miRNA-409-5p靶向TGF-β1介导电针重激活原位肝癌裸鼠免疫抑制NK细胞的机制研究

With the emergence of "Wei Zexi scandal", the tumor immunotherapy is widely challenged with its primary obstacle of expensive medical expenses. There is important practical significance on how to exert the advantages of "simple, effective and economic" in acupuncture, also as an alternative medicine supplement to immune regulation and a reasonable intervention in survival with tumor in intermediate and terminal stage cancer.However, there are reports on acupuncture to improve immune function of cancer patients via acupuncture at present, lacking of clear explanation of its mechanism of action.The preliminary research confirmed that electroacupuncture had the effect of delaying the growth tendency of tumor, and obtained miRNA-409-5p, which was aimed at inhibiting the tumor-suppressing factor TGF-β1.Immunosuppression is an important reason for the low function of anti-tumor immune response. TGF-β1 can down-regulate the activity of key effector, NK cells which block the formation of immunosuppression microenvironment. In this study, we use HepG2 orthotropic hepatocellular carcinoma nude mice under the guidance of healthy qi and exterior-interior meridian acupoints selection theory, and by means of the key link of slowing tumor growth tendency via electroacupuncture, inhibition of TGF-β1 on miRNA-409-5p signal pathway, exploring the mechanism of immunosuppressive condition of NK cells associated with electroacupuncture to explain the scientific connotation of positive regulation of tumor immune function by acupuncture.

“魏则西事件”的出现，使肿瘤免疫治疗受到多方质疑，昂贵的医疗费用是其推广应用的首要障碍。如何发挥针灸“简验效廉”的优势，将其作为免疫调节的补充替代手段，合理介入中晚期癌症患者“带瘤生存”的过程中，有重要的实践意义；目前已有针灸改善肿瘤患者免疫功能的报道，但其作用机制尚不明确。课题组前期证实电针能延缓肿瘤生长趋势，效应的产生与基因芯片筛选得到的miRNA-409-5p，靶向抑制TGF-β1的分子通路关系密切。免疫抑制是抗肿瘤免疫应答功能低下的重要原因，TGF-β1能下调阻止免疫抑制微环境形成的关键效应物NK细胞的活性。故本研究在中医正气学说和表里经配穴理论指导下，使用HepG2原位肝癌裸鼠为载体，以电针发挥缓瘤生长作用的关键环节“miRNA-409-5p靶向抑制TGF-β1”信号途径为基础，探索电针重激活NK细胞相关肿瘤免疫抑制状态的转导机制，深入阐释针灸正向调控肿瘤免疫功能的科学内涵。

中晚期肝癌病人死亡率高，预后较差，给患者带来身体和经济上的双重负担。肿瘤免疫疗法因其较为高昂的费用，临床推广仍存在阻碍。因此，如何发挥针灸“简验效廉”的优势，作为免疫调节的补充替代手段，合理介入“带瘤生存”的过程中，有重要的实践意义。.本项目对研究目标进行了修正，继续以中医正气学说和表里经配穴理论为指导，使用HepG2肝癌裸鼠为载体，在明确电针延缓荷瘤生长趋势作用的基础上，重新筛选出miR-486-5p作为效应调控的中心环节，并验证了以“miR-486-5p为始动因子，靶向SRSF3”信号途径在电针缓瘤生长下游效应中的转导机制。.结果表明：1.通过小动物活体成像系统及荷瘤体积每日监测，与模型组相比，电针组裸鼠肿瘤的生长趋势更为平缓，差异有统计学意义（P<0.05）。2.对组间荷瘤组织进行测序、差异基因RT-qPCR验证等操作，选择了均为上调表达的3个miRNAs进行深入探索。3.miR-486-5p在HepG2及Huh7两种肝癌细胞系中的细胞功能学实验均表现出正向调控作用，其表达水平与测序结果相一致。4.运用Luciferase实验验证了miR-486-5p与靶基因SRSF3的相关性。5.在更多的肝癌细胞系中测定了miR-486-5p的表达量，并通过PCNA及KI-67的Western blot检测确定了其在增殖表型中的研究价值。6.在以HepG2为主的肝癌细胞系中进行了miR-486-5p靶向调控SRSF3的细胞功能学实验，证实miR-486-5p转染后能下调SRSF3表达。.结论：电针可能是通过miR-486-5p靶向SRSF3对荷瘤增殖表型进行调控，以有效发挥缓瘤生长作用。但仍需通过进一步的动物实验，明确电针作用机制，以验证该科学假说。