Low Expression Of GFI-1 Gene Is Associated With Panobinostat-resistance In Acute Myeloid Leukemia Through Influencing The Level Of HO-1

Objective: This experiment was designed to investigate the role of growth factor independent 1 transcriptional repressor(GFI-1)in acute myeloid leukemia(AML).We found that low expression of GFI-1 could affected the Panobinostat-resistance in acute myeloid leukemia(AML).To improve the treatment outcomes of AML,We further explored the correlation between GFI-1 and heme oxygenase-1(HO-1)upon chemotherapeutic response and the underlying mechanism.Methods: This experiment was divided into two parts: clinical testing and in vitro experiments.In clinical testing,we selected 64 patients in bone marrow biopsies from obtained from newly diagnosed AML patients(excluding AML-M3).Meanwhile,23 donors were selected as a control group.Separately extracted the mononuclear cells in these samples,and extracted their mRNA and proteins or further culture blasts according to experimental requirements.The TCGA database compares the effect of untreated GFI-1 and heme oxygenase-1(HO-1)on overall survival(OS).Real-time PCR and Western blot were used to detect the levels of GFI-1 and HO-1 in mononuclear cells between AML patients and normal controls.Cell apoptosis was detected by flow cytometry,and cell viability was detected by CCK8.In the in vitro experiment,we used GFI-1 siRNA(si-GFI-1)to reduce the levels of GFI-1 in HL-60 and kasumi-1 cell lines,and then used Panobinostat to treat si-GFI-1 group and the control group each 24 hours,respectively.Flow cytometry and CCK8 assays modulate the effect of GFI-1 levels on the effects of Panobinostat in AML cells.Western blot was used to detect the effect of GFI-1 on cell apoptosis induced by Panobinostat.Next we used ZnPP and hemin to further regulate HO-1 levels in AMLcells.Western blot was used to detect the levels of GFI-1,HO-1,HDAC1,HDAC2,HDAC3 and apoptotic proteins in AML cell lines,treated with Panobinostat or combined treatment with si-GFI-1,ZnPP and hemin.Flow cytometry and CCK8 were used to detect changes in apoptosis rate and cell viability of AML cell lines.Next we conducted an exploration of the pathway mechanism and used LY294002 as an inhibitor of the PI3K/Akt pathway.Western blot tested the level of GFI-1,HO-1,and tested protein levels associated with apoptosis and PI3K/Akt pathway when PI3K/Akt was inhibited by LY294002.Apoptosis rate was detected by flow cytometry,and CCK8 was used to detect cell viability.Results: In this study,We detected the transcription levels of GFI-1 in bone marrow biopsies from obtained from newly diagnosed AML patients and followed up the treatment outcomes.After two courses of induction,we selected 32 patients in complete remission(CR)stage and another 32 in relapsed or resistant(relapsed)stage.CR stage and control groups had similar GFI-1 mRNA expression levels which were significantly higher than that of the relapsed stage group.The protein expressions of GFI-1 had the same results.Meanwhile,we tested the mRNA expression of HO-1 in the two groups.There was an inverse correlation between the expression of GFI-1 and HO-1,so such level may be related with GFI-1 in AML patients.In vivo study,with decreasing GFI-1 expression,the mRNA levels of HO-1 were increased.CCK-8 assay showed that reducing HO-1reversed the increased chemoresistance of AML cells in which GFI-1 was knocked down by si-GFI-1,and enhancing HO-1 promoted the sensitivity of Panobinostat.We also found that PI3K-Akt pathway involved in HO-1 induction by GFI-1 and hint that GFI-1could part influence phosphorylation of PI3K/Akt.Conclusion: GFI-1 is a novel index of poor prognosis in AML.It suggested that the resistance of Panobinostat to AML cells at low level of GFI-1 was mainly due to up-regulated level of HO-1 through the PI3K-Akt pathway.Taken together,GFI-1 regulated HO-1probably via the PI3K/Akt pathway.