泛素结合酶E2C通过特异性结合β-微管蛋白参与调控肝细胞癌进展

Ubiquitin-conjugating enzyme E2C regulates the progression of hepatocellular carcinoma by specifically binding to β-tubulin

目的筛选泛素结合酶E2C(UBE2C)的底物蛋白,并探讨其在肝细胞癌进展中的作用。方法构建带有Halotag标签的UBE2C过表达质粒,将UBE2C与Halotag在人高转移肝癌细胞系HCCLM3中融合表达,通过Halolink树脂沉淀UBE2C结合的底物蛋白,并联合SDS-PAGE和质谱对底物蛋白进行鉴定。在人肝癌细胞系Huh-7中过表达UBE2C,通过qRT-PCR、蛋白质印迹分析和细胞免疫荧光实验检测UBE2C过表达对其底物蛋白表达的影响,利用细胞核质分离结合蛋白质印迹分析及TOP flash/FOP flash荧光素酶报告基因实验检测UBE2C过表达对Wnt信号通路的影响,并进一步通过qRT-PCR和蛋白质印迹分析验证UBE2C过表达对Wnt信号通路下游基因表达的影响。结果Halotag共沉淀产物经SDS-PAGE和银染后所获得差异条带的质谱鉴定结果显示,β-微管蛋白是UBE2C结合的底物蛋白。与对照组相比,过表达UBE2C降低了Huh-7细胞中β-微管蛋白在蛋白水平的表达(P<0.01),但对其在mRNA水平的表达没有影响(P>0.05)。同时,过表达UBE2C促进了β-联蛋白入核(P<0.05),提高了TOP flash/FOP flash荧光素酶活性(P<0.01),增强了Wnt信号通路下游基因JNK、细胞周期蛋白D1和基质金属蛋白酶7的表达(P均<0.05)。结论UBE2C通过特异性结合β-微管蛋白降低肝癌细胞中β-微管蛋白水平,从而提高Wnt信号通路活性,促进其下游基因表达,进而参与调控肝细胞癌的进展。

Objective To screen the substrate protein of ubiquitin-conjugating enzyme E2C(UBE2C)and to explore its role in the progression of hepatocellular carcinoma.Methods An overexpression plasmid of UBE2C was constructed with Halotag and was used to infect the highly metastatic human liver cancer cell line HCCLM3.The UBE2C binding substrate protein was precipitated by Halolink resin and identified by sodium dodecyl sulphate-polyacrylamide gel electrophoresis(SDSPAGE)and mass spectrometry.UBE2C was overexpressed in human liver cancer cell line Huh-7;the effect on the expression of its substrate protein was detected by quantitative real-time polymerase chain reaction(qRT-PCR),Western blotting and immunoflurescence staining,the effect on Wnt signal pathway was detected by cell nucleoplasmic separation combined with Western blotting and TOP flash/FOP flash luciferase reporter assay,and the effect on the expression of Wnt signal pathway downstream genes was further verified by qRT-PCR and Western blotting.Results The mass spectrometry analysis of the differential band of the Halotag coprecipitate obtained by SDS-PAGE and silver staining showed that the substrate protein of UBE2C wasβ-tubulin.Compared with the control group,overexpression of UBE2C had no effect on the expression ofβ-tubulin in mRNA level in Huh-7 cells(P>0.05),while it significantly decreased the expression ofβ-tubulin in protein level(P<0.01).Meanwhile,overexpression of UBE2C promoted the entry ofβ-catenin into nucleus(P<0.05),increased the luciferase activity of TOP flash/FOP flash(P<0.01),and enhanced the expression of the Wnt signaling pathway downstream genes(c-Jun N-terminal kinase[JNK], cyclin D1 and matrix metallopeptidase 7) (all P<0.05). Conclusion UBE2C contributes to theprogression of hepatocellular carcinoma through decreasing the expression of β-tubulin in hepatocellular carcinoma cells byspecifically binding to β-tubulin, activating Wnt signaling pathway and promoting expression of downstream genes.