摘要
探讨磷脂酰肌醇3-激酶(PI3K)信号通路调节肝癌细胞增殖的机制.用LY294002特异性阻断PI3K信号通路后,人肝癌细胞(SMMC-7721)的增殖明显被抑制.RT-PCR及蛋白质印迹结果显示,LY294002增加了p27蛋白的表达,但不影响p27的mRNA表达.在LY294002处理的细胞中转入p27的RNAi质粒以干扰p27蛋白的表达后,肝癌细胞的增殖能力可部分恢复.放线菌酮(Chx)处理实验表明,阻断PI3K信号通路使p27蛋白的半衰期延长,稳定性增加.进一步研究发现,LY294002可抑制介导p27蛋白降解的关键分子Skp2的mRNA表达,还可缩短Skp2蛋白的半衰期,降低Skp2蛋白的稳定性.但在SMMC-7721中分别转染PI3K下游重要靶分子Akt的持续激活和失活突变体,却并不影响p27蛋白的表达.这些结果表明,PI3K信号通路在转录及翻译后水平调节Skp2的表达而影响p27蛋白的降解,从而调节肝癌细胞的增殖,但Akt并没有参与这种调节.
To investigate the mechanism of PI3K signaling-mediated hepatoma cell growth, specific PI3K inhibitor LY294002 were used to treat hepatocellular carcinoma cell line （SMMC-7721）. LY294002 could inhibit cell proliferation of SMMC-7721. RT-PCR and Western blotting results showed that inhibition of PI3K signaling increased the protein expression of p27, but not mRNA expression. The protein expression of p27 could be inhibited by transfecting p27 SiRNA plasmid in LY294002-treated cells. And the knock-down of p27 protein expression could partly block the cell growth-inhibition induced by LY294002. Chx treatment experiment revealed that LY294002 prolonged the half-life of p27 protein, which increased its stability. In LY294002-treated cells, not only the mRNA expression of Skp2 （which is a critical molecule mediating the degradation of p27 protein） were reduced, but also the half-life of Skp2 protein were shorten. However, the activity alteration of Akt （an important downstream effector of PI3K signaling） by transfecting Akt constitutively active mutant and Akt dominant negative plasmid, did not influence the expression of p27. Taken together, these findings indicated that PI3K signaling regulated cell growth through modulating the degradation of p27 protein via Skp2 in SMMC-7721, however which was Akt independent.
出处
《生物化学与生物物理进展》
SCIE
CAS
CSCD
北大核心
2008年第11期1263-1269,共7页
Progress In Biochemistry and Biophysics
基金
国家自然科学基金资助项目(30570963)
上海市重点学科建设资助项目(B110)~~
