FOXD3/FBP1功能轴激活糖酵解代谢促进胃癌放疗抵抗的研究

Neoadjuvant radiotherapy (NCRT) can reduce local tumor stage, increase the radical resection rate and decrease locoregional recurrences. However, only minority of patients can benefit from NCRT. It is urgent to find the potential biomarker and mechanism involved in radio-sensitivity. Glycolysis is one of the main mechanisms regulating radioresistance. We established a radio-resistant gastric cell line and found FOXD3 was one of the most upregulated genes by RNA-sequence analysis. In our previous study, FOXD3 has been validated as a novel prognosis predictor in gastric cancer. Functional study further indicated that FOXD3 involved in radiosensitivity and glycolysis regulation, and RNA-sequence analysis after silencing FOXD3 further indicated that FBP1 is a potential target of FOXD3. On the basis of the previous research, the present study aimed to research on molecular, cellular, animal levels, as well as using tissue specimens, to further investigate the following scientific problems: ①to clarify the transcriptional relationship between FOXD3 and FBP1; ②to investigate whether FOXD3 regulates radioresistance in gastric cancer through activating glycolysis by repressing FBP1 transcription; ③to intervene the expression of FOXD3 and FBP1 in vitro to provide a new theoretical and experimental basis for reversing radioresistance of gastric cancer in clinics.

新辅助放疗可使胃癌局部降期、增加手术切除率、降低术后复发率，但仅部分患者对放疗敏感，并且放疗也可带来不良反应，因此寻找胃癌放疗抵抗相关的标志物和机制尤其重要。研究提示多种机制参与了放疗耐受的诱导，糖酵解途径的激活是其中重要的机制之一。我们通过筛选培养获得胃癌放疗抵抗细胞株，并用RNA测序发现FOXD3在胃癌放疗抵抗的细胞株中显著上调，我们既往研究发现FOXD3是一个新型的与胃癌预后密切相关的转录因子。前期功能学实验发现FOXD3与胃癌的放射敏感性和糖酵解活性密切相关，沉默FOXD3后RNA测序发现FBP1是FOXD3的一个潜在的靶基因。本课题拟在前期的基础上从分子、细胞、动物和临床标本多个层面进一步明确：①FOXD3对FBP1的转录调控关系；②探究FOXD3是否通过转录调控FBP1激活糖酵解代谢促进胃癌的放疗抵抗；③体外干预相关基因的表达,为临床逆转胃癌放疗抵抗提供新的靶点和依据。

放疗是胃癌的重要治疗手段之一，放疗抵抗常会导致胃癌治疗失败，并且无效的放疗还会增加患者的痛苦。我们前期通过对胃癌放疗抵抗的标本进行转录组测序，发现FOXD3在胃癌放疗抵抗组织中显著上调，高FOXD3表达预示着胃癌根治术后预后不良。随后我们在胃癌细胞株中沉默FOXD3表达后进行转录组测序，利用GSEA软件分析这些差异基因的 GO 功能分类和 KEGG 通路富集发现，FOXD3的敲低伴随着KEGG糖酵解通路的显著抑制（FDR<0.01）。进一步功能实验显示沉默FOXD3表达后可以显著降低胃癌细胞葡萄糖消耗、乳酸生成、ATP生成，Seahorse实验显示沉默FOXD3后显著降低ECAR活性、增加OCR活性，并降低胃癌细胞DNA损伤修复和放疗抵抗能力。过表达FOXD3有相反的作用。在沉默FOXD3的测序结果中我们发现了变化最显著的糖代谢酶FBP1，在胃癌细胞中沉默FOXD3表达后FBP1蛋白和转录水平出现明显的上升，反之过表达FOXD3后FBP1在蛋白和转录水平出现显著降低。生信分析显示在FBP1启动子区有多个FOX转录因子家族结合位点，双荧光素酶报告基团实验显示FOXD3可以剂量依赖的抑制FBP1启动子活性，ChIP实验提示FOXD3可以与FBP1启动子区-377—852区域发生特异性结合。综上所述，FOXD3是影响胃癌放疗抵抗的一个重要调节因子，FOXD3通过激活有氧糖酵解参与了胃癌的放疗抵抗，FBP1是FOXD3调节胃癌放疗抵抗的一个重要的下游靶基因，FOXD3通过转录因子FBP1启动子活性发挥作用。