FEZF1-AS1作为胃癌新型诊断标志物及其经由Sirt1/TSC2/mTOR介导的自噬调控网络在胃癌进程中的机制研究

Recent studies show that long non-coding RNAs (lncRNAs) can participate in tumor progression by regulating autophagy, but the specific regulatory mechanism is still unclear. We previously screened differentially expressed molecules FEZF1-AS1 by tissue microarray, and showed high expressionof of FEZF1-AS1 in tissues and serum of gastric cancer patients, and significantly promoted the proliferation of gastric cancer cells. Bioinformatics software predicted that there was binding site between Sirt1 and FEZF1-AS1. After FEZF1-AS1 interference, the expression of Sirt1 and TSC2 were increased, while mTOR phosphorylation were decreased. Atg1,13 and other autophagy-related genes were increased. We speculate that high expression of FEZF1-AS1 in gastric cancer inhibits mTOR signaling pathway by TSC2, inhibits the occurrence of autophagy, promotes the proliferation and metastasis of tumor cells, and accelerates the malignant progression of gastric cancer. The project also establishes real-time PCR to detect of the expression of FEZF1-AS1 to explore the value of plasma FEZF1-AS1 as a new marker in the early diagnosis and prognosis of gastric cancer. This study will explore the molecular mechanism of gastric cancer and provide new ideas in clinical diagnosis and treatment.

新近研究表明长链非编码RNA（lncRNA）可通过调控细胞自噬参与肿瘤进展，但其具体调控机制尚不明确。我们前期通过组织芯片筛选出差异表达的分子 FEZF1-AS1，胃癌患者组织和血清中呈高表达，且显著促进胃癌细胞增殖能力。通过生物信息学软件预测发现沉默信息调节因子1（Sirt1）与FEZF1-AS1存在结合位点，FEZF1-AS1干扰后，Sirt1和TSC2表达升高，mTOR磷酸化水平降低，而Atg1、13等自噬相关基因表达上调，我们推测，胃癌中高丰度的FEZF1-AS1通过结合sirt1，抑制TSC2对mTOR信号通路调控作用，抑制细胞自噬的发生，促进肿瘤细胞的增殖与转移，加快胃癌的恶性进展。本项目还将建立FEZF1-AS1的Real time-PCR检测方法，探讨血浆FEZF1-AS1作为新的标志物在胃癌早期诊断和预后判断中的价值。本研究将为胃癌分子机制探讨及临床诊疗及提供新思路。

新近研究表明长链非编码RNA（lncRNA）可通过调控细胞自噬参与肿瘤进展，但其具体调控机制尚不明确。前期通过组织芯片筛选出差异表达的分子 FEZF1-AS1。我们拟探讨FEZF1-AS1对胃癌（GC）诊断及预后的临床应用价值及其促进胃癌恶性进展的作用机制。在本项目的资助下，首先建立SYBR GreenⅠ荧光定量PCR检测FEZF1-AS1的方法，并对方法学进行评价。初步分析了GC患者血清标本（89例术前病例、配对的63例术后、25例胃息肉病例及73例健康体检者）及50对胃癌组织及癌旁组织中的表达水平，分析其与临床病理参数间的相关性，结果发现GC初诊患者FEZF1-AS1表达量显著高于胃息肉组及健康体检者组，而术后血清表达水平显著下降，且高表达的FEZF1-AS1与肿瘤大小、TNM分期、淋巴结转移及远处转移有关。FEZF1-AS1受试者工作曲线下面积（AUC）高于CEA、CA199，且三者联合诊断灵敏度最高，提示FEZF1-AS1作为新的标志物在胃癌早期诊断和预后判断中的潜在价值。细胞功能学实验发现，过表达FEZF1-AS1后，细胞增殖、侵袭迁移能力上升，S期细胞增多而凋亡细胞减少；FEZF1-AS1干扰后则结果相反。FISH及核浆分离实验证实FEZF1-AS1主要定位于GC细胞核中；生物信息学软件预测FEZF1-AS1与sirt1蛋白（sirt1）存在互补结合区域，且qRT-PCR及Western blot证实FEZF1-AS1干扰后，sirt1 mRNA和蛋白水平均升高。荧光显微镜下观察发现，FEZF1-AS1敲除后细胞自噬小体形成增多；qRT-PCR及Western blot发现，FEZF1-AS1干扰后自噬相关基因（ATG）mRNA及蛋白水平发生变化。自噬抑制剂3-MA和FEZF1-AS1干扰载体共转时，细胞的增殖加快、可逆转FEZF1-AS1干扰引起的S期细胞减少及凋亡促进现象。本研究提示FEZF1-AS1可能作为GC诊断、治疗及预后的新型标志物，有望为胃癌的基因诊断与发病机制的研究提供新的策略和思路。