摘要
目的:研究SEMA3B作为潜在的肺癌治疗药物诱导细胞凋亡作用的机制。方法:将pc DNA3.1/pc DNA3.1-SEMA3B质粒转染至Cos7细胞,400μg/ml G418筛选后建立稳定表达SEMA3B的Cos7细胞系。利用含有SEMA3B-Cos7细胞的培养上清液、特异性siRNA、实时定量PCR和蛋白免疫印迹方法,检测SEMA3B蛋白对Akt磷酸化及下游基因表达的影响及分子机制。结果:在细胞培养上清及细胞裂解液中稳定表达SEMA3B的Cos7细胞模型建立;利用含有SEMA3B蛋白的Cos7培养上清液及对照组培养上清液处理NCIH1299细胞24小时后,含有SEMA3B蛋白组可显著降低H1299细胞内p Akt-ser^(473)的蛋白表达及下游基因MDM2 mRNA表达,增加Caspase3 mRNA表达;特异性siRNA阻断IGFBP6表达,可部分消除SEMA3B抑制Akt磷酸化的作用,并影响其对MDM2和Caspase3的作用。结论:SEMA3B是通过IGFBP6介导其抑制Akt磷酸化作用及其下游基因表达,而发挥诱导非小细胞肺癌细胞凋亡的作用,进一步提示在非小细胞肺癌中SEMA3B的抑癌作用及其潜在的治疗药物价值。
Objective: To study the role of SEMA3 B as a novel therapeutic agent for non- small cell lung cancer.Methods: Cos7 cells were transfected with the vector pc DNA3. 1 / pc DNA3. 1- SEMA3 B and selected with 400μg / ml G418,following transfection to generate a stable expressed SEMA3B- Cos7 cell line. We employed SEMA3B- Cos7 cell supplemented medium,specific siRNA,RT- PCR and western blot to investigate the role of SEMA3 B on Akt phosphorylation and downstream gene expression of MDM2 and Caspase3 in NCI- H1299 cells. Results: SEMA3 B protein was detected in both the supernatant and cell lysate of SEMA3B- Cos7 cells. SEMA3 B treatment significantly decreased the p Akt- 473 expression and downstream oncogene mRNA expression of MDM2,notably increased the Caspase3 expression in NCI- H1299 cell,following treatment with the supernatant of SEMA3B- Cos7 cells after 24 h.Inhibition of IGFBP6 by siRNA knockdown partly abrogated the SEMA3 B activity on Akt- 473 phosphorylation and the downstream gene expression of MDM2 and Caspase3. Conclusion: The induced apoptosis effect of SEMA3 B is mediated by IGFBP6- dependent suppression of Akt phosphorylation activity. The results further support the tumor suppressive role of SEMA3 B in non- small cell lung cancer and its potential as a therapeutic agent.
出处
《现代肿瘤医学》
CAS
2016年第12期1854-1857,共4页
Journal of Modern Oncology
基金
国家自然科学基金项目资助(编号:81160253)
内蒙古自治区自然科学基金资助(编号:2011MS1158)
