| Background：Because of the significant effect of proliferation suppression, apoptosis promotion andchemotherapy side effects alleviation for hematologic malignancies including lymphoma,Dexamethasone (Dex) is widely used in clinical treatment. Interferon (IFN) is a pleiotropiccytokine, which is effective in the cure of lymphoma. Nevertheless, the mechanism remainselusive. It’s known that it has antitumor effects only when Dex binds to GlucocorticoidReceptors (GR); moreover, STAT is one of target genes of IFN. Bioinformatics analysisshows that both GR and STAT might bind to SARI (suppressor of AP-1regulated byinterferon) promoter region. SARI is a tumor suppressor gene discovered recently, which ishighly expressed in normal cells rather than the corresponding tumor cells. It couldspecifically suppress tumor cells proliferation without killing normal cells, which providesit a bright prospect in the field of tumor biotherapy. Up to now, the molecular mechanism ofits antitumor effect is still not clarified and the upstream gene of SARI hasn’t been reported.Objective：To explore the effects of Dexamethasone and Interferon on lymphoma cellsproliferation and SARI expression and to investigate the possible molecular mechanism.Methods：(1) Human lymphoma cell line (Namalwa, JeKo-1) and monocytic cell line (THP-1)were treated with GR agonist Dex alone or combined with GR antagonist Mifepristone(Mfp)of different doses. Cell proliferation was measured by CCK-8assay. SARI and its targetgenes p53and p21expression were detected by RT-PCR, Real time-PCR and Western blotat transcriptional and translational level.(2) GR binding sites in the SARI promoter region were analyzed by bioinformatcis. SARI promoter deletion constructs were constructed with molecular cloning technique.After having been transfected with the constructs, cells were treated with Dex alone orcombined with Mfp. Subsequently, SARI promoter viability was measured by luciferaseassay. Whether GR could directly bind to SARI promoter region was confirmed by EMSAand ChIP in extracellular and intracellular ways, separately. With the binding site mutated,SARI promoter viability was surveyed by luciferase assay.(3) Namalwa cells were injected subcutaneously or intravenously to establish twokinds of Nude mice tumor xenograft model. Each kind was treated with PBS or Dex andappearance of hindlimb paralysis or death would be the endpoint. Tumor tissue weight wasweighed and volume was measured. Blood smear and tumor tissue slice were used toevaluate the success of the model establishment. Lymphocytes from peripheral blood andtumor tissue were collected to extract total RNA and total protein, which were assayed byRT-PCR, Real time-PCR and Western Blot. SARI protein was also checked byimmunohistochemisty(4) After transfected with siSARI, cells were treated with Dex to examine SARI, p53and p21expression. The cell viability was assayed by CCK-8. AP-1activity was detectedby EMSA.(5) Lymphoma cells (Namalwa and JeKo-1) and leukemia cells (Jurkat and THP-1)were treated with IFN-α/β for24h. Cell viability was measured by CCK-8assay. Theexpression of SARI at mRNA level and protein level were tested by RT-PCR, Realtime-PCR and Western blot. The transcription factor binding sites in SARI gene promoterregion were predicted using bioinformatics methods.Results：(1) Dex decreased lymphoma cells (Namalwa, JeKo-1) proliferation and induce SARImRNA and protein expression dose-dependently, which were reversed by Mfp.Nevertheless, neither Dex nor Mfp had significant effect on cell proliferation and SARIexpression of THP-1cells, which served as control cell line.(2) Dex increased SARIpromoter activity, which was attenuated by Mfp.(3) GR could directly bind to a cis-actingelement of SARI promoter region, ER9(-178TGTCCTccctcccctAGAACA-158).(4) Dexcould significantly repress Namalwa lymphoma proliferation; prolong the survival time ofnude mice and upregulate SARI mRNA and protein expression in tumor tissue and lymphoma cells from blood of the xenograft nude mice.(5) With SARI inhibited by siRNA,Dex could no longer inhibit lymphoma cells proliferation; the AP-1activity and its targetgene p53/p21expression were rescued as well.(6) Lymphoma cells (Namalwa and JeKo-1)were effectively killed by both IFN-α and IFN-β, meanwhile, the expression of SARImRNA and protein were induced by these two IFNs. However, IFN-α/β had no significanteffect on cell viability and SARI expression of leukemia cells (Jurkat and THP-1).Transcription factor STAT may bind to SARI gene promoter region.Conclusions：(1) GR is one of the upstream genes that could directly regulate SARI.(2) GR ligandDexamethasone activated GR could upregulate SARI gene expression and suppresslymphoma cells proliferation, which could be reversed by GR antagonist Mfp.(3)Upregulation of SARI may be one of the novel mechanisms by which Dex plays ananti-lymphoma effect. GR-SARI signaling pathway might be a novel target for lymphomabiotherapy and a biomarker to test the sensitivity of Dex.（4）Dex could inhibit lymphomacells proliferation and regulate AP-1activity and its target genes expression in aSARI-dependent manner.(4) The induction of SARI by IFN-α/β may be mediated by STAT. |
PDF Full Text Request