SETD4对脂多糖诱导的AML12细胞IL-6转录的调控作用

孙江; 李月; 牛世贤; 黄梦怡; 钟玙沄; 赵舒祺; 罗海华; 姜勇; 刘靖华

doi:10.13418/j.issn.1001-165x.2018.03.013

中国临床解剖学杂志 ›› 2018, Vol. 36 ›› Issue (3) : 282-287. DOI: 10.13418/j.issn.1001-165x.2018.03.013

实验研究

SETD4对脂多糖诱导的AML12细胞IL-6转录的调控作用

孙江，　李月，　牛世贤，　黄梦怡，　钟玙沄，　赵舒祺，　罗海华，　姜勇，　刘靖华

作者信息 +

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

SUN Jiang, LI Yue, NIU Shi-xian, HUANG Meng-yi, ZHONG Yu-yun, ZHAO Shu-qi, LUO Hai-hua, JIANG Yong, LIU Jing-hua

Author information +

文章历史 +

摘要

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。

Abstract

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 pcDNA3.0/HA-SETD4 and pGL3.0/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 LPS-induced IL-6 transcription in AML12 cells.

导出引用

孙江,李月,牛世贤,黄梦怡,钟玙沄,赵舒祺,罗海华,姜勇,刘靖华. SETD4对脂多糖诱导的AML12细胞IL-6转录的调控作用[J]. 中国临床解剖学杂志. 2018, 36(3): 282-287 https://doi.org/10.13418/j.issn.1001-165x.2018.03.013

SUN Jiang, LI Yue, NIU Shi-xian, HUANG Meng-yi, ZHONG Yu-yun, ZHAO Shu-qi, LUO Hai-hua, JIANG Yong, LIU Jing-hua. Effect of SETD4 on LPS-induced IL-6 transcription in AML12 cells[J]. Chinese Journal of Clinical Anatomy. 2018, 36(3): 282-287 https://doi.org/10.13418/j.issn.1001-165x.2018.03.013

参考文献

[1] Dillon SC, Zhang X, Trievel RC, et al. The SET-domain protein superfamily: protein lysine methyltransferases[J]. Genome Biology, 2005, 6(8):227-240.
[2] Dan L, Kuo AJ, Chang Y, et al. Lysine methylation of the NF-κB subunit RelA by SETD6 couples activity of the histone methyltransferase GLP at chromatin to tonic repression of NF-κB signaling[J]. Nat Immunol, 2011, 12(1):29-41.
[3] Xia M, Liu J, Wu X, et al. Histone methyltransferase Ash1l suppresses interleukin-6 production and inflammatory autoimmune diseases by inducing the ubiquitin-editing enzyme A20[J]. Immunity, 2013, 39(3):470-481.
[4] 叶萍, 蔡军伟, 付晓霞, 等. p38信号通路对SETD4在细胞中的表达和定位的影响[J]. 中国病理生理杂志, 2012, 28(5):878-883.
[5] 雷烨铭, 崔航, 钟玙沄, 等. SETD4蛋白在Bac-to-Bac杆状病毒系统的表达[J]. 中国临床解剖学杂志, 2015, 33(4):426-429.
[6] 黄穗, 黄梦怡, 钟玙沄, 等. SETD4基因敲除小鼠的构建及鉴定[J]. 中国临床解剖学杂志, 2016, 34(2):202-207.
[7] Faria JA, Corrêa NC, de Andrade C, et al. SET domain-containing protein 4 (SETD4) is a newly identified cytosolic and nuclear lysine methyltransferase involved in breast cancer cell proliferation[J]. J Cancer Sci Ther, 2013, 5(2):58-70.
[8] Dai L, Ye S, Li HW, et al. SETD4 regulates cell quiescence and catalyzes the trimethylation of H4K20 during diapause formation in artemia[J]. Mol Cell Biol, 2016, 37(7):MCB.00453-16.
[9] Damas P, Ledoux D, Nys M, et al. Cytokine serum level during severe sepsis in human IL-6 as a marker of severity[J]. Ann Surg, 1992, 215(4):356-362.
[10]Orphanides G, Reinberg D. A unified theory of gene expression[J]. Cell, 2002, 108 (4): 439-451.
[11]Villard J. Transcription regulation and human diseases[J]. Swiss Medical Weekly, 2004, 134(39-40):571-579.
[12]Cohn O, Feldman M, Weil L, et al. Chromatin associated SETD3 negatively regulates VEGF expression[J]. Sci Rep, 2016, 6(11):37115.
[13]Francisca AP, Victoriano M, Cayuela ML. Application of the dual-luciferase reporter assay to the analysis of promoter activity in Zebrafish embryos[J]. Bmc Biotechnol, 2008, 8(1):81-92.
[14]Brasier AR, Tate JE, Habener JF. Optimized use of the firefly luciferase assay as a reporter gene in mammalian cell lines[J]. Biotechniques, 1989, 7(10):1116-1122.
[15]Zhang Y, Reinberg D. Transcription regulation by histone methylation: interplay between different covalent modifications of the core histone tails[J]. Genes Dev, 2001, 15(18):2343-2360.
[16]Cao R, Zhang Y. The functions of E(Z)/EZH2-mediated methylation of lysine 27 in histone H3[J]. Curr Opin Genet Dev, 2004, 14(2):155-164.