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目的研究抑癌基因PTEN转染对人膀胱癌细胞BIU-87中PI3K-Akt信号通路的作用。方法将携有PTEN基因的重组真核表达质粒pBp-PTEN转化大肠杆菌DH5α并扩增,抽提纯化质粒并进行酶切鉴定,pBp-PTEN体外转染BIU-87细胞(pBp-PTEN-BIU87),筛选稳定转染的细胞并扩增培养,以转染了空质粒pBp的BIU-87细胞(pBp-BIU87)和正常BIU-87细胞为对照,用RT-PCR检测PTEN的表达情况。将PTEN基因的真核表达质粒转染膀胱癌细胞BIU-87(pBp-PTEN-BIU87),以BIU87细胞和pBp-BIU87细胞为对照,应用Wester blot方法检测三组细胞P110(PI3K的催化亚单位)、磷酸化P110、Akt和磷酸化Akt表达的情况。结果3种细胞P110和Akt蛋白总水平没有变化,但磷酸化P110和磷酸化Akt在PTEN转染细胞中的表达明显弱于对照组。结论PTEN是通过对PI3K-Akt信号传导途径的负调控而抑制肿瘤的形成。PTEN-PI3K-Akt途径可能是PTEN抑癌作用的重要机制。
Objective To investigate the effect of tumor suppressor gene PTEN on the PI3K-Akt signaling pathway in human bladder cancer cell line BIU-87. Methods The recombinant eukaryotic expression plasmid pBp-PTEN carrying PTEN gene was transformed into E. coli DH5α and amplified. The plasmid was purified and identified by restriction enzyme digestion. PBp-PTEN was transfected into BIU-87 cells (pBp-PTEN-BIU87) , And stably transfected cells were screened and expanded. BIU-87 cells (pBp-BIU87) transfected with empty plasmid pBp and normal BIU-87 cells were used as controls. The expression of PTEN was detected by RT-PCR. The eukaryotic expression plasmid of PTEN gene was transfected into bladder cancer cell line BIU-87 (pBp-PTEN-BIU87). BIU87 cells and pBp-BIU87 cells were used as control. Wester blot was used to detect the expression of PI10 ), Phosphorylated P110, Akt, and phosphorylated Akt. Results The total levels of P110 and Akt did not change in all three cells. However, the expression of phosphorylated P110 and phosphorylated Akt in PTEN transfected cells was significantly weaker than that in control cells. Conclusions PTEN inhibits tumor formation through the negative regulation of the PI3K-Akt signaling pathway. PTEN-PI3K-Akt pathway may be an important mechanism of PTEN tumor suppressor.