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【目的】探讨生育酚结合蛋白(TAP)对前列腺癌细胞的生长调节及其分子机制。【方法】四甲基偶氮唑盐生长试验(MTT)测定细胞增殖情况,体外集落形成试验测定各细胞的致癌能力,高效液相层析法检测细胞内维生素E的浓度,利用基因转染、基因沉默、免疫印迹、Northern blot,RT-PCR、荧光定量PCR、免疫沉淀等方法研究TAP对前列腺细胞生长的作用及机制。【结果】TAP mRNA水平在前列腺癌细胞LNCaP、PC-3、DU-145、CWR22R中均较正常前列腺细胞RWPE-1低。TAP可促进癌细胞保留维生素E并增强其抗前列腺癌增殖的效应。无维生素E作用下,转染TAP可抑制前列腺癌细胞LNCaP、DU-145的生长,第6天细胞数比对照组分别减少35.8%与42.4%;LNCaP细胞克隆形成率下降54.3%(P<0.05)。利用siRNA在良性前列腺细胞HPr-1中沉默TAP基因,培养9d后,细胞数比对照组增加124.3%(P<0.01)。TAP通过抑制磷酸肌醇PI3激酶信号,而非通过影响细胞周期或雄激素受体信号发挥作用。免疫沉淀实验表明TAP通过抑制PI3K的亚单位p110与p85的相互作用,进而干扰PI3K-Akt信号通路;持续激活Akt的活性可削弱TAP对前列腺癌细胞生长的抑制能力。【结论】TAP不仅能促进前列腺癌细胞摄取和增强维生素E的抗前列腺癌效应,还可通过非维生素E途径发挥作用,可能是防治前列腺癌的有价值的分子靶点。
【Objective】 To investigate the regulation of tocopherol binding protein (TAP) on the growth of prostate cancer cells and its molecular mechanism. 【Method】 MTT assay was used to determine the proliferation of cells. The in vitro colony formation assay was used to determine the carcinogenicity of each cell. The concentration of vitamin E in the cells was determined by high performance liquid chromatography (HPLC) Gene silencing, Western blotting, Northern blot, RT-PCR, real-time quantitative PCR, immunoprecipitation and other methods to study the effect of TAP on the growth of prostate cells and its mechanism. 【Results】 The levels of TAP mRNA in prostate cancer cells LNCaP, PC-3, DU-145 and CWR22R were lower than those in RWPE-1 normal prostate cells. TAP can promote cancer cells to retain vitamin E and enhance its anti-prostate cancer proliferation. Transfection of TAP could inhibit the growth of prostate cancer cells LNCaP and DU-145 under the condition of no vitamin E transfection. The number of cells on day 6 decreased by 35.8% and 42.4% respectively compared with the control group, and the colony formation rate of LNCaP cells decreased by 54.3% (P <0.05 ). The silencing of TAP gene in HPr-1 of benign prostatic cells by siRNA reduced the number of cells by 124.3% (P <0.01) after 9 days. TAP acts by inhibiting phosphoinositide PI3 kinase signaling, but not by affecting cell cycle or androgen receptor signaling. Immunoprecipitation experiments showed that TAP could inhibit the PI3K-Akt signaling by inhibiting the interaction between PI3K subunit p110 and p85. The sustained activation of Akt attenuated the inhibitory effect of TAP on the growth of prostate cancer cells. 【Conclusion】 TAP can not only promote the uptake of prostate cancer cells and enhance the anti-prostate cancer effect of vitamin E, but also through the non-vitamin E pathway, which may be a valuable molecular target for the prevention and treatment of prostate cancer.