蟾毒灵调控蛋氨酸代谢抑制胰腺癌干细胞侵袭转移的功能机制研究

Pancreatic cancer is highly metastatic, there is no effective treatment yet. Previous studies have shown that the pancreatic cancer stem cells play an important role in the invasion and metastasis of pancreatic cancer. Accoding to the “Ai Du” theory, Chinese traditional medicine assumes “Ai Du Running” contributes to tumor metastasis, and detoxifying herbs have the effect to inhibit tumor metastasis.Our previous study found that bufalin, an effective component of detoxifying herbs Toad cake, can inhibit the proliferation of pancreatic cancer stem cells. Bufalin not only can reduce the proportion of pancreatic cancer stem cell among the drug resistance of pancreatic cancer cell Miapaca2/GEM, but also can inhibit the metastasis of the subcutaneous transplanted tumor in nude mice in vivo. In the meantime, it is also found that the increased expression of methionine adenosyltransferase (MAT2A), a key enzymes of methionine metabolism, in pancreatic cancer stem cells.The expression of MAT2A gene is involved in the development of malignant tumo. Therefore, this project is to confirm that bufalin can inhibit the invasion and metastasis of pancreatic cancer stem cells, by simulating the methionine deficient environment. In our experiment, in order to explore the intervention mechanism of methionine metabolism of bufalin by regulating the expression of MAT2A and to clarify the possible mechanism of bufalin inhibiting invasion and metastasis of pancreatic cancer stem cells, the RNAi and MS-PCR technique will be used to verify the function of MAT2A of pancreatic cancer stem cells.In the further, the research will provide theoretical support for the clinical application of detoxifying herbs for the treatment of invasion and metastasis of pancreatic cancer and provide a solid theoretical basis for the ”Ai Du” theory from the molecular level.

胰腺癌具有高度的侵袭转移性，目前缺乏有效治疗手段。既往研究显示胰腺癌干细胞在胰腺癌侵袭转移过程中发挥了重要作用。中医“癌毒”学说认为癌毒流注是肿瘤转移的原因，解毒中药具有抑制肿瘤转移的功效。我们前期研究发现解毒中药蟾酥有效成分蟾毒灵具有抑制胰腺癌干细胞的特性，能降低胰腺癌耐药细胞Miapaca2/GEM中肿瘤干细胞的比例，还能抑制皮下移植瘤裸鼠的体内转移。同时发现蛋氨酸代谢关键酶甲硫氨酸腺苷转移酶（MAT2A）在胰腺癌干细胞中表达升高，而MAT2A基因表达广泛参与恶性肿瘤的发生发展。本课题将通过模拟蛋氨酸缺乏环境，验证蟾毒灵抑制胰腺癌干细胞侵袭转移能力，探索蟾毒灵通过调控MAT2A表达干预蛋氨酸代谢的功能机制，阐明蟾毒灵抑制胰腺癌干细胞侵袭转移的可能作用机制，为解毒中药抑制胰腺癌侵袭转移的临床应用提供理论支持，从分子水平为中医“癌毒”学说提供坚实的理论基础。

胰腺癌是预后极差的恶性肿瘤，目前手术仍然是胰腺癌患者治愈的唯一手段，但是由于侵袭转移能力极强，只有10-20%的患者有机会进行根治性手术切除。以吉西他滨为主的化疗方案，也很容易出现耐药，临床疗效欠佳。蟾毒灵是中药蟾酥的主要有效成分，我们的前期研究表明蟾毒灵可以抑制胰腺癌干细胞的侵袭转移，其作用机制仍需进一步研究。近年来的研究热点表明，蛋氨酸代谢参与了恶性肿瘤的侵袭转移，因此我们从蛋氨酸代谢相关基因表达来研究蟾毒灵对胰腺癌的抗肿瘤机制，以寻找潜在的药物作用靶点。本项目证实了蛋氨酸代谢关键酶MAT2A参与了抑制胰腺癌CSCs的侵袭转移和自我更新能力的分子作用机制，蛋氨酸剥夺和敲除MAT2A能够显著抑制胰腺癌干细胞STAT3蛋白的甲基化，通过靶向干扰蛋氨酸代谢对胰腺癌干细胞移植瘤裸鼠模型有抑制侵袭转移的作用。另外我们还发现，对蟾毒灵干预后的胰腺癌细胞株进行转录组测序及生物信息学分析，蟾毒灵在胰腺癌中的抗肿瘤作用与肿瘤免疫微环境相关。体外实验表明蟾毒灵干预和蛋氨酸限制可影响胰腺癌免疫微环境中髓源性细胞的积累。通过生物信息学分析预测了蟾毒灵作用于胰腺癌中的关键基因，比如IFIH1、BST2、SAMD9、DDX60等，揭示了中药蟾酥有效成分蟾毒灵于胰腺癌中的抗癌机制以及潜在的药物作用靶点，为可能的胰腺癌药物研发提供证据，同时将在发展和拓展中医药治疗肿瘤的领域中发挥显著的应用价值。