The Expression Of HGF In Acute Leukaemia And Its Mechanism

Objective:To investigate the expression and functions of hepatocyte growth factor(HGF)in human acute myelocytic leukemia(AML)cells.Methods:The m RNA and protein level of HGF and its related genes(c-Met、HGFA、HAI-1、HAI-2、Matriptase)in AML cells was detected by q RT-PCR and Western-blot.The expression level of these genes in the remission patients group,untreated patients group and healthy control group was evaluated.The relationship between the HGF pathway gene expression profiles to clinical data was analyzed.In Kasumi and HL-60 cells,HGF was knocked down using sh RNA,the effectiveness of gene silence was confirmed by q RT-PCR and western-blot.The influence of HGF knock-down on the biological behaviors of AML cells was evaluated.CCK-8 assay and colony formation assay were performed to detect the proliferative ability of AML cells.Flow cytometry was used to detect cell cycle and apoptosis.The morphology of apoptotic cells was observed using DAPI staining method.Cell invasion potent was evaluated by Transwell chamber method.The downstream signal transduction of HGF pathway was analyzed by western-blot method.Result:1.The relative expression median of HGF in untreated group,remission group and healthy control group was 0.008096、0.001271、0.002598 respectively.The expression of HGF in untreated group is higher than remission group and healthy control group(p <0.05).The median relative expression of c-Met in untreated group,remission group and healthy control group was 0.0004972、0.003988 and 0.0002308 respectively.The expression of c-Met in untreated group is higher than remission group and healthy control group(p <0.05).The m RNA level of HGFA in untreated group is higher than in healthy control group.The m RNA level of HAI-2 in untreated group is lower than in healthy control group.No obvious difference has been observed in the expression patterns of HAI-1 and Matriptase amoung all 3 groups.No direct relationship of HGF expression with the gender,age,hemoglobin,blood platelet and the first remission status of the patients has been found.High level of HGF has been detected in patients with high WBC,or with adverse prognosis,the p value is 0.0405、0.009 respectively.HAI-2 expression is high in patients with high WBC(p=0.03902),HAI-1 level is high in patients with adverse prognosis(p=0.0462).2.Stable HGF knock-down Kasumi-1、HL-60 cell lines were established,the m RNA and protein levels of HGF were reduced significantly in knock-down cells.3.Silence of HGF gene expression showed influence on the growth,apoptosis and invasion behaviors.a.The cell growth inhibition rate is 54.16% in HGF knock-down Kasumi cells and 49.04% in HGF knock-down HL-60 cells compared with untransfected cells.b.Knock down of HGF resμlted in inhibited colony formation ability.The colony formation inhibition rate is 34.47% in Kasumi cells and 57.14% in HL60 cells.c.Silence of HGF gene in both cells induced cell cycle arrest in G2/M phase.d.The apoptotic cell percentage increased significantly in HGF sh RNA transfected cells(p <0.05).e.FN adhesive assay and transwell chamber experiment also demonstrate that knock-down of HGF lead to decreased migration and invasion abilities.f.Western-blot resμlts showed that in Kasumi cells,silence of HGF gene down regulated expression of Bad、Bcl-XL、Bcl-2、CDK1、Cyclin B、MMP2、MMP9、p-c-Met、p-AKT、p-Erk、p-m TOR,upregμlated expression of cleaved caspase9、cleaved caspase3、Bax、P21、p-Chk1、p-cdc25、p-PTEN.No significant change has been observed in the protein level of t-Akt、Bak、t-Raf、MMP7、pro-caspase8.In HGF sh RNA transfected HL60 cells,protein amount of Bcl-2、p-PTEN、p21 showed little change,while the expression of Bax and p-GSK3 beta was enhanced.Conclusions:(1)High levels of HGF and c-Met are expressed in AML cells.The expression levels are related to prognosis and the amount of WBC.(2)HGF is possible to induce proliferation and invasion of AML cells via PI3K-AKT and MAPK/ERK pathway.Downregμlation of HGF may lead to cell cycle arrest at G2/M phase,induction of caspases-mediated apoptosis and decreased invasion capability via PI3K-AKT and MAPK/ERK pathway.