骨髓干细胞和肺癌干细胞差异表达miRNA调控Notch信号通路介导双黄升 白颗粒改善骨髓抑制治疗非小细胞肺癌

Lung cancer is by far the leading cause of cancer death among both men and women.Surgery is the treatment of choice for only 20% of NSCLC patients. Adjuvant chemotherapy regimens are engaged for stage I or II NSCLC. Chemotherapy drugs attack cancer cells by interfering with the different steps involved in cell division. But normal cells in bone marrow that divide frequently are also affected. When the bone marrow is suppressed, it is unable to supply the body with enough blood cells. Our previous studies have revealed that the traditional Chinese medicine shuang huang sheng Bai ke li (SHSB granule) are effective in treating NSCLC by simultaneous reliving chemotherapy-induced bone marrow suppression while inhibiting the growth of lung cancer cells, possibly via dual regulation of Notch-c-Myc-Cyclin D-CDK4/6 signaling pathway. To explore the underlying mechanisms of this dual regulation, we assessed the miRNA profiling of bone marrow stem cells and lung cancer stem cells in cyclophosphamide (CTX)-induced bone marrow suppression model of A549 xenograft nude mice. We discovered dramatically differentially expressed miRNAs in both bone marrow stem cells and lung cancer stem cells. On the basis of these observations, in this project, we will investigate the effect of those differentially expressed miRNAs on the expression of Notch-c-Myc-cyclin D-CDK4/6 signaling pathway molecules and the proliferation of bone marrow stem cells and lung cancer stem cells. We will apply a multidisciplinary approach to further identify the major anti-tumor component from SHSB granule, with the intent of developing new targets to optimize anti-lung cancer regimens.

中药制剂双黄升白颗粒能够通过促进骨髓干细胞增殖与分化改善非小细胞肺化疗所致的骨髓抑制，同时对肺癌细胞的生长具有一定的抑制作用，但其双向调控细胞增殖的分子机制尚不明确。我们的前期工作表明：双黄升白颗粒既能上调骨髓干细胞中众多Notch信号转导通路分子及其下游基因c-Myc、Cyclin D、CDK4/6的表达，又能下调该信号通路分子在肺癌组织的表达，从而以细胞特异性方式调控细胞周期以达到缓解骨髓抑制治疗小细胞肺癌的作用。这种细胞特异性作用可能与双黄升白颗粒差异调控骨髓干细胞与肺癌干细胞miRNA表达有关。本项目拟在前期工作基础上深入研究差异表达的miRNA对Notch信号通路及其下游靶标表达的影响，从而确认对该通路分子表达起主要调控作用的miRNA，进而筛选双黄升白颗粒中有效的单药成分，为今后进一步分离纯化缓解放化疗所致骨髓抑制和/或抑制肺癌细胞增殖的有效药物提供细胞与分子。

化疗获得性耐药是制约化疗效果的直接因素，因此，逆转化疗耐药或增强化疗敏感性至关重要。本课题旨在研究双黄升白颗粒及其两种主要生物活性成分黄芪甲苷(AS)和黄精皂苷(SRP)对骨髓干细胞损伤抑制以及对肺癌干细胞的化疗敏感性的影响。.由于预实验显示双黄升白颗粒的有效组分AS并不是通过调节肿瘤干细胞来增强A549细胞的DDP化疗敏感性，这与实验预期存在差异。因此，我们将实验的重点放在双黄升白颗粒及其有效组分对骨髓干细胞的影响研究上。.我们构建了肺癌异种移植联合环磷酰胺诱导的骨髓抑制小鼠模型。发现双黄升白颗粒及其有效组分AS和SRP均能显著逆转CTX诱导的白细胞和骨髓有核细胞的下降，且不影响肺癌的化疗效果。双黄升白颗粒能上调骨髓干细胞Notch信号通路分子及其下游靶标分子c-Myc、CyclinD、CDK4/6表达的影响。AS和SRP治疗可减轻CTX诱导的细胞损伤，抑制miR-142-3p的表达。miR-142-3p的过表达部分逆转了AS和SRP在CTX诱导的BMHSCs损伤中的治疗效果。进一步的机制探索发现HMGB1是miR-142-3p的靶基因，而mirR-142-3p负调控HMGB1的表达。AS和/或SRP可以治疗CTX化疗后的骨髓抑制症，其作用可能与其调节BMHSCs中miR-142-3p和HMGB1表达的作用有关。.本研究为研发以miRNA为靶标的新型抗肿瘤药和提纯双黄升白颗粒中的有效抗肿瘤成分提供契机，有助于促进中医药精髓研究的科学化与国际化。