RKIP基因与卵巢上皮性癌转移的关系

Relationship between raf kinase inhibitor protein and metastasis of ovarian carcinoma

目的探讨一种新的转移抑制基因——RKIP基因与卵巢癌转移的关系。方法应用免疫组化、RT-PCR及蛋白印迹法对卵巢肿瘤的组织标本（卵巢癌组织22份，卵巢交界性肿瘤组织8份，卵巢良性肿瘤组织10份）及卵巢癌细胞系小RKIP基因的表达情况进行检测。将含正义和反义RKIPcDNA的表达载体导入卵巢癌细胞系SKOV3，蛋白印迹法检测转染前后细胞中丝裂原激活的蛋白激酶／细胞外信号调节激酶的激酶（MEK）、细胞外信号调节激酶（ERK）活性的变化。应用四甲基偶氮唑蓝法、双层软琼脂集落形成实验、体外黏附实验、体外侵袭实验、流式细胞仪等观察RKIP基因对卵巢癌细胞生物学行为的影响。结果（1）22份卵巢癌组织中RKIP的表达以阴性（6份，27％）、弱阳性（9份，41％）为主，而10份卵巢良性肿瘤和8份交界性肿瘤组织以强阳性（分别为4份、2份）、阳性（分别为4份、5份）为主。（2）RKIP基因转染后，含正义cDNA的ssRKIP细胞的磷酸化MEK和磷酸化ERK蛋白表达水平下调，ssRKIP#1和ssRKIP#4细胞磷酸化MEK蛋白的相对表达含量分别为0．35和0．34，两者磷酸化ERK蛋白的相对表达含量分别为0．48和0．46。（3）ssRKIP细胞的增殖能力明显低于未转染细胞（P〈0．01）。（4）锚定非依赖性生长能力：ssRKIP#1、ssRKIP#4细胞的集落形成数（83．7±5．7、106．0±9．2）较其对照空载体pcDNA3．1（＋）转染细胞（158．3±14．6）明显减少，分别比较，差异均有统计学意义（P〈0．01）。（5）体外黏附能力：ssRKIP#1、ssRKIP#4细胞的黏附率[分别为（68．3±0．8）％、（64．1±0．9）％]明显低于未转染细胞[（100．0±1．1）％]，分别比较，差异均有统计学意义（P〈0．01）。（6）体外侵袭能力：ssRKIP#1、ssRK]P#4细胞的穿膜细胞数[分别为（24±5）、（25±4）个]明屁低于未转染细胞[（68±5）个]，分别比较，差异均有统计学意义（P〈0．01）。（7）ssRKIP细胞呈G1期细胞比例增加和G2＋S期细胞比例下降。结论RKIP基因不仪对卵巢癌细胞的侵袭、转移有抑制作用，且对其生长也有抑制作用。

Objective To investigate the relationship between raf kinase inhibitor protein （ RKIP）,a novel metastasis suppressor gene, and metastasis of ovarian carcinoma. Methods Immunohistochemistry, RT-PCR, and western blot analysis were performed to examine the expression of RKIP in clinical samples of ovarian tumors and five human ovarian carcinoma cell lines. Stable cell lines over-expressed or deleted of RKIP were cloned to investigate the function of RKIP in ovarian cancer cells. The recombinant plasmids expressing sense （ss） or antisense （as） RKIP cDNA or empty vector was transfected into ovarian cancer cell line SKOV3 by lipofectamine. The expression level of mitogen-activated protein kinase/extracellular signal-regulated kinase kinase （MEK） and extracellular signal-regulated kinase （ERK） in ovarian cancer cells were detected by western blot analysis. Assays of cell proliferation, soft-agar colony formation, cell adhesion, and cell invasion in vitro were used to examine the malignant pbenotypes of the transfected cells. Flow cytometric analysis was performed to observe the effect of RKIP on cell cycle distribution before and after transfection. Results （ 1 ） The expression levels of RKIP protein in ovarian carcinoma tissues frompatients were found to be reduced than those in ovarian benign tumor and borderline tumor. SKOV3 clones stably expressing full-length recombinant ssRKIP, asRKIP, and their respective empty vector were obtained. （2） RKIP was able to block basal levels of MEK and ERK in ovarian cancer cells. The expression level of phosphorylation MEK in ssRK/P#1 and ssRKIP#4 cells were 0. 35, 0. 34; while the expression level of phosphorylation ERK in ssRKIP # 1 and ssRKIP # 4 cells were 0. 48 and 0. 46. （ 3 ） Abilities of cell proliferation in the ssRKIP vector-transfected cells were decreased compared with that in the non-transfected cells （ P 〈 0. 01 ）. （4） Anchorage-independent growth in the ssRKIP#1 and ssRKIP#4 cells （ 83.7 ± 5.7, 106. 0± 9. 2 ） were decreased compared with that in the empty vector-transfected cells （158.3 ±14. 6, P 〈 0. 01 ）. （5） Cell adhesion in the ssRKIP#1 and ssRKIP#d cells [ （ 68.3± 0. 8 ） % , （ 64. 1 ± 0. 9 ） % ] were decreased compared with that in the non-transfected cells [ （ 100. 0 ± 1.1 ） %, P 〈0. 01 ]. （6） Cell invasion in the ssRKIP#1 and ssRKIP#4 cells （24 ± 5, 25 ±4） were decreased compared with that in the nontransfected cells （ 68± 5, P 〈 0. 01 ）. （ 7 ） ssRKIP cells had a significant increase in the G1 phase and decrease in the G2 ＋ S phase. Conclusion RKIP could inhibits the metastasis, but also the growth of ovarian cancer cells.