骨桥蛋白在肝癌细胞中转录后调控的研究

A study on posttranscriptional regulation of osteopontin in hepatoma cell

目的分离鉴定在肝癌细胞HepG2和Hep3B中与骨桥蛋白(OPN)5′非翻译区(5′-UTR)结合后在转录后水平调节OPN mRNA的稳定性的mRNA结合蛋白。方法利用亲和纯化的方法分离和纯化OPN 5′UTR结合蛋白,质谱分析消化后的片段以鉴定蛋白;Hep3B细胞瞬时转染真核细胞翻译延长因子1A(EF1A)siRNA抑制EF1A,HepG2细胞瞬时转染(pcDNA3.1+EF1A)质粒过表达EF1A,Western blot检测OPN蛋白的表达,real-time PCR检测OPN mRNA半衰期的变化。结果质谱分析法确认此结合蛋白为EF1A。在HepG2细胞中过表达EF1A能通过加速OPN mRNA的降解而降低OPN表达;在Hep3B细胞中利用siRNA技术下调EF1A的表达能增强OPN mRNA的稳定性,进而增强OPN的表达。结论 EF1A作为一个去稳定因素作用于OPN 5′UTR,至少部分决定了Hep3B和HepG2细胞中OPN的表达。

Objective To isolate and identify mRNA-bingding protein regulating stability of osteopontin（OPN） mRNA at posttranscriptional regulation level after combination with 5′ untranslated region（5′-UTR） of OPN in HepG2 and Hep3B hepatoma cells.Methods Affinity purification method was applied to isolated and purified OPN 5′-UTR-binding proteins,the digested fragment was subjected to mass spectrometry analysis for protein identifying.Eukaryotic translation elongation factor 1A（EF1A） siRNA was transiently transfected into Hep3B cells to inhibit EF1A,（pcDNA 3.1＋EF1A） plasmid was transiently transfected into HepG2 cells to over express EF1A,and then the expression of OPN protein and changes of OPN mRNA half-life in cells was detected by Western blot and real-time PCR,respectively.Results Mass spectrometry analysis confirmed the binding protein as a eukaryotic translation EF1A.Over-expressing EF1A in HepG2 cells decreased OPN protein expression via accelerating degradation of OPN mRNA,and down-regulating EF1A expression using siRNA technique in Hep3B cells enhanced stability of OPN mRNA,and thereby promoted the expression of OPN protein.Conclusion EF1A interacts with OPN 5′UTR as a destabilizing factor and at least partially determines the expression of OPN in Hep3B and HepG2 cells.