基于PI3K/AKT信号通路探讨消痰解郁方抑制乳腺癌前病变的效应基础与分子机制

Studies have shown that PI3K/AKT signaling pathway is one of the key signal pathways for the development of breast cancer. The XiaotanJieyu Prescription created by the task team has an satisfactory clinical effect in treating breast lesions. Previous studies have confirmed: PI3K/AKT signaling pathway has been activated in the precancerous stage of breast cancer; XiaotanJieyu Prescription can induce apoptosis in breast cancer precancerous cell line MCF-10AT and have a clear therapeutic effect on rat model and the mechanism and impact is related with the Signal path. Based on the above study, following hypothesis is proposed: PI3K/AKT signaling pathway plays an important role in the precancerous stage of breast cancer. Suppose XiaotanJieyu Prescription activates PI3K/AKT by acting on some key receptors in precancerous cells of breast cancer Signaling pathway, thus affecting the occurrence and development of the disease. Therefore, in this study, to explore the action mechanism of XiaotanJieyu Prescription, gene chip technology, bioinformatics analysis and protein pathway inhibitors and other technologies are used in vitro experiments to screen out the key receptors in activating PI3K/AKT signaling pathway in breast precancerous lesions. And then the study observes and verifiesthe effect of XiaotanJieyu Prescription on breast precancerous lesions and its effect on key receptor-related molecules in vivo and vitro experiments.This study provides a theoretical basis for the early diagnosis and treatment of breast cancer.

PI3K/AKT信号通路是乳腺癌发生发展的关键信号通路之一。前期研究证实：PI3K/AKT信号通路在乳腺癌前病变阶段存在过度激活；课题组创制的“消痰解郁方”用于临床干预乳腺癌前病变疗效显著，基础研究证实其可显著诱导乳腺癌前病变细胞株MCF-10AT凋亡并改善乳腺癌前病变大鼠预后，作用机制可能与抑制过度激活的PI3K/AKT信号通路相关。基于此，本项目提出假说：PI3K/AKT信号通路的异常激活在乳腺癌前病变阶段具有关键作用，消痰解郁方可通过抑制PI3K/AKT信号通路干预乳腺癌前病变的发生发展。为此，本项目拟通过基因芯片、生物信息学分析及蛋白通路抑制剂等技术明确乳腺癌前病变中激活PI3K/AKT信号通路的上游受体，并通过细胞实验以及动物实验揭示消痰解郁方对乳腺癌前病变的具体作用机制，为乳腺癌早期诊断与治疗提供理论依据。

PI3K/AKT是乳腺癌发生发展的关键信号通路之一。前期研究证实：该通路在乳腺癌前病变阶段存在过度激活；课题组创制“消痰解郁方”用于临床干预乳腺癌前病变疗效显著，基础研究证实其可显著诱导乳腺癌前病变细胞株MCF-10AT凋亡，作用机制可能与抑制过度激活的PI3K/AKT信号通路相关。为此本课题提出假说：消痰解郁方通过作用于乳腺癌前病变细胞上的某个差异基因激活P13K-AKT信号通路，从而影响乳腺癌前病变的发生发展。基于此，本项目展开以下研究：1.明确PI3K/AKT信号通路相关差异基因；2.消痰解郁方干预MCF-10AT细胞的机制；3.差异基因通过PI3K/AKT信号通路干预MCF-10AT的研究；4.消痰解郁方对乳腺癌前病变大鼠模型的干预作用。结果发现：1、通过生信分析筛选出PI3K/AKT信号通路相关差异基因WT1、SATB1及PI3K下游基因MME， WIF1，KCNE4，KCNK15等5个差异基因。2.用CCK8、Q-PCR、细胞周期、凋亡、迁移及侵袭等方法并与MCF-10A（正常人乳腺细胞）相比较筛选出显著差异基因WT1，并通过敲低WT1基因进行了细胞实验验证。结果表明：WT1基因能有效抑制MCF-10AT细胞的增殖，促进细胞的凋亡。3.消痰解郁方通过降低WT1差异基因抑制MCF-10AT细胞的增殖、促进细胞的凋亡，与对照组有显著差异（P＜0.01）。4.消痰解郁方对大鼠乳腺癌前病变模型有明确治疗作用。病理结果显示：与模型对照组比较，TAM组、消痰解郁方低中高剂量剂量组大鼠乳腺组织癌变发生率降低(P<0.05)。Western Blot与Q-PCR结果显示：与模型组比较，消痰解郁方各剂量组、TAM组可以下调乳腺癌前病变模型大鼠乳腺组织PI3K及p-Akt蛋白的表达，上调PTEN表达，其机制与影响PI3K-AKT信号传导通路、诱导细胞凋亡有关。综上，本研究明确了PI3K-AKT信号通路与乳腺癌前病变的关系；筛选出通路上显著差异基因WT1，并从体内、外实验研究验证消痰解郁方对乳腺癌前病变的干预作用；从PI3K-AKT信号通路揭示该方有效治疗乳腺癌前病变、阻断乳腺癌发生发展的作用机理，为 “消痰解郁方”在乳腺癌的防治中的指导作用提供现代分子生物学依据。