FAM83D调控BMI-1/RNF2/Snail复合物影响EMT在食管鳞癌细胞放射抵抗中的作用及机制研究

Radiotherapy is one of the main treatments for esophageal squamous cell carcinoma. Radioresistance is the bottleneck that impacts the curative effect of radiotherapy, but its exact mechanism has not been clarified at present. Our previous study had reported that FAM83D, as one of oncogene, highly expressed in esophageal squamous cell carcinoma, and played an important role in cell apoptosis, cloning efficiency and invasion induced by radiation. In addition, we also found that the expression of BMI-1, RNF2 and Snail was increased with the increase of FAM83D expression by exposure to irradiation and the three proteins interacted with each other. However, the relationship between FAM83D and radioresistance has not been proved at home and abroad. All the genes of BMI-1, RNF2 and Snail are key transcription factors of EMT and involved in the radioresistance of tumor. Based on the above studies, we hypothesized that FAM83D may induce EMT of esophageal squamous cell cancer via the BMI-1/RNF2/Snail complex, which may be an important mechanism of radioresistance. To verify this hypothesis, this project intends to observe the effect of FAM83D on EMT and radioresistance, and its mechanisms for esophageal squamous cell carcinoma through immunoprecipitation and Laser confocal technology in tissues, cells and animals levels. We will clarify the radioresistant molecular mechanism to provide a novel target for the reversal of radioresistance.

放射治疗是食管鳞癌的主要治疗手段，放射抵抗是制约放疗疗效的瓶颈，但机制尚不明确。我们前期发现FAM83D在食管鳞癌组织中高表达，参与放射诱导的食管鳞癌细胞的凋亡、克隆形成及侵袭过程，且X线作用后随着FAM83D表达的增加，BMI-1、RNF2和Snail表达也增加，且三者之间相互作用。但FAM83D与食管鳞癌细胞放射抵抗的关系，迄今国内外未见报道。BMI-1、RNF2和Snail是上皮间质转化（EMT）的关键转录因子，均参与放射抵抗的形成。我们推测FAM83D可能通过BMI-1/RNF2/Snail复合物诱导食管鳞癌EMT的发生，这成为放射抵抗的重要基础。为证实这一假说，本项目拟采用免疫共沉淀、激光共聚焦等技术，从组织-细胞-动物三个层次，观察FAM83D对食管鳞癌细胞EMT发生及放射抵抗的影响及具体调控机制。试图从分子水平阐明食管鳞癌细胞放射抵抗作用发生的机制，为逆转放射抵抗提供新靶点。

食管癌是危害人类健康的常见恶性肿瘤，其主要病理类型为鳞状细胞癌，放射治疗是食管鳞癌的主要治疗手段。研究发现肿瘤侵袭转移过程中上皮-间质转化（epithelial-mesenchymaltransition，EMT）是肿瘤细胞发生放疗抵抗的主要机制之一。FAM83D是一种癌基因，与肿瘤的侵袭与转移关系密切，可能与介导EMT有关。本实验旨在研究沉默食管鳞癌细胞中FAM83D的表达后，细胞增殖与侵袭能力及放射敏感性的变化，为探讨FAM83D对食管鳞癌细胞放射敏感性的调控作用及机理提供依据。.我们已经按预定计划开展了研究工作，分别从临床研究、体外实验和体内实验三个方面进行了相关研究。在临床标本中，发现与癌旁正常粘膜组织相比，食管鳞癌组织内FAM83D蛋白表达量明显增高，且FAM83D与Ecadherin蛋白表达具有显著的负相关关系，FAM83D与vimentin蛋白表达具有显著的正相关关系；食管鳞癌组织中FAM83D蛋白的表达水平与患者与肿瘤大小、淋巴结的转移及TNM分期关系密切；与FAM83D阴性表达者相比，阳性表达者的总生存率明显降低。在体外实验，构建了FAM83D基因shRNA慢病毒稳转食管癌细胞株，发现FAM83D shRNA组细胞在培养48 h、72 h后细胞的吸光度值明显低于对照组、空载组；FAM83D shRNA组细胞的放射敏感性、穿膜细胞数、细胞凋亡率均高于对照组、空载组；且与对照组、空载组相比，FAM83D shRNA组细胞中Bcl-2、Mcl-1蛋白的表达水平显著下降，Cleaved caspase-3蛋白的表达水平明显上升而p-Akt、p-GSK-3β、Snail蛋白的表达水平均显著下降。在体内实验，发现与空载组（NC）相比，FAM83D shRNA组裸鼠体内肿瘤生长速度减慢，瘤重量与瘤体积均明显降低，照射后效果更明显，表明采用RNA干扰技术降低肿瘤细胞中FAM83D蛋白表达水平，在动物体内实验证实确实发挥了放射增敏作用。.目前课题研究工作已经完成结题，基本达到预期研究目标。