肺癌是最常见的癌症和致死率最高的癌症之一，肺癌患者的死亡人数约占所有癌症死亡人数的四分之一。近年来，肺癌的发病率呈上升趋势，其中非小细胞肺癌（Non-small cell lung cancer, NSCLC）的发病率占80%~ 85%。由于早期症状不明显，约70%的NSCLC患者确诊时已为肺癌晚期，5年生存率仅为19%。因此，对NSCLC发生发展机制和治疗策略的研究十分重要。大量研究证据显示，乏氧微环境（Tumor microenvironment, TME）对肿瘤发展进程和抗肿瘤治疗效果具有深远的影响。乏氧是实体瘤的常见特征，瘤体组织氧浓度通常低于2%。与正常细胞在乏氧条件下进入凋亡不同，乏氧肿瘤细胞往往通过激活乏氧诱导因子-1（Hypoxic inducible factor-1, HIF-1）途径逃脱细胞死亡，该途径与血管生成、代谢、免疫逃逸以及肿瘤细胞生存和增殖有关。因此，乏氧被认为是肿瘤存活和进展的重要刺激物。而HIF-1通路的激活也被认为是导致癌细胞许多侵袭性生物学行为和患者临床预后差的原因之一。在这样的研究背景下，建立一套可实时监测NSCLC进程中组织乏氧状态，并可以有效评价抗肿瘤药物对肿瘤组织乏氧状态影响的系统就显得很有意义。 大量研究表明，HRE（Hypoxia response elements，HRE）作为HIF-1靶向的乏氧反应基因增强子调控元件，可以提供与HIF-1α亚基相互作用的DNA结合位点，进而促进乏氧反应基因启动子区域的转录起始复合物形成，启动靶基因转录，参与介导细胞对乏氧的反应。利用这一原理，将HRE与光学报告基因启动子相结合构建的载体在共转染细胞后，可以利用报告基因表达的变化监测细胞HIF-1乏氧响应活性的变化。

鉴于此，本项目以非小细胞肺癌为研究对象，选择乏氧响应元件可诱导的荧光素酶报告基因载体HRE-LUC，建立乏氧响应型A549细胞体系，可视化追踪非小细胞肺癌A549细胞和小鼠肿瘤模型中HIF-1α表达活性的改变，以及乏氧抑制剂和抗肿瘤药物对肿瘤乏氧的影响，为活体动物水平监测非小细胞肺癌的乏氧状态，评价乏氧相关抗肿瘤治疗效果提供有效思路。主要研究结果如下：

1. 构建了pGL2-HRE-Luc质粒与pGL3-RLuc质粒共转染的双荧光素酶报告基因瞬转体系（简称为HRE-LUC/RLUC-A549细胞系），并进行了细胞水平和分子水平实验验证，实验结果显示该体系可以用于监测A549细胞HIF-1α表达活性的变化。结果证明A549细胞中HRE-LUC可在乏氧诱导下表达，细胞中HIF-1α和VEGFa 的mRNA表达水平和蛋白表达水平同样受到乏氧诱导。这些实验结果显示HRE-LUC可以用于监测A549细胞中乏氧诱导下HIF-1α信号通路的响应变化。

2. 利用HRE-LUC/RLUC-A549双荧光素酶报告基因瞬转体系监测了HIF-1α抑制剂（PX-478和LW6）和抗肿瘤药物（褪黑素和芹菜素）对乏氧环境中A549细胞HIF-1α活化水平的影响，随后进行了分子细胞水平的验证实验。结果表明褪黑素与PX-478在非小细胞肺癌A549细胞系中可以抑制乏氧条件下HIF-1α和VEGFa的转录和翻译，能够显著的抑制肿瘤乏氧通路从而抑制肿瘤生长进程。

