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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。
AIM: To investigate the mechanism of SEMA3B as a potential therapeutic agent for inducing apoptosis of lung cancer cells. METHODS: Cos7 cells were transfected with pcDNA3.1 / pcDNA3.1-SEMA3B plasmid and Cos7 cells stably expressing SEMA3B were screened by 400μg / ml G418. The effect of SEMA3B protein on Akt phosphorylation and downstream gene expression and its molecular mechanism were detected by using culture supernatant containing SEMA3B-Cos7 cells, specific siRNA, real-time quantitative PCR and Western blotting. Results: Cos7 cells stably expressing SEMA3B in cell culture supernatant and cell lysate were established. After treated with the culture supernatant of Cos7 containing SEMA3B protein and NCIH1299 cell culture supernatants for 24 hours, SEMA3B protein group Significantly reduced the expression of p Akt-ser ~ (473) and MDM2 mRNA in H1299 cells and increased the expression of Caspase3 mRNA; blocking the expression of IGFBP6 with specific siRNA partially abolished the effect of SEMA3B on Akt phosphorylation Effect on MDM2 and Caspase3. Conclusion: SEMA3B can induce the apoptosis of non-small cell lung cancer cells through the inhibition of Akt phosphorylation and its downstream gene expression through IGFBP6, further suggesting that the tumor suppressor effect of SEMA3B and its potential in non-small cell lung cancer Treatment of drug value.