基于microRNA差异表达调控PI3K/AKT信号通路对益气健脾化积方逆转胃癌化疗耐药的机制研究

YiQi JianPi Huaji formula is the experience foumula of clinical curative effects in treatment of gastric carcinoma.It is composed of 12 kinds of Chinese traditional medicines, many clinical experiments show its effect on gastric cancer patients after chemotherapy was repeated, our previous study shows Yiqi Jianpi Huaji formula combined 5-fluorouracil could obviously inhibit the drug resistant, increasing apoptosis cells rate, and significantly increase the sensitivity of drug resistant cells to 5-fluorouracil. The project on the basis of previous research, proposed by the following method to discuss Yiqi Jianpi Huaji formula reversal of gastric cancer cells to chemotherapy, and clarify its mechanism, This work provide reliable theoretical basis for improving clinical efficacy..1 Establishment of drug resistant to gastric cell lines ,Study the effect of Chinese medicine to the drug resistance of gastric cancer cells. and to explore the molecular mechanism through PI3K/AKT signaling pathway based on the difference of expression of miRNA.2 to further explore the Yiqi Jianpi Huaji formula in the reversal of secondary drug resistance in nude mice model of drug resistant gastric cell line，study the side effect on drug resistant gastric cancer mice xenograft model.

益气健脾化积方是由12种中药组成的临床治疗胃癌有确切疗效的经验方，为多年临床实践经验所得，多项临床实验显示它对临床胃癌反复化疗后病人效果明显，我们前期的研究显示益气健脾化积方与氟尿嘧啶联合应用能明显抑制胃癌耐药细胞的增殖，增加耐药细胞的凋亡率，并显著增加耐药细胞对氟尿嘧啶的敏感性。本项目拟在前期研究的基础上，通过以下方法进一步探讨益气健脾化积方逆转胃癌细胞对化疗药的耐药性，并阐明其机制，为临床提高胃癌化疗疗效提供可靠的理论依据。①建立胃癌耐药细胞系，研究益气健脾化积方对胃癌耐药细胞的影响，并探讨其诱导microRNA差异表达调控PI3K/AKT/PTEN分子作用机制；②通过耐药细胞系荧光胃癌原位裸鼠模型，进一步探讨益气健脾化积方在逆转继发耐药中的作用机制。

胃癌是全世界死亡率较高的恶性肿瘤之一。健脾益气化积方是临床长期实践治疗胃癌证实有效的经验方，尤其和化疗药联用疗效显著，具有益气健脾，行气和胃，软坚散结的功效。我们前期预实验结果发现益气健脾化积方可以通过调控多药耐药相关基因的表达逆转胃癌细胞的多药耐药性，使胃癌细胞对氟尿嘧啶的敏感性显著上升。故我们考虑益气健脾化积方与胃癌细胞的多药耐药可能密切相关。关于多药耐药的多重机制来说，目前miRNA及基因治疗成为新的热点。我们使用液相色谱-质谱联用技术测定益气健脾化积方有效活性成分，确定中药复方的无细胞毒剂量。检测益气健脾化积方对耐药相关miRNA表达的影响。Western blot法检测筛选出益气健脾化积方对细胞信号通路的影响及相关机制。LC-MS结果显示益气健脾化积方里含有迷迭香酸、莪术醇、莪术二醇、白术内酯III、橙皮苷、芍药苷、甘草苷、阿魏酸、琥珀酸等9种有效活性成分。我们采用CCK-8法和流式细胞法结果提示低剂量的益气健脾化积方抑制SGC7901/5-Fu细胞增殖，低剂量的益气健脾化积方联合5-Fu能诱导SGC7901/ 5-Fu细胞阻滞在G0/G1期，导致细胞凋亡增加。Affymetrix miRNA基因芯片结果显示SGC7901/5-Fu胃癌细胞中47个miRNA显著上调，而55个miRNA显著下调，我们发现miR-6785-5p、miR-642a-3p、miR-4516在胃癌耐药细胞中表达上调，而miR-21-3p表达下调，使用中药益气健脾化积方后发现这4个miRNA的表达反向改变，其中miR-6785-5p、miR-642a-3p、miR-4516表达明显下调，而miR-21-3p表达明显上调。Western blot实验提示SGC7901/5-Fu耐药细胞比敏感细胞PTEN表达下调，p-AKT表达上调，中药作用后反向改变，提示其通过PI3K/AKT/PTEN通路发挥逆转耐药作用。加入miR-642a-3p mimic能够抑制中药益气健脾化积方对PTEN和p-AKT表达调控，CCK-8实验和Western blot实验验证了上述结果，与前期的预期结果一致。