HNF1α作用于HMGB1调控食管鳞癌细胞放射敏感性的机制与应用研究

Radiotherapy is one of the important treatments for esophageal squamous cell carcinoma. Radiosensitivity determines the curative effect of radiotherapy, and most of the experts agreed with that the principal reasons for poor sensitivity are related to DNA damage repair. HNF1α is a kind of transcription factor with homologous structure domain, involved in DNA damage repair process. In our preliminary study from immunohistochemical, we detected that HNF1α staining was mainly localized in the nuclei, highly expressed in esophageal squamous cell carcinoma, however, its expression was weak or absent in a normal esophagus epithelium and Barrett’s metaplasia. In addition, the expression of HNF1α at mRNA and protein levels was obviously higher in the esophageal squamous cell carcinoma cell lines than in the normal human esophageal epithelial cell line. The proliferation activity of tumor cells was significantly inhibited after knockdown the HNF1α gene, while the radiosensitivity of the cells was markedly enhanced and the apoptosis was effectively induced. The trend was more obvious after X-ray irradiation. Our previous study also had reported that the expression of HNF1α was enhanced after radiation, followed by down-regulation of HMGB1 protein in the nucleus and up-regulation of it in the cell plasma. The release of HMGB1 from the nucleus to the cytoplasm can be effectively inhibited by adding the HMGB1 transposition inhibitor ethyl pyruvate (EP) into the cell culture medium. The number of γH2AX spots in the nucleus also decreased significantly, accompanied by the decrease of HMGB1 release into cytoplasm, which made us hypothesize that the expression of HNF1α in esophageal squamous cell carcinoma may regulate the transposition of HMGB1 from nuclear to plasma, and then induce DNA damage repair to play an important role of radioresistance. However, the relationship between HNF1α and radiosensitivity has not been seen at home and abroad. In order to verify this hypothesis, we intends to explore the effect of HNF1α on DNA damage repair and signal transduction pathway for esophageal squamous cell carcinoma by advanced technologies such as immunoprecipitation and laser confocal in tissues, cells, and animals experiments. This project will reveal the regulatory mechanism of HNF1α for radiosensitivity to esophageal squamous cell carcinoma from a new perspective, which will become the key of systematic research, and provide theoretical basis and a new therapeutic target for improving the clinical effect of radiotherapy.

放射治疗是食管鳞癌的重要治疗手段，放射敏感性决定着放疗疗效，敏感性差的主要原因大多认为与DNA损伤修复有关。HNF1α是一类含同源结构域的转录因子，参与DNA损伤修复。在前期免疫组化结果中，我们发现HNF1α主要在食管组织的细胞核表达，并且食管鳞癌中HNF1α蛋白的表达水平明显增加，而在癌旁正常食管粘膜组织及Barrett化生组织中不表达或弱表达。此外，在食管鳞癌细胞中HNF1α在mRNA和蛋白的表达水平均明显高于人正常食管上皮细胞，我们采用siRNA技术敲低HNF1α基因后，肿瘤细胞的增殖活性显著下降，而细胞放射敏感性明显增强，并有效诱导了细胞凋亡的发生，X线照射后这种趋势更加明显；研究还发现照射后HNF1α表达水平增强，且伴随着HMGB1蛋白在细胞核中的表达下降而细胞浆中的表达升高，在细胞培养液中加入HMGB1转位抑制剂丙酮酸乙酯（EP）后，能够有效抑制HMGB1由细胞核向细胞浆释放。随着HMGB1向细胞浆释放的减少，γH2AX在细胞核内斑点数量也明显降低，由此推测食管鳞癌细胞中HNF1α的表达可能通过调控HMGB1蛋白的核浆转位，进而诱导DNA损伤修复从而导致放射抵抗发生。然而，HNF1α与放射敏感性的关系，迄今国内外尚未见报道。为证实我们的研究假说，拟采用免疫共沉淀、激光共聚焦等先进技术从组织-细胞-动物三个层面上，观察食管鳞癌细胞中HNF1α对照射后DNA损伤修复及信号转导通路的影响，从新的视角揭示HNF1α对细胞放射敏感性的调控机制，并成为系统性研究的关键切入点，为提高放疗疗效提供理论依据和新靶点。

食管癌是常见的消化道恶性肿瘤，大部分食管癌患者就诊时为中晚期已失去手术治疗机会，需行放射治疗,而放疗后5年局部未控或复发率高达 50%～70%，治疗效果不尽人意。放射敏感性决定着放疗疗效，敏感性差的主要原因大多认为与DNA损伤修复有关。HNF1α是一类含同源结构域的转录因子，参与DNA损伤修复。我们通过质谱分析、免疫共沉淀、激光共聚焦等先进实验技术，围绕HNF1α-HMGB1进行了深入研究，发现并验证了HNF1α、HMGB1可作为提高放疗疗效的分子靶标,血浆外泌体HMGB1可以作为预测食管鳞癌放射敏感性的肿瘤标志物,并从HNF1α/PI3K/AKT信号轴方向阐明食管癌放射抵抗的分子机制，此外，射线照射诱导的肿瘤源性外泌体HMGB1通过激活PI3K/AKT/FOXO3A通路招募了γH2AX蛋白，进而启动食管鳞癌细胞射线照射导致DNA损伤后的修复过程，最终诱导放射抵抗产生。本研究从新的视角揭示HNF1α、外泌体HMGB1对食管鳞癌放射敏感性的调控机制，为提高放疗疗效提供理论依据和新靶点。