SETD4对脂多糖诱导的AML12细胞IL-6转录的调控作用

Effect of SETD4 on LPS-induced IL-6 transcription in AML12 cells

目的探讨SETD4（SET-domain containing protein 4）对脂多糖（LPS）诱导的AML12（小鼠肝脏细胞系）细胞IL-6的转录调控作用。方法构建SETD4表达质粒和IL-6启动子荧光素酶报告基因质粒,共转染小鼠肝脏细胞系AML12,使用双荧光素酶报告基因技术检测LPS刺激后IL-6启动子荧光素酶活性;单独转染SETD4表达质粒上调AML12细胞SETD4表达,检测LPS刺激后IL-6 mRNA水平变化。结果双酶切鉴定及核酸测序证实重组质粒pcDNA3.0/HA-SETD4、pGL3.0/IL-6 promoter的构建成功。pcDNA3.0/HA-SETD4与pGL3.0/IL-6 promoter共转染AML12细胞,LPS刺激后SETD4对IL-6启动子荧光素酶活性没有影响;上调SETD4表达后,可以促进LPS诱导的IL-6 mRNA转录。结论成功构建SETD4真核表达质粒和IL-6启动子荧光素酶报告基因质粒;SETD4对LPS诱导的AML12细胞IL-6的转录发挥正调控作用。

Objective To explore the effect of SETD4（SET-domain containing protein 4） in IL-6 transcription in AML12 cells（murine hepatocyte line） stimulated with LPS. Methods Recombination plasmids encoding SETD4 or IL-6 promoter luciferase were constructed and cotransfected into AML12 cells.After LPS stimulation, the luciferase activity of IL-6 promoter was detected by Dual-luciferase reporter assay system. The LPS-induced IL-6 mRNA in AML12 cells with SEDT4 over-expression was also measured.Results The plasmids of pc DNA3.0/HA-SETD4 and p GL3.0/IL-6 promoter were constructed successfully,evidenced by double restriction enzyme digestion identification and DNA sequencing. The results showed that SETD4 have no effect on luciferase activity when IL-6 promoter luciferase plasmid and SETD4 plasmid were cotransfected into the AML12 cells following LPS stimulation. Over-expression of SETD4 could up-regulate the LPS-induced IL-6 mRNA transcription in AML12 cells. Conclusion SETD4 expression plasmid and IL-6 promoter luciferase plasmid were constructed successfully. SETD4 may play a positive effect on LPSinduced IL-6 transcription in AML12 cells.