3. 建立了A549-HRE- LUC稳定转染细胞系，在此基础上建立非小细胞肺癌皮下A549-HRE-LUC肿瘤模型，并对褪黑素抗肿瘤治疗过程中的肿瘤乏氧微环境的变化进行了在体成像和连续监测。治疗过程中可以监测到肿瘤部位荧光信号逐渐低于对照组，治疗结束后给药组肿瘤体积均小于对照组，说明褪黑素不仅能够抑制肿瘤的生长进程，还可以抑制肿瘤的乏氧。

Accounting for about a quarter of all cancer deaths, lung cancer is one of the most deadly types of cancer. In recent years, the incidence of lung cancer has been on the rise, especially non-small cell lung cancer (NSCLC) which accounts for 80% to 85%. Because early symptoms are not obvious, about 70% of patients with NSCLC have advanced lung cancer when diagnosed, and the 5-year survival rate is only 19%. Therefore, it is very important to study the pathogenesis and treatment strategies of NSCLC. A large number of studies have shown that the tumor microenvironment (TME) has a profound impact on the tumor development process and the efficacy of anti-tumor therapy. Hypoxia is a common feature of solid tumors, and the tissue oxygen concentration is usually less than 2%. Unlike normal cells, which enter apoptosis under hypoxic conditions, hypoxic tumor cells often escape cell death by activating the hypoxic inducible factor-1 (HIF-1) pathway, which is associated with angiogenesis, metabolism, immune escape, and tumor cell survival and proliferation. Therefore, hypoxia is considered to be an important stimulus for tumor survival and progression. Activation of HIF-1 pathway is also thought to be one of the reasons for many aggressive biological behaviors of cancer cells and poor clinical outcomes of patients. Under such a research background, it is of great significance to establish a system that can monitor and evaluate the effect of anti-tumor drugs on tumor tissue hypoxia status in real time. A large number of studies have shown that hypoxia response elements (HRE), as a regulatory element of the enhancer of hypoxia response gene targeted by HIF-1, can provide a DNA binding site to interact with the HIF-1α subunit, thereby promoting the formation of transcription initiation complex in the promoter region of hypoxia response gene. Based on this principle, the vector constructed by combining HRE and optical reporter gene promoter can be used to monitor the change of HIF-1 hypoxia response activity.

 

In view of this, this project took NSCLC as the research object, selected the luciferase reporter gene vector HRE-LUC to established a hypoxia responsive A549 cell system, and tracked the changes of HIF-1α expression activity visually in A549 cells and mouse tumor models. So that can providing an effective way to monitor the effects of hypoxia inhibitors and antitumor drugs on tumor hypoxia at the level of living animals. The main findings are as follows:

 

1. Constructed a double luciferase reporter gene transient system co-transfected with pGL2-HRE-Luc plasmid and pGL3-RLuc plasmid and verified by cellular and molecular experiments. The results showed that the system could be used to monitor the change of HIF-1α expression activity in A549 cells. The results showed that the expression of HRE-LUC in A549 cells could be induced by hypoxia, the mRNA and protein expression levels of HIF-1α and VEGFa in A549 cells were also induced by hypoxia. These results suggest that HRE-LUC can be used to monitor changes in the response of HIF-1α signaling pathway in A549 cells induced by hypoxia.

 

2. Monitored the effects of HIF-1α inhibitors (PX-478 and LW6) and anti-tumor drugs (melatonin and apigenin) on HIF-1α activation in A549 cells in hypoxia environment by using the HRE-LUC/ RLUC-A549 double luciferase reporter gene transient system. The results showed that melatonin and PX-478 can inhibit the transcription and translation of HIF-1α and VEGFa in A549 cell line under hypoxia conditions.

 

3. Established a stable transfection cell line of A549-HRE-LUC to establish a subcutaneous A549-HRE-LUC tumor model, and monitored the changes of hypoxia microenvironment during melatonin anti-tumor therapy. In the course of treatment, the fluorescence signal at the tumor site was gradually lower than that in the control group, and the tumor volume in the administration group was smaller than that in the control group after treatment, indicating that melatonin could not only inhibit the growth process of the tumor, but also inhibit the hypoxia of the tumor.

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