Rap1蛋白在卵巢浆液性癌组织中的表达及抑制细胞增殖的研究

Rap1 expression in serous ovarian cancer and its inhibitory effect on cell proliferation

目的探讨卵巢浆液性癌组织中Rap1蛋白表达及其在浆液性癌细胞增殖中的作用。方法 Western blot检测并比较Rap1在浆液性卵巢癌组织、卵巢良性组织和正常卵巢组织中的表达;应用RNA干扰技术构建3个Rapl shRNA真核表达载体,将3个重组质粒、空质粒、阴性质粒分别转染进卵巢癌细胞株SKOV3,通过Western blot对重组质粒进行有效性筛选,G418筛选建立SKOV3-Rap1-RNAi稳定转染细胞株;MTT法检测Rap1表达缺失的SKOV3细胞增殖能力的变化。结果与卵巢良性组织和正常卵巢组织相比,浆液性卵巢癌组织中Rap1呈现高表达(P<0.05)。经双酶切和DNA测序证实成功构建了3个重组质粒pSUPER.retro.neo/GFP-Rap1i-#1,-Rap1i-#2及-Rap1i-#3;瞬时转染提示上述3个质粒的Rap1抑制效率分别是50%、66%、19%,遂最终采用pSUPER.retro.neo/GFP-Rap1i-#2转染并用G418筛选出单克隆细胞Rap1i-1和Rap1i-2,其Rap1蛋白抑制率分别为70%和48%,选取Rap1i-1细胞进一步检测Rap1表达缺失的SKOV3细胞增殖能力的变化,发现其增殖能力较对照组更强(P<0.05)。结论 Rap1蛋白在卵巢浆液性癌组织中呈高表达并抑制细胞增殖。

Objective To determine the expression of Rap1 and its role in the malignant growth in serous ovarian cancer. Methods Rap1 expression was evaluated through Western blot analysis in serous EOC tissues, benign ovarianadenoma tissues and normal ovarian tissues. Three Rapl shRNA eukaryotic expression vectors were constructed with RNA interference technology. Three recombinant plasmids, empty vector and negative plasmid were transfected into ovarian cancer cell line SKOV3 respectively through Lipofectamine 2000, then identified by restriction endonuclease digestion and DNA sequencing. The Rap1 siRNA efficiency was determined through monoclonal screening with G418 followed by Western blotting. Two strains of SKOV3-Rap1-RNAi cell lines were established. And malignant proliferation was evaluated by MTT assay. Results EOC tissues presented significantly higher Rap1 expression than benign ovarian tumors and normal ovarian tissues （P〈0.05）. For the 3 recombinant plasmids, pSUPER.retro.neo/GFP-Rap1i-#1, -Rap1i-#2 and -Rap1i-#3, their inhibitory efficiency in transient transfection was 50%, 66% and 49%, respectively. So the second one was used to transfect SKOV3 cells, and 2 monoclones of Rap1i cells （ Rap1i-1 and Rap1i-2 with inhibitory rate of Rap1 of 70% and 40% respectively） were established through pSUPER.retro.neo/GFP-Rap1i-#2 transfection followed by G418 screening. Rap1i-1 cells demonstrated enhanced proliferation as compared with control （P〈0.05）. Conclusion Rap1 is highly expressed in serous ovarian cancer, and exerts inhibitory effect on the proliferation of the cancer cells.