PAX2激活lnc-HIF1A-1调控乏氧信号途径对食管癌放射敏感性的影响及机制研究

Hypoxia can induce radioresistance, which may affect radiotherapy efficacy and prognosis. However, regulatory mechanisms of hypoxia signaling pathway are not completely clear. Previously, we have reported that PAX2, a key transcriptional factor in development, is overexpressed in esophageal cancer tissues and plays a critical role in the progression of esophageal cancer. And PAX2 affected the radiosensitivity of esophageal cancer cells. By lncRNA microarray, we found that PAX2 could activate multiple lncRNAs, including lnc-HIF1A-1. And Inhibition of lnc-HIF1A-1 downregulated the expression of HIF-1α expression in esophageal epithelial cells. Therefore, we postulate that PAX2 is likely to modulate hypoxia signaling pathway and radiosensitivity by activating lnc-HIF1A-1 in esophageal epithelial cells. This project will utilize multiple molecular biological techniques at the cellular level and animal level to investigate: 1) The mechanism of PAX2 in transcriptional regulation of lnc-HIF1A-1 expression; 2) The effect of lnc-HIF1A-1 on the radiosensitivity of esophageal cancer cells and xenografts by modulating HIF-1α expression and hypoxia signaling pathway. This project will contribute to our understanding of PAX2/lnc-HIF1A-1 pathway in modulating the radiosensitivity of esophageal cancer cells, which can provide novel targets and therapeutic strategies in esophageal cancer radiosensitization.

乏氧可诱导食管癌放射抵抗，影响放疗疗效及预后，但乏氧信号途径的调控机制仍不完全清楚。项目组前期对食管癌中异常表达的发育调节转录因子PAX2的功能进行了研究，发现其能影响食管癌细胞的放射敏感性；运用基因芯片技术发现PAX2能调节数百个lncRNA的表达，其中lnc-HIF1A-1受到PAX2激活后能调节乏氧诱导因子HIF-1α的表达及其转录活性。据此我们推测：PAX2可能通过转录调控lnc-HIF1A-1的表达，调节乏氧信号途径，影响食管癌细胞的放射敏感性。本项目拟在前期工作基础上通过多种分子生物学手段探索：1）PAX2转录调控lnc-HIF1A-1的分子机制；2）lnc-HIF1A-1通过调控HIF-1α及乏氧信号途径，对食管癌细胞及移植瘤放射敏感性的影响。本研究有望揭示PAX2/lnc-HIF1A-1通过调控乏氧信号途径对食管癌放射敏感性的影响及机理，为食管癌放射增敏提供新的靶点与策略。

食管癌是全球死亡率最高的恶性肿瘤之一，食管癌细胞对电离辐射的抗拒性是影响其放疗疗效和预后的重要问题。前期研究发现Pax2过表达可影响食管癌放射敏感性，但其具体机制尚不明确。课题组研究发现，Pax2在ESCC中过表达，且其表达水平与肿瘤TNM分期、淋巴结转移、淋巴管浸润相关。下调Pax2的表达及过表达Pax2能够显著促进或抑制食管癌细胞的增殖和克隆形成能力。进一步通过Western blot分析发现，Pax2与P21的表达存在相关性，提示Pax2抑制食管癌细胞的生长。采用Boyden chamber、Western blot发现，Pax2促进转移调控因子MMP2和MMP9的表达水平，促进TE-1及Eca-109细胞的转移能力。mRNA基因芯片、CHIP、real-time PCR等方法显示，Pax2与IL-5的mRNA表达水平密切相关，Pax2是IL-5的上游调控因子，上调IL-5的表达水平可促进Eca-109细胞转移能力。从而，我们证实Pax2介导IL-5调控食管癌转移的机制。另一方面，我们采用生物信息学研究方法，发现lncRNA-HIFA-1在食管癌、肺鳞癌等多种肿瘤组织中差异表达，lnc-HIF1A-1在食管癌中影响的通路包括：TGFb Family Member，Cytokines等。最后，我们研究发现，过表达Pax2能增加食管癌细胞放射抗拒性，高通量的ArrayStar Human LncRNA microarray及real-timePCR显示，Pax2过表达显著上调lncRNA-HIFA-1的表达。此外，下调TE-1细胞中lncRNA-HIFA-1的表达能显著降低HIF-1α的表达水平。. 总之，本课题研究了PAX2激活lnc-HIF1A-1后对HIF-1α及乏氧信号通路进行调节，及其影响食管癌细胞放射敏感性的机制。本课题有望为食管癌的放射增敏提供新的靶点和策略。