Study On The Expression Of IL-15 Induced By IL-6 And LPS And Identification Of The Promoter Regulatory Elements In HaCaT Cells

Objective (1) To explore the effect and mechanism of IL-6 and LPS on the expression of IL-15 in HaCaT cells; (2) To construct series of luciferase reporter gene vectors containing IL-15 promoter regions, analyze the activity of IL-15 gene promoters and identify the key regulatory elements.Method (1) HaCaT cells were cultured and treated with IL-6 and LPS. Real-time PCR was used to study the expression of IL-15 mRNA and Western-Blot was used to study the expression of IL-15 protein. We also analyzed signaling pathways induced by IL-6 and LPS by Western-Blot, and then used the specific inhibitors to selectively inhibit ERK and AKT phosphorylation to discusse the relationship between the expression of IL-15 and these relative signaling pathways. (2) Seven DNA fragments of IL-15 promoter were isolated from genomic DNA of HaCaT cells by PCR. After digested with restriction enzymes, IL-15 gene family promoters were inserted into the luciferase reporter vector pGL3-Basic to construct series of luciferase reporter gene vectors containing IL-15 promoter regions.The recombinant constructs were transiently transfected into HaCaT cells and were respectively treated with IL-6 and LPS. Then, the activity of IL-15 promoter was detected by dual-luciferase reporter gene system to identify the key regulatory elements.Results (1) In HaCaT cells, IL-6 and LPS both induced the expression of IL-15 mRNA and protein, and induced the phosphorylation of ERK1/2 and AKT. When the phosphorylation of ERK1/2 or AKT was blocked by the specific inhibitors, the expression of IL-15 induced by IL-6 and LPS decreased. (2) Series of IL-15 gene promoters were amplified by PCR, and the identification of PCR and double digestion of recombined plasmid was completely correct. The experiment of transient transfection showed that the largest IL-15 promoter region has the significant activity after the stimulation of IL-6 and LPS.Conclusion (1) The expression of IL-15 up-regulated by IL-6 and LPS is associated with MAPKs-ERK1/2 and PI3K-AKT signaling pathways in HaCaT cells. (2) Series of IL-15 gene promoter luciferase reporter gene vectors have been constructed successfully.