肝癌中高表达的新基因LAPTM4B对细胞增殖及成瘤性的影响

Effects of the novel gene, LAPTM4B, highly expression in hepatocellular carcinoma on cell proliferation and tumorigenesis of NIH3T3 cells

目的 :研究在肝细胞癌中高表达的新基因LAPTM4B对小鼠NIH3T3细胞生物学行为的影响 ,探讨LAPTM4B基因的生物学功能。方法 :构建真核表达质粒 pcDNA3 TM 4B ,利用脂质体稳定转染LAPTM 4BcDNA至NIH3T3细胞中 ,用RT -PCR、Northern和Western杂交法筛选和鉴定转染后LAPTM4B基因高表达的单克隆细胞株 ,研究其与细胞增殖相关的生物学特征的变化。结果 :与转染空载体的对照组相比 ,转染了LAPTM4BcDNA的NIH3T3细胞表面发生明显变化 ,微绒毛的数量和长度增加 ,多分枝且缠绕成团。转染细胞在纤黏连蛋白、人工基膜和层黏连蛋白基质上的黏附 /铺展能力增强。生长曲线显示转染细胞的生长速率增高。流式细胞仪检测显示转染细胞的S期细胞数比未转染细胞显著增多 ,而G0 ～G1期细胞数减少。Western杂交显示CyclinE蛋白在转染细胞中的表达显著增加。转染细胞对血清的依赖性下降并具有成瘤性。结论 :LAPTM 4B基因能影响细胞增殖的调节 ,促进NIH3T3细胞的增殖 ,并使NIH3T3细胞具有成瘤性 ,在肿瘤发生过程中具有重要的作用。

Objective: To study the biological effects of the novel gene LAPTM4B high expressed in hepatocellular carcinoma on cell proliferation and tumorigenesis of NIH3T3 cells. Methods: Eukaryotic expression plasmid pcDNA3 TM4B was constructed and transfected into mouse NIH3T3 cell line using Lipofectamin 2000 mediating gene transfer technique. Monoclonal transfected cells with high expression of LAPTM4B gene were identified and selected by RT-PCR, Northern blot and Western blot method. Cell surface morphology was detected by scanning electronic microscopy (SEM). The cell attachment /spreading on various matrices was examined. The cell growth curves were measured by acid phosphatase method. Cell cycle was detected by flow cytometry (FCM). The expression level of Cyclin E protein was examined using Western blot. Results: The cell surface of the LAPTM4B transfected cells showed abundant tube/finger like microvilli, which were distinguished significantly from the MOCK cells. The cell attachment/spreading of transfected cells increased on fibronectin, matrigel and laminin. The growth rate of transfected cells was faster than that of the MOCK cells. FCM analysis indicated that the amount of the transfected cells in S phase was increased significantly. Cyclin E protein expression of transfected cells was much higher than that of the MOCK cell. The serum dependence of transfected cells was decreased. The fibrosarcoma was formed at a tumorigenic rate of 50% in NIH mice inoculated with LAPTM4B transfected cells.Conclusion: LAPTM4B gene promotes the cell proliferation by involving in the regulation of cell cycle control and causes tumorigenesis of NIH3T3 cells, indicating that it plays important roles in tumorigenesis.