RESUMEN
<p><b>BACKGROUND</b>The transcription factor, repressor of GATA-3 (ROG), can simultaneously suppress the expression of T helper cells (Th1 and Th2) cytokines. Since the suppression of Th2 cytokines by GATA-3 is well understood, it is postulated that there are other molecular targets of ROG that can suppress the expression of the Th1 cytokines. We hypothesized that ROG might suppress the stimulators of T lymphocyte cytokines such as CD3, CD28, and inducible costimulator (ICOS), or indirectly enhance the expression of cytokine suppressors such as T lymphocyte-associated antigen-4 (CTLA-4) and CD45. The objective of this study was to clarify the molecular targets of ROG involved in suppressing Th1 or Th2 cytokines.</p><p><b>METHODS</b>Real-time quantitative PCR (RT-PCR) and Western blotting were performed to evaluate the mRNA and protein levels of CD3, CD28, ICOS, CTLA-4, and CD45 in Th1 and Th2 cells during various levels of ROG expression. Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of interferon-γ (IFN-γ) and interleukin (IL)-4 in culture media of Th1 and Th2 cells.</p><p><b>RESULTS</b>The results showed that the mRNA and protein levels of ROG were relatively low in Th1 and Th2 cells (P < 0.01). After ROG-pcDNA3.1 transfection, the mRNA and protein level of ROG was significantly elevated, while the expression of ICOS, IFN-γ, and IL-4 was markedly down-regulated (P < 0.01). Conversely, transfection of ROG-siRNA led to inhibition of ROG expression and up-regulation of ICOS, IFN-γ and IL-4 (P < 0.01). However, the expression levels of CD3, CD28, CTLA-4 and CD45 did not change in either ROG-pcDNA3.1 or ROG-siRNA-transfected Th1 and Th2 cells (P > 0.05).</p><p><b>CONCLUSION</b>It is concluded that ROG can inhibit the expression of Th1 and Th2 cytokines by down-regulating the expression of ICOS, which might be a potential molecular target for asthma treatment.</p>
Asunto(s)
Animales , Masculino , Ratones , Western Blotting , Antígenos CD28 , Metabolismo , Complejo CD3 , Metabolismo , Linfocitos T CD4-Positivos , Metabolismo , Antígeno CTLA-4 , Metabolismo , Células Cultivadas , Citocinas , Metabolismo , Ensayo de Inmunoadsorción Enzimática , Proteína Coestimuladora de Linfocitos T Inducibles , Metabolismo , Interferón gamma , Metabolismo , Interleucina-4 , Metabolismo , Antígenos Comunes de Leucocito , Metabolismo , Ratones Endogámicos BALB C , Reacción en Cadena en Tiempo Real de la Polimerasa , Proteínas Represoras , Genética , Metabolismo , Linfocitos T , Metabolismo , Células TH1 , Metabolismo , Células Th2 , MetabolismoRESUMEN
Objective: To investigate the regulatory role of LKB1 gene on expression of vascular endothelial growth factor (VEGF) in lung cancer cell line A549. Methods: DNA fragment encoding LKB1 protein was amplified by Nest-PCR from human fetal brain cDNA library and was sub-cloned into the eukaryotic expression vector pcDNA3. 1. The recombinant vector was transferred into A549 cells by Lipofectamine and screened by G418. RT-PCR and Western blot were used to study the expression of LKB1, SP1 and VEGF in A549 cells. Transcription factor SP1 was silenced by small interference RNA (siRNA); RT-PCR and Western blot were applied to examine the changes of SP1 and VEGF gene. Results: DNA sequencing analysis revealed that the open reading frame of LKB1 gene was successfully cloned into the expression vector. Stable cell line of A549 expressing exogenous LKB1 was constructed. LKB1 remarkably suppressed SP1 and VEGF expression. SiRNA targeting SP1 effectively decreased the expression of SP1 and SP1 silencing caused remarkable down-regulation of VEGF expression. Conclusion: LKB1 can negatively regulate the expression of VEGF by negatively regulate the expression of transcription factor SP1.