PTEN对K562细胞mTOR调控的研究

Regulatory effects of PTEN gene on mTOR signaling pathway in K562 cell line

目的:探讨肿瘤抑制基因PTEN在人慢性粒细胞白血病细胞株K562中对哺乳动物雷帕霉素靶蛋白(mammalian target of rapamycin,mTOR)的调控作用,以及雷帕霉素(rapamycin,RAPA)对K562细胞增殖抑制的影响。方法:通过实时荧光定量PCR(real-time fluorescent quantification PCR,RFQ-PCR)法检测甲磺酸伊马替尼(imatinib mexylate)干预K562细胞后BCR/ABL、PTEN、mTOR mRNA的表达水平及相互关系;Western印迹法检测甲磺酸伊马替尼干预和以腺病毒为载体感染野生型PTEN(Ad-PTEN-GFP)后K562细胞PTEN、Akt和p-Akt的蛋白表达水平;用MTT和FCM方法检测RAPA对不同腺病毒感染组K562细胞增殖及凋亡的影响。结果:格列卫干预K562细胞后,BCR/ABL和mTOR mRNA表达下调,PTEN mRNA表达上调;与感染Ad-GFP组相比,感染Ad-PTEN-GFP组的mTOR mRNA表达下调;Ad-PTEN-GFP感染与10nmol/L RAPA联合作用于K562细胞能够发挥协同作用,对K562细胞的增殖抑制率明显高于单独作用组。结论:PTEN是mTOR的上游调控基因,能够抑制mTOR的表达。RAPA与野生型PTEN一起可以协同抑制K562细胞的增殖。

Objective ： To investigate the regulatory effects of tumor-suppressing gene PTEN on mammalian target of rapamycin （mTOR） signaling pathway in human chronic myeloid leukemia （CML） K562 cells and the effect of rapamycin on the growth inhibition and apoptosis induction of K562 cells in vitro. Methods：The mRNA levels of BCR/ABL, PTEN, and mTOR in K562 cells were detected by real-time fluorescent quantitative PCR （RFQ-PCR） after treatment with imatinib mesylate. The protein expression levels of PTEN, Akt, and p-Akt were measured by Western blotting after imatinib mesylate treatment or transfection of recombinated adenovirus-PTEN vector containing green fluorescent protein （Ad-PTEN-GFP） into K562 cells. MTr assay was used to test the effect of rapamycin on the proliferation of K562 cells and flow cytometry （FCM） was performed to evaluate the influence of rapamycin on apoptosis of K562 cells in different adenovirus infected groups. Results：The mRNA expression levels of BCR/ABL and mTOR were down-regulated and the expression of PTEN mRNA was up-regulated after imatinib mesylate interference. The expression of mTOR mRNA was decreased in K562 cells after the cells were transfected with wild type PTEN gene compared with Ad-PTEN-GFP transfection group. Ad-PTEN-GFP transfection combined with rapamycin had synergistic effect in inhibiting the proliferation and promoting apoptosis of K562 ceils. The is inhibitory effect was significantly higher in combined treatment group than that in single treatment groups. Conclusion： PTEN is an upstream regulatory gene of mTOR expression. It could inhibit the expression of mTOR. Rapamycin has sygergistic effects with wild type PTEN in inhibiting the proliferation of K562 cells.