lncRNA ATB/miR-429调控自噬在食管鳞癌发生发展中的作用及分子机制

Over the past decade, more and more attention has been paid to the study on the regulation of long noncoding RNAs (lncRNAs) on tumor proliferation and metastasis. Our study previously reported that lncRNA ATB (lnc-ATB) promoted the invasion and migration of esophageal squamous cell carcinoma (ESCC). In the previous study, we found that lnc-ATB enhanced the proliferation and autophagy of ESCC by targeting and inhibiting miR-429. On the basis of the bioinformatic analyses and preliminary experimental results, we suggested that lnc-ATB/miR-429 promotes proliferation and autophagy of ERCC by regulating SESN3 and mTOR and AMPK signaling pathways. To testify our hypothesis, RNA immunoprecipitation (RIP), luciferase reporter assay, protein mass spectrometry assay, co-immunoprecipitation (CO-IP), in vivo tumor formation and metastasis assays are conducted (1) to elucidate the molecular mechanism of lnc-ATB as a competing endogenous RNA (ceRNA) in miR-429 regulation through competing with SESN3 mRNA; (2) to provide sight into the precise mechanism of lnc-ATB/miR-429/SESN3 axis promotes ESCC autophagy via inhibiting mTORC1 signaling and activating AMPK signaling. Our study not only expands the comprehensive understanding of complicated functions and molecular mechanisms of lnc-ATB in ESCC progression, but also provide a novel potential biomarker for ESCC prognosis and a gene target for new therapies.

近年来长链非编码RNA（lncRNAs）在肿瘤增殖和转移中的调控研究受到越来越多重视。我们课题组首次发现lncRNA ATB (lnc-ATB)能促进食管鳞癌组织（ESCC）的侵袭转移。前期研究发现lnc-ATB通过结合并抑制miR-429表达促进ESCC增殖和自噬。根据生物信息学分析及预实验结果，我们猜测lnc-ATB/miR-429通过靶基因SESN3及mTOR和AMPK通路促进ESCC自噬。因此，本项目拟通过RNA免疫沉淀、荧光素酶报告系统、质谱分析、免疫共沉淀和体内动物实验等技术来验证：①lnc-ATB作为ceRNA与SESN3竞争性结合miR-429的调控机制；②阐明lnc-ATB/miR-429/SESN3途径通过调控mTOR和AMPK通路促进ESCC细胞自噬的分子机制。本项目旨在拓展对lnc-ATB功能全面认识，更为lnc-ATB成为ESCC预后判断和治疗新靶点提供实验依据。

食管癌发病率在世界范围内居实体肿瘤的第 7 位。我国食管癌新发病人占全球50%以上，其中90%的为食管鳞状细胞癌。因此，深入研究食管鳞癌发生发展的分子机制，以及靶向药物的开发具有重要的临床价值。我们课题组首次发现lncRNA ATB(lnc-ATB)、SNHG22能促进食管鳞癌组织（ESCC）的侵袭转移。前期研究发现lnc-ATB通过结合并抑制miR-429表达促进ESCC增殖和自噬。因此，我们设计lnc-ATB/miR-429通过靶基因SESN3及mTOR和AMPK通路促进ESCC自噬。主要研究内容包括：1.观察 lnc-ATB 、SNHG22和 miR-429 在 ESCC 组织中表达和临床意义；2.lnc-ATB 、SNHG22和 miR-429 对 ESCC 细胞增殖和自噬的影响；3.lnc-ATB、SNHG22 作为 ceRNA 抑制 miR-429，进而调控 SESN3 表达的分子机制；4.lnc-ATB、SNHG22/miR-429/SESN3 途径通过调控 mTORC1 和 AMPK 通路促进 ESCC细胞自噬。本项目通过RNA免疫沉淀、荧光素酶报告系统、质谱分析、免疫共沉淀和体内动物实验等技术验证了：①lnc-ATB、SNHG22作为ceRNA与SESN3竞争性结合miR-429的调控机制；②阐明lnc-ATB、SNHG22/miR-429/SESN3途径通过调控mTOR和AMPK通路促进ESCC细胞自噬的分子机制。结果发现：1. lnc-ATB 和SNHG22有负相关的高表达细胞株；2.抑制SNHG22后对食管鳞状细胞癌细胞进展产生相关影响；细胞自噬标记物LC3B蛋白荧光表达水平明显增强。3. miR-429能够与SNHG22结合而抑制食管鳞状细胞癌细胞。4. 动物实验验证lnc-ATB、SNHG22/miR-429/SESN3信号通路存在于食管鳞状细胞癌细胞。5.病理组织标本ESCC肿瘤组织中lnc-ATB增高、SESN3的表达明显降低。6. lnc-ATB、SNHG22/miR-429/ SESN3途径可通过mTOR/AMPK通路调控ESCC细胞自噬，主要参与了mTOR、MAPK、P53和泛素化蛋白降解等信号通路。通过本研究拓展对lnc-ATB功能全面认识，更为lnc-ATB、SNHG22成为ESCC预后判断和治疗新靶点提供重要实验依据.