相关性。进行磷酸激酶阵列以检测JunN末端激酶(JNK)磷酸化。SYT11JNK,通过蛋白质印迹和免疫沉淀测定证实了MKK7复合物的形成。我们研究了SYT11对GC增殖和转移的影响,实时细胞图像分析,粘附试验,侵袭试验,球状体形成,小鼠异种移植试验,和肝转移。
结果:SYT11在527例GC患者的转录组分析中在GC的茎样分子亚型中高表达。此外,SYT11是组织学分类的弥漫性GC的潜在预后生物标志物。SYT11作为支架蛋白,结合在JNK1磷酸化中起作用的MKK7和JNK1信号分子。反过来,JNK激活导致信号级联,导致cJun激活和下游基因血管生成素样2(ANGPTL2)的表达,血小板反应蛋白4(THBS4),Vimentin,和交界粘附分子3(JAM3),在上皮间质转化(EMT)中发挥作用。SNU484细胞感染SYT11shRNA(shSYT11)显示球体形成减少,小鼠肿瘤形成,和肝转移,提示SYT11的致癌作用。此外,SYT11反义寡核苷酸(ASO)在我们的小鼠异种移植模型中显示出抗肿瘤活性,并在SNU484和MKN1细胞中具有抗增殖作用。
结论:SYT11可能是弥漫型GC患者的潜在治疗靶点和预后生物标志物,SYT11-ASO可用于茎样分子亚型弥漫性GC的治疗剂开发。
BACKGROUND: Identifying biomarkers related to the diagnosis and treatment of gastric cancer (GC) has not made significant progress due to the heterogeneity of tumors. Genes involved in histological classification and genetic correlation studies are essential to develop an appropriate treatment for GC.
METHODS: In vitro and in vivo lentiviral shRNA library screening was performed. The expression of Synaptotagmin (SYT11) in the tumor tissues of patients with GC was confirmed by performing Immunohistochemistry, and the correlation between the expression level and the patient\'s survival rate was analyzed. Phospho-kinase array was performed to detect Jun N-terminal kinase (JNK) phosphorylation.
SYT11, JNK, and MKK7 complex formation was confirmed by western blot and immunoprecipitation assays. We studied the effects of SYT11 on GC proliferation and metastasis, real-time cell image analysis, adhesion assay, invasion assay, spheroid formation, mouse xenograft assay, and liver metastasis.
RESULTS: SYT11 is highly expressed in the stem-like molecular subtype of GC in transcriptome analysis of 527 patients with GC. Moreover,
SYT11 is a potential prognostic biomarker for histologically classified diffuse-type GC.
SYT11 functions as a scaffold protein, binding both MKK7 and JNK1 signaling molecules that play a role in JNK1 phosphorylation. In turn, JNK activation leads to a signaling cascade resulting in cJun activation and expression of downstream genes angiopoietin-like 2 (ANGPTL2), thrombospondin 4 (THBS4), Vimentin, and junctional adhesion molecule 3 (JAM3), which play a role in epithelial-mesenchymal transition (EMT). SNU484 cells infected with SYT11 shRNA (shSYT11) exhibited reduced spheroid formation, mouse tumor formation, and liver metastasis, suggesting a pro-oncogenic role of
SYT11. Furthermore, SYT11-antisense oligonucleotide (ASO) displayed antitumor activity in our mouse xenograft model and was conferred an anti-proliferative effect in SNU484 and MKN1 cells.
CONCLUSIONS: SYT11 could be a potential therapeutic target as well as a prognostic biomarker in patients with diffuse-type GC, and SYT11-ASO could be used in therapeutic agent development for stem-like molecular subtype diffuse GC.