基于Bcl-2/CytC-lncRNA调控网络探讨消癌解毒方抗肿瘤作用机制研究

Xiaoai Jiedu Prescription anti-cancer medication developed by traditional Chinese medicine master Prof. Zhou Zhongying, and its efficacy has been validated through long-term clinical trial. Previous studies have summarized the experience and diagnostic methods of differentiation tumor from the angle of “cancer toxin”, and proved the existence of cancer toxin and the anti-cancer efficacy and wide application prospects of Xiaoai Jiedu Recipe. Currently, there is an urgent need to perform systematic research of this medication. Based on previous research, this project takes Bcl-2/CytC apoptotic pathway as a target, approaches the problem from lcnRNA high-throughput genetic sequencing related gene expression, and takes human hepatoma SMMC-7721 nude mouse tumor tissue and human hepatoma SMMC-7721 cell line as the research target. The aim is to observe the effect of Xiaoai Jiedu Prescription on apoptosis-related genes, especially caspase-3, bcl-2, bax, P53, etc. Meanwhile, this project looks for more apoptosis-related genes as the target, obtains the lcnRNA gene sequence related with cancer toxin and Xiaoai Jiedu Prescription, and selects apoptosis-related differentiated lcnRNA for qRT-PCR validation, bioinformatics analysis, lcnRNA transfection, qRT-PCR and western-blot in order to validate key genes. These methods can uncover the anti-cancer mechanism of Xiaoai Jiedu Prescription from lncRNA regulatory network on macro- and molecular level and provide new ideas for developing TCM anti-tumor medication.

消癌解毒方是国医大师周仲瑛教授癌毒理论指导下, 临床长期应用有效的抗癌方。前期研究总结了从癌毒辨治肿瘤的经验及诊疗方法，证实了癌毒的客观存在及该方的抗癌疗效，是一种具有较好应用前景的抗癌方，急需对其进行深入系统的研究。本项目以前期研究为基础，以Bcl-2/CytC凋亡信号通路为靶点，以lcnRNA高通量测序检测相关基因表达入手，以人肝癌SMMC-7721裸鼠瘤体组织以及人肝癌SMMC-7721细胞株为研究载体，观察消癌解毒方干预后凋亡相关基因的影响，重点关注caspase-3, bcl-2、bax、cytc等，同时寻找更多凋亡相关基因作为靶标，获得癌毒与消癌解毒方相关的lnRNA基因序列，挑选凋亡相关差异lcnRNA进行qRT-PCR验证及生物信息学分析，lcnRNA转染、qRT-PCR及western-blot对关键基因进行验证，在整体及分子水平从lncRNA调控网络揭示该方抗肿瘤机制。

本课题以前期研究为基础，选择Bcl-2/CytC凋亡信号转导通路作为治疗肿瘤的新靶点，运用细胞生物学、分子生物学技术，通过体内构建C57BL/6J小鼠H22-Luc移植瘤模型，检测各组抑瘤率、HE染色、Tunel染色检测病理及凋亡，ELISA法检测炎症相关因子、western blot法检测关键蛋白，LncRNA基因芯片检测LncRNA表达，应用GO和KEGG生物信息学分析，找出消癌解毒方抗癌机制相关的LncRNA。选取差异表达LncRNA进行实时定量PCR检测，选择Srrd进行干扰及过表达。体外选用人肝癌HepG2细胞、人肝癌SMMC-7721细胞进行实验。CCK-8检测细胞抑制率，光镜进行形态学观察，流式细胞仪检测细胞凋亡。选用人肝癌HepG2细胞进行转染，实时定量PCR检测干扰及过表达后的情况，荧光显微镜观察转染效率，CCK-8检测干扰及过表达后细胞抑制率，流式细胞仪检测干扰及过表达后细胞凋亡，划痕试验检测干扰及过表达后细胞迁移。.研究结果表明：消癌解毒方对C57BL/6J小鼠H22-Luc移植瘤模型的抑瘤作用明显，同时对体外培养的肿瘤细胞也有明显的抑制作用。消癌解毒方对移植瘤小鼠外周血IFN-γ、IL-1β、IL-2、IL-6、IL-8、IL-10、IL-12、IL-16、NF-kB、VEGF、TNF-α、TGF-β表达以及对凋亡相关蛋白Bcl-2、Caspase-3、Caspase-9、Jak-2、STAT3、ATF-2的表达都有影响，对瘤体差异LncRNA表达谱有调控，差异表达主要涉及细胞凋亡、肿瘤生长、炎症、氧化应激等方面。消癌解毒方能诱导肿瘤细胞凋亡，干扰及过表达差异LncRNA srrd对细胞增殖、凋亡、迁移都有影响。消癌解毒方能调控Bcl-2/CytC凋亡信号通路，同时还能影响炎症相关指标发挥抗癌作用。.本研究为消癌解毒方的临床应用提供微观实验依据，同时也为进一步探讨消癌解毒方的分子机制，丰富中医“癌毒”理论的内涵打下基础。