热应激状态下热休克因子1依赖的肝癌细胞外泌体miRNA调节巨噬细胞促进射频消融术后复发机制研究

Since early 1990,radiofrequency ablation (RFA) has been widely used to treat liver tumor and developed rapidly. For small hepatocellular carcinoma, (HCC), randomized control trials indicated that RFA yielded similar long-term survival as compared to resection. However, the residual cancer rate and recurrence rate remain high after RFA, Even after curative RFA, residual cancer could be detected in 3%-52% of patients and recurrence rate has been reported up to 53%-81%. It is widely reported that tumor associated microenvironment plays the key role in the process of hepatocellular carcinoma (HCC) initiation and metastasis. However, the role of tumor associated microenvironment on the recurrece of HCC after RFA are fewly reported. Increasing evidences suggest that exosomes have emerged as a novel mode of intercellular communication. In our previously study, we found and reported that high peritumoral macrophage density and heat shock factor 1 (HSF1), which correlated with large tumor size, presence of intrahepatic metastasis, and high TNM stage, were independent prognostic factors for both overall survival and time to recurrence. Notably, serum exosome in HCC patients was found increased sharply after RFA therapy. .. Exosomes have been shown to act as shuttles between cells by transmitting signals. In addition to lipids,nucleic acids and proteins, double-stranded DNA, mRNA and miRNA have also been detected in exosomes. In vitro study showed that under heat stress, the exsome increased significantly with high HSP90 expression, which can be inhibited when the expression of HSF1 was knocked down. In vitro and in vivo study both suggested that the exosome secreted by HCC cells under heat stress have more opportunities to contact and fusion with macrophage. .，and the cytokine secretion, such as TGFβ, increased sharply. In pilot study, the expression of HSF1 in peritumoral liver tissue of nude mice with orthotopic xenograft HCC after RFA therapy. The level of HSP90 in nude serum increased sharply. It is very interested that the macrophage in peritumoral liver tissue increased obviously after RFA treated. It is reported HSF1 facilitates malignant transformation, cancer cell survival and proliferation in model systems. Based on our data and reported studies, we presented that HSF1 and downstream key molecules are the important elemants for the reconstruct of microenvironment, which in favor of HCC growth and metastasis. In further study, we will adopted Mass Spectrometry laser confocal fluorescence microscopy, flow cytometry, chromatin immunoprecipitation, co-immunoprecipitation, to verified the mechanism why HSF1 increases exosome secretion and tageted to macrophage, the role of HSPs in the exosome targeted to macrophage, which kind of miRNA play the key role of exosome to regulate tumor suppressor gene of macrophage.With nude mice orthotopic xenograft HCC receiving RFA treatment, we explore the role of inhibitor of HSPs, the inhibitor of miRNA, and macrophage specific cytotoxic drug, on the recurrence after RFA treatment, exploring the possible methods to decrease the recurrence rate and increase overall survival.

射频消融在小肝癌治疗中已取得接近手术切除的总体生存效果，但较高的局部复发率迫切需要深入探索并适当干预。肿瘤和微环境间有效通信对残癌发生发展作用巨大。外泌体作为细胞间通信有效载体参与了肿瘤进展。前期研究发现热应激状态下肝癌细胞分泌的表达有热休克蛋白（HSPs）的外泌体数量增加，其所包含的miRNA表达谱亦变化显著，且有靶向癌旁巨噬细胞（Mφ）特性。体内实验提示热应激下分泌的外泌体能显著增加肝癌细胞的肝内成瘤和肺转移。沉默热休克因子1（HSF1）表达后热应激下外泌体数量减少，表达的HSPs显著降低，对Mφ的调节能力明显减弱。下一步将着力探讨HSF1调节外泌体数量和功能的机制，HSPs在外泌体靶向Mφ中的作用和相应受体鉴定，确认外泌体中哪种miRNA抑制Mφ中的抑癌基因表达，进而导致细胞因子谱表达向有利于肝癌发展的方向转变。寻找针对关键环节给予联合抑制减少射频消融术后复发的方法。

射频消融在小肝癌的治疗中已取得接近手术切除的总体生存效果，但较高的局部复发率值得探索并给予适当干预。肿瘤和微环境间有效通信和相互作用对残癌发生发展作用巨大。前期研究提示热应激条件下肝癌细胞增殖和侵袭潜能增加，HSF1表达显著升高，并伴有HSP90的增加。通过对外泌体的检测发现热应激状态下肝癌细胞外泌体中HSP90、PD-L1增加是GOLM1依赖的。Co-IP提示GOLM1和HSP90以及PD-L1相互作用，激光共聚焦证明GOLM1和外泌体标志物CD63以及HSP90和PD-L1空间共定位。Cre-LoxP外泌体体外追踪系统观察到肝癌细胞外泌体进入到巨噬细胞。人肝癌组织标本检测发现肝癌组织中GOLM1表达和巨噬细胞分布显著正相关。将肝癌细胞外泌体加入到巨噬细胞培养基中，HSP90和PD-L1表达增加，当用GW4869阻断外泌体分泌，发现能显著减少巨噬细胞中HSP90和PD-L1增加的幅度。同时发现巨噬细胞表型从M1向M2转化。EMT的多个关键标志物E-cadheirn、Snail、Slug以及Vimetin检测结果提示上皮向间质转化。.不同的肝癌细胞系中热应激状态下均检测到外泌体miR-302c -3p表达水平降低。人肝癌组中标本检测发现和癌旁肝组织相比，肝癌组织中miR-302C-3p表达显著降低，并在有复发的肝癌组织中进一步降低。KM plotter数据库分析结果提示miR-302C-3p表达与患者预后良好相关。通过对预测所得miR-302C-3p潜在靶基因和VEGF上调的基因取交集，得到5个miR-302C-3p的候选靶基因：ABCA1、PTPRD、CREBRF、MEX3A、MYO1D，肝癌和癌旁肝组织检测以及生存分析提示只有MEX3A高表达与肝癌患者预后不良相关，这提示MEX3A或为miR-302C-3p抑制血管生成的靶基因。进一步通过对肝癌组织中MEX3A和miR-302C-3p的检测我们发现，二者显著负相关。在体外实验中给与miR-302C-3p干预后肝癌细胞中的MEX3A表达在不同时间点均显著降低。将HUVECs分别转染miR-302c-3p mimics或inhibitors以及相应的NC来验证其对HUVECs血管形成能力，迁移能力和单层细胞通透性的影响证明miR-302c-3p具有抑制血管生成和调节血管通透性的作用。