鞘糖脂微结构域1相关磷酸化蛋白基因影响高转移人前列腺癌细胞体外生物学行为的机制探讨

目的 探讨鞘糖脂微结构域1相关磷酸化蛋白(PAG1)基因对高转移人前列腺癌细胞系PC-3M-1E8细胞体外生物学行为影响的机制.方法 异丙基硫代半乳糖苷(IPTG)诱导JM109细菌中GST-Raf1-Ras结合域(GST-Raf1-RBD)融合蛋白的表达,其中Raft-RBD能特异性的与活性Ras(GTP-Ras)结合,聚丙烯酰胺凝胶电泳和考马斯亮蓝染色观察融合蛋白诱导表达结果.GST-pull down实验检测稳定转染PAG1基因、转染空载体及未转染的PC-3M-1E8细胞中活性Ras水平.Western blot检测Ras信号通路相关蛋白表达变化.用异硫氰酸四甲基罗丹明(TRITC)耦联的鬼笔环肽标记F-肌动蛋白,激光扫描共聚焦显微镜观察肿瘤细胞形态和细胞内F-肌动蛋白排列变化.收集人前列腺及前列腺腺癌组织标本,通过免疫组织化学PV9000法检测PAG1蛋白的表达.结果 GST-Raft-RBD融合蛋白诱导表达结果显示,经IPTG诱导后在相对分子质量约33 000处出现较亮的GST-Raf1-RBD融合蛋白条带.GST-pull down结果显示PC-3M-1E8细胞稳定转染PAG1基因后,活性Ras蛋白表达明显减少,总Ras蛋白表达无明显变化.Western blot检测结果显示PC-3M-1E8细胞稳定转染PAG1基因后,磷酸化胞外信号调节激酶(ERK)表达明显减少,总ERK表达无明显变化;细胞周期素D1(cyclin D1)表达明显减少;p21WAF1/CIP1蛋白及磷酸化蛋白激酶B(p-Akt)表达无明显变化.TRITC耦联的鬼笔环肽标记F-肌动蛋白结果显示稳定转染PAG1基因的PC-3M-1E8细胞F-肌动蛋白骨架结构紊乱,丝网状结构不明显,细胞呈长梭形,细胞表面伪足明显较少.免疫组织化学检测结果显示正常前列腺组织PAG1蛋白表达阳性率较高,前列腺腺癌组织中随着Gleason分级的增加,PAG1蛋白表达阳性率降低.结论 PC-3M-1E8细胞中PAG1表达上调能降低Ras活性,抑制ERK活化,进一步抑制cyclin D1的表达而引起细胞周期阻滞,抑制细胞增殖.PAG1表达上调可引起PC-3M-1E8细胞运动、侵袭和转移能力降低.正常前列腺组织中PAG1蛋白表达阳性率较高,前列腺腺癌组织中PAG1蛋白的表达与肿瘤细胞的分化程度及恶性程度相关.

Influence of phosphoprotein associated with glycosphingolipid microdomains 1 on biologic behavior of human prostatic cancer cell line in-vitro

Objective To investigate the mechanism of influence of phosphoprotein associated with glycosphingolipid microdomains 1(PAG1) on the biological behavior of human prostatic cancer cell line PC-3M-1E8. Methods The expression of GST-Raf1-RBD recombinant protein, a specific binding domain of active Ras(GTP-Ras) , was induced by IPTG in JM109 bacterium. SDS-PAGE and coomassie brilliant blue staining were performed using cleaved product of the bacterium to determine the expression of the fusion protein. GST-pull down essay was designed to detect the level of active Ras in PGA1 and vector transfected,respectively and in the native PC-3M-1E8 cells. Western blotting was used to detect the expression levels of downstream proteins of Ras signal pathway which may be related to the function of PAG1. Phalloidine labeled by tetramethylrhodamine-5-(and-6) isothiocyanate (TRITC)was used for the staining of intracellular F-actin,Laser passing confocal microscopy was adopted for observing change of the cell morphology and the arrangement of F-actin. Results After IPTG induction, the GST-Raf1-RBD recombinant protein, with a molecular weight of 33 000, was noticed to be highly expressed in JM109 bacterium. GST-pull down assay revealed that the expression level of active Ras markedly decreased after PAGl-transfection while the total Ras remained unchanged. The expression of p-ERK and cyclin D1 in the PAG1-transfected cells decreased in accordance with the level of active Ras. However, the expression of p21WAF1/CIP1 and p-Akt didn't show any variation. Additionally, the structure of F-actin was turbulent and the pseudopodia of cells diminished conspicuously after PAG1-transfection. There was a high expression of PAG1 in normal human prostate tissue, however,the positive rate of PAG1 immuno-staining decreased in cases of prostatic adenocarcinoma,correspondent with increasing of the grading index of cell differentiation established in the Gleason grading system for the diagnosis of prostate adenocarcinoma. Conclusions An over-expression of PAG1 in PC-3M-1E8 cells effectively suppresses the activation of Ras and ERK, as well as the cyclin D1 expression, leading to an inhibition of the proliferation ability of tumor cells. The turbulence of F-actin and reduction of pseudopodia of cells result in an impairment of cell mobility, invasiveness and metastatic capability. In human prostate and prostatic adenocarcinoma tissues, the expression of PAG1 is related to the cellular differentiation and malignancy.