hMOB2调控Hippo信号通路抑制肝癌细胞迁移和侵袭机制的研究

We have previously demonstrated that hMOB2 inhibited migration and invasion of the hepatocellular carcinoma (HCC) cells through regulating the formation of stress fibers, filopodia and lamellipodia, and induction of phenotype from epithelial-mesenchymal transition (EMT) to mesenchymal-epithelial transition (MET) states, and up-regulation of both mRNA and protein of YAP, and pYAP and pTAZ, and down-regulation of the protein level of TAZ and c-myc protein were shown in hMOB2-overexpressing HCC cells. Meanwhile, we also showed that the levels of hMOB2, YAP/TAZ and their activity correlated with HCC progression and HCC cells migration potential. Based on our previous study, this item will be carried out from the HCC tissue samples, in vivo tumor spontaneous metastasis and experimental metastasis model, and the HCC cell lines with different migration capacity by using the lentiviral-mediated gene overexpression, knock-down by RNAi, and knock-out by CRISPR/Cas9 gene editing systems to clarify the function and the mechanism of hMOB2 in the inhibition of HCC cell migration and invasion and the tumor metastasis resulted from negative regulation of EMT by decrease the activity of the YAP/TAZ transcriptional activation through it combined with NDR1/2 kinase directly or indirectly to activate the Hippo signaling pathway. This study will not only be helpful to improve our understanding the Hippo signaling pathway involved in the regulation of tumor metastasis, but also provide theoretical basis and a novel potential therapeutic target for the anti-metastasis treatment of cancer.

前期研究发现，hMOB2可以通过调控张力纤维、片状和丝状伪足的形成抑制肝癌细胞的迁移和侵袭，hMOB2过表达可诱发肝癌细胞发生EMT/MET转换，上调肝癌细胞中YAP的mRNA、蛋白水平以及pYAP和pTAZ，下调细胞中TAZ和c-myc蛋白表达；而且hMOB2、YAP/TAZ的表达及其活性与肝癌发生、肝癌细胞的迁移能力相关。本课题拟在前期工作基础上，采用慢病毒介导的基因过表达、干扰表达以及CRISPR/Cas9基因编辑等方法，以肝癌组织样本、小鼠动物模型和不同迁移能力的肝癌细胞为研究对象，阐明hMOB2通过与NDR1/2激酶结合直接或者间接地激活Hippo信号通路，抑制YAP/TAZ转录激活的活性，负调控EMT，抑制肝癌细胞迁移和侵袭以及肿瘤转移的分子机制。本研究不仅有助于完善我们对Hippo信号通路参与肿瘤转移调控的理解，也有可能为发展相应的抗肝癌转移治疗措施提供理论依据和新靶标。

前期研究发现，MOB2可以调控肝癌细胞的细胞周期进程、显著抑制肝癌细胞的迁移和侵袭，为了研究MOB2过表达和干扰表达抑制肝癌细胞的迁移和侵袭的可能机制，本课题采用慢病毒介导的基因过表达、干扰表达以及基因编辑等方法，主要从三个方面开展研究：首先在肝癌组织样本、不同转移能力的肝癌细胞以及小鼠动物模型中证实了MOB2的表达与肿瘤恶化程度和转移的关系，明确了MOB2可能参与肝癌的发生、发展过程；其次在细胞水平上明确了MOB2过表达和干扰表达均可以显著抑制不同转移能力的肝癌细胞的迁移和侵袭、并通过不同的方式调控细胞自噬过程，初步揭示了MOB2影响细胞的迁移能力很可能是通过调控细胞自噬过程来实现的；最后在细胞水平上明确了MOB2可以对Hippo信号通路中相关分子的表达及其活性具有调控作用，一方面，MOB2在调控MOB1与LATS1和NDR1/2的选择性相互作用中发挥重要作用，另一方面，MOB2可以通过调控YAP和TAZ的活性影响肝癌细胞的迁移与细胞自噬，证实了MOB2干扰表达可能通过NDR1/2-YAP通路影响肝癌细胞的迁移与细胞自噬，沉默YAP和TAZ可能通过促进细胞自噬而抑制EMT的进程而抑制细胞的迁移；此外，本研究结果还显示MOB2、TAZ/YAP可以影响肝癌细胞中RNA的m6A修饰水平，调控细胞中与RNA的m6A修饰相关基因的表达，是否进一步参与调控自噬相关基因以及EMT相关基因的表达尚需进一步研究。本研究结果揭示了MOB2可以直接或间接地调控Hippo信号通路的效应分子YAP/TAZ的表达及其活性，调控相关基因的表达，负调控EMT，并进一步影响Rho GTP 酶活性而影响肌动蛋白细胞骨架的装配，影响肝癌细胞的迁移、侵袭和细胞自噬以及肿瘤转移的分子机制。本研究不仅有助于完善我们对Hippo信号通路参与肿瘤转移调控的理解，也有可能为发展相应的抗肝癌转移治疗措施提供理论依据和新靶标。