外泌体miRNAs在肝癌复发转移中的作用及作用机制研究

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide, especially in china. In the past 30 years, the long-term survival after radical treatment of HCC had no distinct improvement, mainly because of the high recurrence/metastasis rate with no effective early warning and intervention measures. Until now, the molecular mechanism of HCC recurrence/metastasis is still not fully elucidated. Abundant evidences showed that exosomes miRNAs participate in the regulation of a series of important process in the development of tumor, and can be used for tumor diagnosis and treatment, suggesting important roles of exosomes miRNAs in development of HCC. Previously, we successfully purified exosomes form HCCLM3 (with high metastatic potential) cell medium, and confirmed that HCCLM3 exosomes can enhance the migration/invasion of SMMC-77121 and Bel-7402 cells with low metastatic ability. By comparing the miRNAs profiles of HCCLM3 and SMMC-7721 exosomes, we screened and identified 43 metastasis related exosomes miRNAs. Among them, miR-301b and miR-598 had aberrant expression, miR-301b was significantly overexpressed in HCCLM3 exosomes and high expression of miR-598 was found in SMMC-7721 exosomes. Further study showed that miR-301b inhibition and miR-598 overexpression can inhibit the invasion and metastasis of HCCLM3 cells. These data indicate the critical roles of exosomes miRNAs in recurrence and metastasis of HCC. However, up to now, the roles of exosomes miRNAs in the recurrence and metastasis of HCC and its molecular mechanism are not yet reported in the literature. Based on the basis of former research, we first screen and identify exosomes miRNAs with high diagnostic efficiency for early warning and diagnosis recurrence and metastasis of hepatocellular carcinoma, by using patients’ serum and miRNAs array analysis. Then, through lentivirus-mediated miRNAs overexpression or knockdown, the biological function of selected exosomes miRNAs were investigated by using growth curves, colony formation assay, wound healing assay, Transwell invasion assay and in situ xenograft nude mouse model. Combination bioinformatics, antibody array and dual-Luciferase reporter assay, we screen and identify the key downstream target of exosomes miRNAs, and elucidate their molecular mechanism. Finally, with in situ xenograft nude mouse model, the potential application value of exosomes mediated miRNAs targeted therapy was evaluated. In conclusion, the present study will elucidate the diagnostic efficiency, biological function and molecular mechanism of exosomes miRNAs in regulating the recrurrence/metastasis of HCC, which could provide novel idea and experimental fundation for developing new strategies for early diagnosis, prevention and treatment of recurrence/metastasis.

复发转移是制约肝癌整体治疗水平提高的关键,目前尚缺乏有效的预警和干预措施. 外泌体miRNAs可调控肿瘤发生发展中的一系列重要进程.我们前期证实HCCLM3细胞外泌体可增强低侵袭转移肝癌细胞的迁徙运动能力;我们通过miRNA芯片筛选出侵袭转移相关外泌体miRNAs,其中miR-301b和miR-598可体外调控肝癌细胞的侵袭转移.目前外泌体miRNAs在肝癌复发转移方面尚无相关文献报道. 本项目拟利用肝癌患者血清和miRNAs芯片筛选可用于诊断预警肝癌复发转移的外泌体miRNAs;体内体外深入研究外泌体miR-301b、miR-598在肝癌发生发展中的作用及其分子机制;利用裸鼠原位肝癌模型,探讨外泌体介导的miRNAs靶向治疗在肝癌复发转移干预中的潜在应用价值. 本研究旨在阐明外泌体miRNAs在肝癌复发转移过程中的作用及其分子机制,为临床预警、防治HCC复发转移提供新的研究思路和实验基础

肝癌是严重威胁我国人民身体健康的常见恶性肿瘤。术后复发转移是目前制约肝癌整体治疗水平提高的关键,尚缺乏有效的预警和干预措施。外泌体miRNAs可调控肿瘤发生发展中的一系列重要进程。本研究中，我们采用基因芯片技术检测复发和无复发患者血清外泌体miRNAs表达谱，通过大样本验证，筛选获得12个和肝癌术后复发转移显著相关的外泌体miRNAs。其中miR-3170在复发肝癌患者血清表达值显著高于慢性肝病患者，良性肝脏肿瘤患者和正常人群。ROC曲线分析发现miR-3170预警肝癌复发的准确性和AFP相当，但其敏感性显著优于AFP；miR-3170和AFP联合预警肝癌复发可显著提高预警的敏感性至88.9%。上述结果显示miR-3170可作为肝癌术后复发转移预警标志物。此外，miR-425-5p在肝癌中呈高表达，其表达水平与肝癌临床病理特征和预后显著相关；体内外研究证实，miR-425-5p可促进肝癌复发转移；进一步研究证实，miR-425-5p可通过靶向SCAI调控ITGB1-Fak/Src-RhoA/CDC42、PTEN-AKT和TIMP2-MMP2/MMP9信号通路，促进肝癌细胞EMT进而促进肝癌的侵袭转移。此研究有助于阐明肝癌术后复发转移分子机理，为肝癌术后复发转移防治提供有效治疗靶点。