SPOP促进SETD2蛋白的降解并增强肾癌细胞的体外成瘤与增殖能力

SPOP promotes SETD2 degradation to enhance in vitro tumorigenic ability and proliferation in renal cell carcinoma

目的:新近发现E3泛素化连接酶的成员之一SPOP在肾癌中作用关键,我们前期的研究表明SPOP在肾癌中表达上调且通过活化β-Catenin的活性来促进肾癌细胞的侵袭与转移。本研究继续研究SPOP在肾癌成瘤与增殖中的作用,并探索其潜在的分子机制。方法:siRNA靶向沉默肾癌细胞内SPOP的表达后,采用克隆形成实验及MTT实验检测细胞的体外成瘤与增殖能力的变化;运用realtime-PCR及western blot技术探索SPOP在肾癌细胞内发挥生物学功能的潜在靶分子;采用免疫组织化学方法检测SPOP与其靶分子SETD2在人肾癌组织中的表达情况,并行相关性分析。结果:SPOP siRNA显著减低肾癌细胞的体外成瘤及增殖能力;SPOP的变化不影响肾癌细胞内SETD2 mRNA的表达,但当采用蛋白合成抑制剂cycloheximide预先阻断细胞内SETD2蛋白的合成后,SPOP siRNA能明显延缓SETD2蛋白的降解。进一步检测人肾癌组织标本中SPOP与SETD2的表达结果证实,SPOP与SETD2蛋白的表达呈显著的负相关(r=-0.41,P<0.05)。结论:SPOP能增强肾癌细胞的体外成瘤与增殖能力,其潜在的机制可能为促进抑癌蛋白SETD2的降解。

Objective： Recent studies show SPOP,an E3 ubiquitin ligase component,is critical for the pathogene- sis of renal cell carcinoma （ RCC ）. We previously demonstrated that SPOP was abnormally upregulated in RCC and promoted its invasion and metastasis. In the present study ,we aim to investigate the role of SPOP in tumorigenesis and proliferation of RCC, and dissect the potential molecular mechanism. Methods： Speficial siRNA was transfected into RCC cell to silence the expression of SPOP, then colony formation and MT/＇ assay were performed to determine the tu- morigenesis and proliferation of cells in vitro. Realtime - PCR and western blot were used to study the biological func- tions of SPOP in RCC and to dissect the potential targeted molecular. Immunohistochemistry was performed to detect the expression of SPOP and its targeted molecular, and their association was further determined by Pearson correlation assay. Results：SPOP siRNA significantly reduced the abilities of tumorigenesis and proliferation in RCC cells. The change of SPOP expression did not affect the mRNA level of SETD2, but when protein synthesis was block by cyelo- heximide, SPOP siRNA dramatically increased SETD2 protein level and decreased its degradation. In SPOP high - ex- pression RCC tissues, the expression level of SETD2 was low, and vice versa. The negative correlation was further con- firmed by Pearson＇s correlation analysis in a cohort of 48 RCC tissues. Conclusion ： SPOP can significantly enhance the tumorigenesis and proliferation of RCC cells, and the potential mechanism of action is likely to promote the degra- dation of SETD2 orotein.