结肠癌中ATP5J基因诱导化疗药物抵抗和细胞迁移能力增加的机制研究

ATP5J is one of the subunits of ATP synthetase.Currently,the studies about ATP5J are mainly focusing on cardiovascular diseases. The relationship between ATP5J and cancer has not been reported yet.Our previous studies had demonstrated that the expression of ATP5J in colon cancer was higher than that in normal tissues.Further results indicated that down-regulation of ATP5J expression by small RNA interference would decrease the migration ability of colon cancer cells, and increase their sensitivity 5-Fu treatment.Meanwhile,up-regulation of ATP5J expression by stable transfection of plasmid would increase the migration ability of colon cancer cells, and decrease their sensitivity to 5-Fu treatment.These results suggested that ATP5J might correlate with metastasis and resistance of colon cancer.However,the underlying mechanisms are still unclear. Therefore we will do our best to clarify the potential mechanisms of ATP5J-induced resistance in colon cancer cells by detecting the apoptotic signaling, stability of mitochondrial membrane, activity of ATP synthetase,and the levels of ROS in the current program.Simultaneously,we also will explore the possible mechanisms of ATP5J-induced cell migration by detecting cellular skeleton protein and activity of Rho kinase pathway. We hope that our studies could enhance the people's recognition about the function of ATP5J in colon cancer cells as well as its mechanisms, which mightbe supply a potential study area for improving the prognosis of colon cancer in the next future.

ATP5J是ATP合成酶的组成亚单位之一。目前ATP5J的研究主要集中在心血管疾病领域，而它和肿瘤之间的关系尚无正式报道。我们先前的研究发现结肠癌中ATP5J的表达明显高于正常组织，进一步的结果揭示下调结肠癌细胞中ATP5J的表达，会阻碍该细胞的迁移并使之对5-Fu的处理更加敏感，而上调该基因的表达则会促进肿瘤细胞迁移并对5-Fu治疗抵抗。这些结果提示ATP5J可能和肿瘤的转移、耐药有关，但是其中的机制目前并不清楚。因此在本项目中我们将从凋亡信号通路相关蛋白、线粒体膜的稳定性、ATP合成酶的活性及氧自由基（ROS）水平等方面来探讨ATP5J诱导结肠癌细胞对化疗药物抵抗的可能机制，同时我们也将从细胞骨架蛋白的表达、Rho激酶信号通路的活化等方面来探讨ATP5J影响肿瘤细胞迁移能力的潜在机制，加深对ATP5J在结肠癌中的功能及机制的了解，为以后改善和提高结肠癌的治疗效果提供一个潜在的研究方向。

ATP5J是ATP合成酶的组成亚单位之一。目前有关研究主要集中在心血管疾病，而关于ATP5J和肿瘤之间的关系尚无正式报道。我们在原先研究的基础上发现ATP5J高表达后DLD1细胞特异性对5-Fu的抵抗。上调ATP5J的表达可以提高线粒体膜的电位，增加线粒体膜的稳定性，减少细胞色素C的释放。但我们进一步研究显示五个细胞在Bcl-2、Bcl-XL、Bak、Bax、Caspase-3、Caspase-8、Caspase-9、PARP、MDR、TS、MLH1、tubulin、vimentin、β-连接蛋白、E-cadherin以及胶原酶MMP-2的基础表达水平均没有差异。另外我们还发现ATP5J表达水平与细胞内ATP合成酶的活性或ROS的活性无明显相关性。接着我们收集细胞的RNA标本，行DNA微列阵分析，其中DLD1/A2高表达ATP5J（对照载体DLD1/C6），DLD1/SA4低表达ATP5J（对照载体DLD1/CA6）。数据显示在SA4 VS CA6这对细胞中有显著差异表达的基因有254个，在A2 VS C6中也有类似的结果。为减少无关基因的干扰，我们进一步筛选了差异表达在两倍以上的基因作为候选基因。最终在A2 VS C6中上调而在SA4 VS CA6中下调的基因有22个；在A2 VS C6中下调而在SA4 VS CA6中上调的基因有45个。我们选取了ZNF516、CRTAM、CD44、CST1、CST1、EEF1A2、SLPI和C10orf53八个基因对DNA微列阵结果进行PCR验证。除SLPI表达无明显差异外，其他7个基因的表达情况和DNA微列阵结果基本符合。我们进一步选择TMTC1(NM_175861)做下一步验证工作。 首先我们通过Western免疫印迹法证实TMTC1在DLD1/A2中表达降低而在DLD1/SA4中表达增高，然后我们将自有的pGT60-TMTC1质粒转染至DLD1细胞得到稳定高表达TMTC1的细胞克隆(DLD1/ TMTC1)。但是进一步的MTT结果显示该细胞克隆对5-Fu的敏感性与DLD1细胞无明显差异，同样高表达TMTC1对于DLD1细胞的迁移能力没有影响，这提示高表达TMTC1并不能模拟ATP5J表达改变所引起的细胞状态，后者的具体机制还有待进一步的研究。