Expression Level of u-PAR in Different Invasive PC-3M Cell Subclones

Objective: To select a target molecule associated with invasive potential in PC-3M cell. Methods: Cell subclones were isolated from PC-3M cell line with the method of limited dilution and their invasive ability characterized by monolayer invasion assay. The expression of u-PAR in the cell subclones at mRNA and protein levels was assayed respectively by non-competitive quantitative RT-PCR and immunohistochemical assay. Results: The expression level of u-PAR in highly invasive cell subclones was higher than that of lower invasive subclones. Conclusion: The higher expression level of u-PAR is associated with the relative strong invasive ability of PC-3M subclones. It is indicated that the u-PAR might be a promising target molecule for inhibiting invasion of highly invasive PC-3M cell subclones.

应用mRNA差异显示技术克隆肿瘤转移相关基因TMSG-1

应用mRNA差异显示技术,对比研究前列腺癌细胞系PC-3M具有不同转移潜能的亚系,克隆差异表达片段,经Northern杂交验证其在不同转移潜能的人类肿瘤细胞系中的表达差异,测序,EST拼接获得cDNA全长序外,RT-PCR验证拼接结果并重复测序再次证实.结果显示,TMSG-1在前列腺癌细胞系PC-3M不转移亚系2B4高表达,而在高转移亚系1E8低表达,二者差异显著.TMSG-1cDNA全长序列2kb,开放读码框编码含230aa的蛋白质,GenBank Blastn检索与已知基因无显著同源性.对TMSG-1所编码的氨基酸序列进行功能预测提示:TMSG-1是一个跨膜蛋白,有3个跨膜区、3个蛋白激酶C磷酸化位点、2个酪蛋白激酶Ⅱ磷酸化位点和1个N末端肉豆蔻酸酰化位点.在6种人类常见肿瘤组织中,RT-PCR结果显示TMSG-1在前列腺癌中表达最强,肺癌转移灶较肺癌原发灶表达显著降低,低分化结肠癌较高分化结肠癌表达显著降低,在复发性乳腺癌、卵巢癌、低分化胰腺癌（均无转移）中均有表达.9例胃癌原发灶标本中,TMSG-1与β-actin RT-PCR扩增产物的灰度扫描（CNT×mm2）比值显示:TMSG-1在已发生转移的胃癌标本中的表达与无转移的胃癌标本相比有下降的趋势,t检验显示其差异有显著性（P<0.05）.结果表明,TMSG-1基因表达水平的降低与肿瘤转移潜能的提高具有相关性.