骨桥蛋白抑制FHIT表达促进结肠癌细胞增殖存活

摘要

Objective To construct an OPN eukaryotic expression vector,and evaluate its effects on proliferation and survival in SW480 colon cancer cells in vitro,exploring the possible mechanism. Methods OPN gene was cloned into the eukaryotic expression vector pEGFP Nl,after sequencing, the vector was then transfected into SW480 cells. The OPN gene and protein expression in transfected cells were demonstrated by RT PCR and Western blotting respectively. The impact on proliferation in transfected SW480 cells were investigated by CCK 8 method,anchorage independent growth was measured using soft agar assay. Western blot ting was used to analyzing the protein of fragile histidine triad gene(FHIT) and protein kinase B(PKB/Akt). Results The levels of OPN gene and protein in colon cancer SW480 cells were distinctly increased after transfection with pEGFP Nl/OPN. The OD450 value of OPN transfection group was higher than negative control group showed by CCK 8 method(P-<0. 05). They could also sig nificantly increase anchorage independent growth in vitro. FHIT protein was significantly increased in SW480 cells transfected with pEGFP Nl/OPN. Conclusion In SW480 colon cancer cells,OPN was involved in promotion proliferation and survival,by regula ting the FHIT protein expression.%目的 构建骨桥蛋白(OPN)基因真核表达载体,转染人结肠癌细胞SW480,研究其对SW480细胞增殖及生存能力的作用机制.方法 构建重组表达载体pEGFP-N1/OPN,转染人结肠癌SW480细胞.采用RT-PCR及免疫印迹法(Western blotting)分别检测SW480细胞OPN基因及蛋白表达量;Cell Counting Kit- 8(CCK-8)测定细胞增殖;软琼脂克隆形成实验观察细胞的锚定非依赖性生长能力.Western blotting检测脆性组氨酸三联体基因(FHIT)和蛋白激酶B(PKB/Akt)蛋白在SW480细胞中的表达.结果 重组质粒pEGFP-N1/OPN转染SW480后,OPN基因获得很好转录,OPN蛋白表达增高,细胞增殖率(光吸收值)显著高于转染阴性组(P＜0.05),细胞软琼脂克隆形成速度增快,数量明显多于对照组和空载体组(P＜0.05);FHIT蛋白在实验组表达降低,Akt蛋白在各组中的表达无显著性差异.结论 OPN基因通过抑制FHIT基因表达,促进SW480细胞增殖、独立存活能力.