极光激酶A调控PKC/MMPs信号通路促进结直肠癌EMT的分子机制研究

Tumor metastasis is primary factor to induce the death of patient with cancer. Recent studies have reported that aurora kinase A was expressed in many tumors and was related with the progress of tumor. There were few reports about aurora kinase A regulating epithelial mesenchymal transition, invasion and metastasis of tumor cells, but it has not been reported in colorectal cancer. In our earlier study, activated protein kinase C could induce a-SMA, MMP-9 and inhibit E-cadherin and promote invasion, metastasis in colorectal cancer cells. The further study showed inhibitors of aurora kinase A blocked protein kinase C induced invasion, metastasis and expression of MMP-9. It was shown that sustained activating MMPs were critical in regulating EMT of tumor cells. It indicated that aurora kinase A probably involved in the EMT of colorectal cancer cells. Accordingly, we put forward the hypothesis "aurora kinase A promoted EMT, invasion and metastasis of colorectal cancer cells by regulating PKC induced MMP-9". To confirm the hypothesis, we want to establish the relationship of aurora kinase A, expression of MMPs and EMT related index by genetic intervention and EMSA. And we clarified the effect and mechanism of aurora kinase A to EMT, invasion and metastasis of colorectal cancer cells to obtain new targets of intervention.

肿瘤转移是导致恶性肿瘤患者死亡的首要因素。新近研究提示极光激酶A在多种肿瘤中过表达，与肿瘤进展密切相关，但尚未见极光激酶A调控肿瘤细胞EMT转化和侵袭转移的相关报道。我们前期研究发现,在结直肠癌细胞中激活蛋白激酶C可促进细胞侵袭和迁移，并抑制E-钙粘蛋白表达和上调a-SMA与MMP-9的表达；进一步实验示极光激酶A抑制剂阻止PKC诱导的细胞侵袭和迁移，降低PKC诱导的MMP-9表达。目前已知MMPs持续激活在调控肿瘤细胞EMT转化中起关键作用，提示极光激酶A可能参与结直肠癌EMT转化过程。据此，提出"极光激酶A通过调控PKC诱导生成的MMP-9从而促进结直肠癌细胞EMT转化及侵袭转移"这一研究假设。为证实该假设，本课题拟通过基因干预和EMSA等方法探讨极光激酶A与MMPs表达水平和EMT相关指标的关联性,阐明极光激酶A对结直肠癌细胞EMT转化和侵袭转移的影响与机制，以期获得新的干预靶点。

肿瘤转移是导致恶性肿瘤患者死亡的首要因素。新近研究提示极光激酶A在多种肿瘤中过表达，与肿瘤进展密切相关，但尚未见极光激酶A调控肿瘤细胞EMT转化和侵袭转移的相关报道。通过本课题探索极光激酶A对PKC介导生成MMPs的调控作用及其信号传导通路，探讨结直肠癌中极光激酶A与MMPs的表达水平和EMT相关指标的相关性，阐明极光激酶A对结直肠癌细胞EMT转化及侵袭转移的影响及其机制。利用显微镜、Scrape-wound migration assay、Chamber invasion assay检测细胞形态和肿瘤细胞侵袭、迁移能力；利用细胞免疫荧光技术、Western blotting、zymography、EMSA等方法检测EMT相关蛋白的表达以及核转录因子的活性。实验结果显示：在不同浓度PKC激活剂TPA诱导下结直肠癌细胞形态学发生了变化，由上皮细胞样转变为间叶细胞样；并且TPA诱导下结直肠癌细胞侵袭、迁移能力增高。TPA诱导下上皮细胞标志蛋白EMT相关蛋白E-cadherin表达降低，间质细胞标志蛋白a-SMA、vimentin表达增加，并且观察到TPA诱导极光激酶A、MMP-9表达也增加。PKC抑制剂可逆转上述TPA诱导的EMT转化，也抑制了极光激酶A的表达。极光激酶A特异性抑制剂VX680、Reversin均可抑制TPA诱导的肿瘤细胞侵袭迁移能力、EMT相关蛋白表达，逆转EMT转化；并且极光激酶A特异性抑制剂抑制EMT蛋白相关核转录因子NF-κB、AP-1活性。极光激酶A-siRNA转染肿瘤细胞可抑制肿瘤细胞侵袭、迁移能力，逆转EMT转化。我们证实了极光激酶A通过转录因子NF-κB、AP-1调控MMP-9的生成，促进PKC介导的结直肠癌EMT转化过程的分子机制，鉴定出涉及肿瘤进展、促进EMT的分子标志物，开发出具有高度特异性、选择性的抑制剂提供了有力的理论基础。