以GSK-3β为靶标的miRNAs调控肝细胞癌转移侵袭的分子机制及中药的干预作用

The metastasis and invasion of hepatocellular carcinoma is one of the key and difficult points of the prevention and treatment of liver cancer. Our preliminary study showed that the expression level of Biejiajian Pills could effectively regulate and control the expression of key protein molecule GSK-3β in the Wnt signaling pathway so as to achieve the purpose of anti-liver metastasis and invasion. Based on the good work of Biejiajian Pills in anti-liver cancer research and the research progress which miRNAs are closely related with tumor, miRNAs differential expression and regulation of GSK-3β is taken as the breakthrough point in this study. The different metastatic potential hepatoma cell lines and nude mice human hepatocellular carcinoma models are taken as the research object. We plan to high throughput screen out the diffiential expressed miRNAs using miRNAs chip, plasmid transfection,luciferase,gene silencing and PCR technology,et al and study the regulatory mechanism of miRNAs closely related with the invasion and metastasis of liver cancer targeting on GSK-3β. On this basis, we plan to observe the effect of Biejiajian Pills on the differential expression on miRNAs and making further discussion on the molecular mechanism of anti-liver cancer of Biejiajian Pills.Our study can be of important clinical significance to screen the effective diagnosis, treatment and prognosis indexes of hepatocellular carcinoma and to find new therapeutic targets as well as having great contribution in further elucidating the scientific connotation of the multi-channel and multi-level anti liver cancer of Biejiajian Pills which provides full theoretical basis for clinical application of Biejiajian Pills.

肝细胞癌的转移侵袭是肝癌防治的重点与难点之一。我们前期研究表明鳖甲煎丸能有效调控wnt信号通路中的关键蛋白分子GSK-3β的表达水平而达到抗肝癌转移侵袭的目的。基于鳖甲煎丸抗肝癌研究的良好工作基础，以及miRNAs与肿瘤密切相关的研究进展，本研究拟以miRNAs差异表达调控GSK-3β为切入点，以不同转移潜能肝癌细胞株及裸鼠人肝细胞癌模型为研究对象，采用miRNAs芯片、质粒转染、荧光素酶、基因沉默、PCR等技术，首先高通量筛选与肝癌侵袭转移密切相关的差异表达的miRNAs，并探讨其对GSK-3β的调控作用，在此基础上进一步观察鳖甲煎丸对miRNAs差异表达的影响，深入研究鳖甲煎丸抗肝癌转移侵袭的分子机理。本研究对筛选肝细胞癌的诊断、治疗及预后的有效指标和寻找新的治疗靶点具有重要的临床意义。同时，有助于进一步诠释鳖甲煎丸多途径、多层次抗肝癌的科学内涵，为该药的临床应用提供充分的理论依据。

本研究以GSK-3β为靶标，以miRNAs调控肝细胞癌转移侵袭为切入点，以鳖甲煎丸抗肝细胞癌的分子机制为研究目标，以不同转移潜能肝癌细胞株及人肝癌细胞裸鼠移植瘤模型为研究对象，采用miRNAs芯片、质粒转染、基因沉默、PCR等技术，首先高通量筛选与肝癌侵袭转移密切相关的差异表达的miRNAs，并探讨其对 GSK-3β的调控作用，在此基础上进一步观察鳖甲煎丸对 miRNAs 差异表达的影响，深入研究鳖甲煎丸抗肝癌转移侵袭的分子机理。.miRNAs高通量芯片筛选结果显示，不同转移潜能肝癌细胞株的miRNAs表达谱存在差异，通过qPCR验证及靶基因预测分析，我们选定miR-215-5p和miR-30a-3p作进一步研究。我们采用双荧光素酶报告基因实验验证GSK-3β为miR-215-5p的直接靶基因，进一步通过质粒转染或慢病毒转染，分别使miR-215-5p和miR-30a-3p在肝癌细胞中过表达或抑制表达后，CCK8、Transwell实验、划痕实验和裸鼠肝癌模型实验等结果表明，miR-215-5p可通过抑制其靶基因GSK-3β的表达促进肝细胞癌的增殖、转移和侵袭，而miR-30a-3p则可通过抑制其靶基因COX-2的表达来起到抗肝细胞癌的作用。.在此基础上，进一步研究了鳖甲煎丸对肝细胞癌中PI3K/AKT/GSK-3β信号通路和差异表达的miRNAs的影响。研究证实鳖甲煎丸对于肝细胞癌中PI3K/AKT/GSK-3β信号通路存在抑制作用。鳖甲煎丸能有效抑制p-AKT、p-GSK-3β蛋白的表达；上调E-cadherin蛋白的表达；抑制N-cadherin、Vimentin及Snail蛋白的表达，来抑制肝癌细胞的转移侵袭，证实鳖甲煎丸可通过抑制GSK-3β的磷酸化来抑制肝癌细胞发生上皮间质转化。同时，qPCR结果显示，鳖甲煎丸可抑制肝癌细胞中miR-215-5p的表达。鳖甲煎丸可能通过抑制肝癌细胞中miR-215-5p的表达而有效调控PI3K/AKT/GSK-3β信号通路中的关键蛋白分子GSK-3β的表达水平而达到抗肝癌转移侵袭的目的。.本研究对寻找肝细胞癌新的治疗靶点具有重要的临床意义。同时，有助于进一步诠释鳖甲煎丸多途径、多层次抗肝癌的科学内涵，为该药应用于抗肝细胞癌的临床研究提供充分的理论依据。