过氧化物还原酶3参与肾透明细胞癌发生与发展的分子机制

Mechanism of PRDX_3 Involved in Development and Progression in Clear Cell Renal Cell Carcinoma

【目的】探讨肾透明细胞癌组织中硫氧还蛋白依赖的过氧化物还原酶3(PRDX_3)的表达与肾透明细胞癌发生发展的关系。【方法】本研究首先在16例肾透明细胞癌组织及癌旁组织中验证了PRDX_3的表达。根据表达量构建了786-O的PRDX_3过表达细胞系及敲低表达细胞系,考察了PRDX_3过表达和敲低表达后,肿瘤细胞的生长情况。通过pull-down试验联合LC-MS/MS技术寻找PRDX_3的相互作用蛋白。初步探究了PRDX_3与过氧化物酶1(PRDX_1)的相互作用关系及PRDX_3参与肾透明细胞癌发生发展的机制。【结果】16例肾透明细胞癌组织样本的western blot检测结果表明,有14例样本PRDX_3下调表达,下调均值1.78倍左右。成功筛选到PRDX_3过表达的786-O单克隆细胞系和对照细胞系[786O-PRDX(_3+)和786O-PRDX(_3-)]和PRDX_3敲低表达的786-O单克隆细胞系和无意义序列对照细胞系(786O-PRDX_3KN和786O-PRDX_3NCi)。qPCR和western blot结果表明,相比786O-PRDX(_3-)组,786O-PRDX(_3+)组在mRNA水平过表达约2.1倍,在蛋白水平过表达约1.8倍;相比786O-PRDX_3NCi组,786O-PRDX_3KN组在mRNA水平低表达约0.48倍,在蛋白水平低表达约0.51倍。CCK-8试验表明,相比786O-PRDX(_3-),786O-PRDX(_3+)细胞生长速率明显降低。但敲低PRDX_3的表达后,敲低组和其对照组细胞的生长速率却无显著性差异。Pull-down试验发现PRDX_3可能与PRDX_1存在相互作用。结合质谱分析及数据库检索,发现PRDX_3通过二硫键与PRDX_1结合并发挥作用,分别通过plink软件鉴定到二者可信的结合位点,初步探讨了PRDX_3参与肾透明细胞癌发生发展的可能机制。【结论】PRDX_3在肾透明细胞癌组织中下调表达,可能参与了肿瘤的发生发展。提高PRDX_3的表达水平,能显著降低肿瘤细胞的生长速率。基于PRDX_3蛋白,可能开发针对肾透明细胞癌的靶向药物。

【Objective】To investigate the relationship between the expression of PRDX3(thioredoxin-dependentperoxide reductase)and the occurrence and development of ccRCC(clear cell renal cell carcinoma).【Methods】Theexpression of PRDX3was first verified in 16 cases of ccRCC tissues and adjacent normal tissues.In the present study,according to the PRDX3over-expression level,we established the stable PRDX3overexpression cell lines and knockdowncell lines in 786-O cell lines.We detected the growth rate of tumor cells after overexpression and knockdown of PRDX3.Interaction proteins with PRDX3were searched by anti-flag pull-down test combined with LC-MS/MS technique.The interaction between PRDX3and PRDX1(peroxiredoxin 1)was preliminarily explored.【Results】The western blot results showed that PRDX3were down-regulated in 14 out of 16 ccRCC tissue samples about 1.78 times.Stable PRDX3overexpression and knockdown cell lines and those control group were successfully established[786O-PRDX3(+)and 786O-PRDX3(-),786O-PRDX3KN and 786O-PRDX3NCi].PRDX3expression in 786O-PRDX3(+)was 2.1 times higher than 786O-PRDX3(-)at mRNA level and 1.8 times at protein level.PRDX3expression in 786O-PRDX3KN was 0.48times lower than 786O-PRDX3NCi at mRNA level and 0.51 times at protein level.The cell growth rate of 786O-PRDX3(+)cell lines was significantly lower than that of 786O-PRDX3(-).Meanwhile,there was no significant difference in786O-PRDX3KN and NCi cell lines.Pull-down results shows that PRDX3may interact with PRDX1through disulfide bond and the binding sites of those two proteins were identified respectively.【Conclusion】PRDX3was down-regulated expression in renal clear cell carcinoma and the interaction with PRDX1may be involved in the occurrence and development of tumor.Increasing the expression level of PRDX3can significantly reduce the growth rate of tumor cells.Based on PRDX3,it is possible to develop targeted drugs for treating renal clear cell carcinoma.