基于外泌体介导miR-23a-3p调控EMT探讨健脾化瘀方抑制肝癌转移机制

The character of hepatocellular carcinoma is high incidence and poor prognosis in China. Jianpi Huayu Decoction is one of the most important proved recipe on HCC. Our previous study revealed that Jianpi Huayu Decoction inhibited the epithelial mesenchymal transition and metastasis in HCC via regulating the Smad3/Smad7 cascade. The preliminary data showed that exosomal miR-23a-3p regulating the Smads pathway and promoting the epithelial-mesenchymal transition in HCC, and Jianpi Huayu Decoction inhibited the expression of exosomal miR-23a-3p. But the underlying mechanism was unknown. We presumed that the exosomal miR-23a-3p regulating the Smads pathway and promoting the epithelial-mesenchymal transition is one of the most important steps of metastasis in HCC, and Jianpi Huayu Decoction inhibiting the procedure was one of the key anticancer mechanisms. So in present study we will explore the regulation of exosomal miR-23a-3p on the transcription of target gene, using the HCC cell lines, nude mice orthotopic models and clinical cases. We will conduct the extraction, identification, transfer and tracking of exosome, high-throughput sequencing of exosomal miRNAs and analysis of target mRNA, the impacts of exosomal miR-23a-3p on the cells and nude mice models, validation in clinical cases. We will explore the anti-tumous effect and the underlying mechanisms of Jianpi Huayu Decoction by studying the inhibition of the exosomal miR-23a-3p that regulated the Smads pathway and promoted the epithelial-mesenchymal transition in HCC. The results will provide more details of the underlying mechanism on the reculation of EMT and metastasis in HCC, reveal the key mechanism of Jianpi Huayu Decoction inhibiting metastasis of HCC, enrich the theoretical basis for antitumor effects of Chinese medicine, and provide a novel therapeutic target.

肝癌发病率高，易侵袭转移和复发。健脾化瘀方是中医治疗肝癌重要验方。前期发现健脾化瘀方调控Smad3/Samd7平衡抑制肝癌上皮间质转化（EMT）和转移。预实验显示miR-23a-3p与肝癌EMT相关，健脾化瘀方抑制其表达，但机制不详。假设：外泌体介导miR-23a-3p调控Smads通路促EMT是肝癌转移重要步骤；健脾化瘀方抑制该调控过程是抗癌关键机制。课题拟以外泌体分泌和miR-23a-3p调控靶基因翻译为切入点，借助肝癌细胞、动物模型和临床病例，按照外泌体提取、鉴定和跟踪，外泌体miRNAs高通量测序和靶基因分析、外泌体miR-23a-3p对细胞和动物模型的影响、临床验证的思路和方法，深入研究健脾化瘀方对外泌体miR-23a-3p调控Smads通路促EMT和转移的拮抗作用和机制。所得结果将完善肝癌EMT和转移调控理论，揭示健脾化瘀方抗癌重要机制，丰富中药抗癌理论基础并提供治疗新靶点。

肝癌发病率高，易侵袭转移和复发。健脾化瘀方是中医治疗肝癌重要验方。前期发现健脾化瘀方调控Smad3/Samd7平衡抑制肝癌上皮间质转化(EMT)和转移。假设:外泌体介导miR-23a-3p调控Smads 通路促EMT是肝癌转移重要步骤；健脾化瘀方抑制该调控过程是抗癌关键机制。课题以外泌体分泌和miR-23a-3p调控靶基因翻译为切入点，借助肝癌细胞、动物模型和临床病例，按照外泌体提取、鉴定和跟踪，外泌体miRNAs高通量测序和靶基因分析、外泌体miR-23a-3p对细胞和动物模型的影响、临床验证的思路和方法，深入研究健脾化瘀方对外泌体miR-23a-3p调控Smads通路促EMT和转移的拮抗作用和机制。研究结果表明，外泌体miR-23a-3p调控Smads通路促EMT和肝癌转移过程。细胞实验研究健脾化瘀方对外泌体和肝癌细胞功能的影响。研究表明健脾化瘀方抑制肝癌细胞间外泌体信号，抑制了肝癌细胞增殖、EMT和侵袭迁移。进一步进行细胞及临床研究健脾化瘀方对miR-23a-3p/Smad通路和肝癌细胞功能的影响，结果表明，健脾化瘀方抑制miR-23a-3p/Smad通路及肝癌细胞增殖、EMT和侵袭迁移。动物模型研究健脾化瘀方对肝癌的作用和机制。研究结果表明，健脾化瘀方可抑制裸鼠皮下移植瘤的生长。健脾化瘀方抑制肝癌裸鼠模型的血清miR-23a-3p水平。进一步研究健脾化瘀方对裸鼠肝癌模型肿瘤组织的EMT和Smad信号通路标志物表达的影响，研究结果表明，健脾化瘀方抑制肝癌裸鼠模型肿瘤组织EMT和Smad通路。所得结果将完善肝癌EMT和转移调控理论，揭示健脾化瘀方抗癌重要机制，丰富中药抗癌理论基础并提供治疗新靶点。