GSK3β对骨肉瘤侵袭转移的影响及其分子机制研究

In recent years, glycogen synthase kinase 3β(GSK3β)as a new cancar therapeutic target is widespread concern, but its role in invasion and metastasis of osteosarcoma and molecular mechanisms are unclear. Our preliminary experimental results show that GSK3β was overexpression in osteosarcoma cells, and in tissue of osteosarcoma pulmonary metastasis was significantly higher, suggesting that GSK3β may be involverd in invasion and metastasis of osteosarcoma. Therefore, this study we will reduce the expression and activity of endogenous GSK3β by the small molecule inhibitors of GSK3β or GSK3β siRNA, and application Trans-well assay, Wound healing assay, colony formation and establishment SCID mouse model of osteosarcoma metastasis, in vitro and in vivo experiments explore that inactivation or silent of GSK3β in the invasion and metastasis of osteosarcoma. We will also further examination that after activity and expression of GSK3β was inhibited, whether GSK3β regulate its downstream signal transduction pathways FAK, PTEN cell motility factor by Western blot, Real-time PCR methods, clarify the mechanism of invasion and metastasis of osteosarcoma. This result will laid the theoretical foundation for open up a new potential therapeutic target in osteosarcoma.

近年糖原合成激酶3β(GSK3β)作为一个新的癌治疗靶点被广泛关注，但其在骨肉瘤侵袭和转移中的作用及分子机制尚不清楚。本课题组前期实验结果显示GSK3β在骨肉瘤细胞中超表达，且在肺转移的骨肉瘤组织中表达水平显著增高，提示GSK3β可能参与骨肉瘤的侵袭、转移过程。故本研究拟采用GSK3β小分子抑制剂或siRNA减少内源性GSK3β表达及活性，通过Trans-well、Wound healing assay、平板克隆形成实验及建立骨肉瘤转移SCID小鼠模型等体内、体外实验，研究GSK3β失活或沉默对骨肉瘤侵袭和转移的影响，进一步通过Western blot，Real-time PCR等方法检测GSK3β抑制后对其下游信号转导通路FAK、PTEN等细胞运动相关因子的影响，阐明其对骨肉瘤侵袭和转移的作用机制，可以为骨肉瘤治疗开辟新靶点奠定理论基础。

近年糖原合成激酶3β(GSK3β)作为一个新的癌治疗靶点被广泛关注，但其在骨肉瘤侵袭和转移中的作用及分子机制尚不清楚。我们在骨肉瘤细胞及肿瘤组织中通过Western blot法及NRIKA法检测GSK3β的表达和活性；在体外及骨肉瘤细胞移植裸鼠检测GSK3β小分子抑制剂对骨肉瘤细胞的迁移、侵袭的影响；我们也通过Western blot、Real-time PCR方法检测GSK3β表达或活性抑制后对其下游FAK、PTEN、MMP2、MMP9细胞运动相关因子的影响。结果显示GSK3β在骨肉瘤细胞中过表达且存在活性调节异常；在转移及非转移骨肉瘤患者肿瘤组织中均可检测到GSK3β表达及活性异常，尤其在转移的骨肉瘤患者的肿瘤组织中；抑制内源性GSK3β活性，在骨肉瘤细胞及MG63-2移植啮齿类动物中抑制了细胞的迁移、侵袭能力；抑制内源性GSK3β的表达明显升高PTEN的mRNA及蛋白表达水平，降低MMP-2、MMP-9的mRNA及蛋白表达水平，FAK在mRNA和蛋白表达水平无显著变化但其磷酸化水平明显降低。我们的这些结果表明GSK3β通过调节PTEN的蛋白稳定性及FAK磷酸化水平，促进细胞分泌MMP2和MMP9，从而影响骨肉瘤的侵袭和转移。初步阐明GSK3β影响骨肉瘤侵袭和转移的作用机制，可以为骨肉瘤治疗开辟新靶点奠定理论基础。