RNAi Silencing HIF-1α Remoulds The Hematopoietic Microenviroment Of Acute Leukemia

Background and Objective:The abnormality of bone marrow hematopoietic microenviroment plays an important role in the genesis and progression of leukemia. Bone marrow stromal cells(BMSCs)are the mostly components of marrow microenviroment, its dysfunction directly cause the formation of abnormal marrow microenviroment. The process of leukemic marrow microenviroment's formation consist of the formation of marrow angigenesis ,abnormal hypoxia microenviroment and the parasecretion of chemotatic factors and adhesion molecules. Hypoxia inducible factor-1α(HIF-1α) is an imprtant transcription factor which play an important role in the formation of tumor and and its stroma.. HIF-1αinvolves the express regulation of VEGF and SDF-1, what's more, it also involves the genesis of angiogenesis,stroma hypoxia microenviroment and the regulation of cell's proliferation and apoptosis. HIF-1αmay be the key regulate factors of the bone marrow microenviroment.So in this study we constructed miR RNAi Vector targeting HIF-1αand silenced the HIF-1αof BMSCs,then studied the affection to proliferation,secreation and adhesion functions of BMSCs. We want to approach the regulate mechanism of HIF-1αin leukemic marrow microenviroment.Methods:1.Isolated and cultured the BMSCs of acute leukemia(AL), examined the expression of HIF-1αmRNA by RT-PCR, examined the expression and distribution of P53, PTEN and HIF-1αby immunohistochemical method, detected the expression of SDF-1 and VEGF by ELISA.2.Constructed the pcDNA?6.2-GW/EmGFP-HIF-1αmiR Vectors targeting HIF-1α, identified it by sequecing methods.Then transfected the vector into AL BMSCs mediated by lipofectamin 2000.Used blasticidine to Screened the positive clones and then expansion them. Examined the expression of HIF-1αmRNA by RT-PCR to identify the silencing effects and choosed the best one for the next procedure.3. Constructed the pLenti6/V5-GW/EmGFP- HIF-1α-miR Vector for lentiviral vector construction, transfected it into the 293FT cells mediated by lipofectamin 2000 and ViraPower? packaging plasmids so as to produce the lentivirus. Used the PT67 cells to Titer the lentivirus.Then infect the AL BMSCs with the lentivirus and screened the positive clones by blasticidine. Examined the expression of HIF-1αmRNA by Realtime RT-PCR and the expression of the HIF-1αprotein by western-blot.4.The experimental objects were divided into 3 groups: the miR RNAi infected group,the miR-neg infected group and the uninfected group.The expression of VEGF,SDF-1 and VCAM-1 were measured by ELISA. Cell proliferation of AL BMSCs was measured by MTT. Constructed the Jurkats-BMSCs cocultured system, detected the adhesion and proliferation abilities of Jurkat cells by cells counting methods. Detected the cell cycle and apoptosis by flow cytometer, examined the migration ability by Transwell.Results:1. Expression level of HIF-1αmRNA and the positive rate of HIF-1αprotein in AL group were higher than the control group(P<0.01), the positive rate of P53 protein was higher than the control group(P<0.01),while the positive rate of PTEN protein was lower than the control. HIF-1αprotein expression of stromal cells was associated with high P53 expression and low PTEN expression. Expression of SDF-1 and VEGF of AL BMSCs were higher than the control group(P<0.01), and the high expression tendency of the two moleculors was concord with the HIF-1αpositive expression.2.We successfully constructed the pcDNA?6.2-GW/EmGFP-HIF-1αmiR Vector and transfected it into the AL BMSCs,the HIF-1αmRNA after transfection was lower than the untransfected group(P<0.01), the results suggested that miR RNAi could silence the HIF-1αexpression.3.We successfully constructed the pLenti6/V5-GW/EmGFP- HIF-1α-miR Vector and produced lentivirus by trnasfeted it into the 293FT cells.The lentivirus could infected the AL BMSCs. The HIF-1αmRNA and protein expression level were lower that the untransfected group(P<0.01), which suggested that the lentiviral vector f or HIF-1αmiR RNAi could efficiently silence the HIF-1αexpression.4.The expression of SDF-1 and VEGF in miR RNAi infected group were 3127.25± 615.42pg/ml and 198.32±54.69pg/m, which were lower that the uninfected group(SDF-1 was 4785.23±568.24pg/ml,VEGF was 312.55±45.68 ).( P<0.01) The proliferation ability of miR RNAi infected group had no difference with the uninfected group(P>0.05).5.After 3 days coculturing with AL BMSCs, the doubling generation time of Jurkat cells in miR RNAi infected group was 50 hours,which is slower than the uninfected group(34hours). The percentage of S phase of Jurkat cells in miR RNAi infected group was reduced compared with the uninfected group,while the G2/M phase was increased( P<0.01).The apoptosis rate of Jurkat cells in miR RNAi infected group was(17.78±4.87) % , which is higher than the uninfected group (6.53±3.28)%)( P<0.01).After 24 hours coculturing with AL BMSCs,the adherent rate Jurkat cells in miR RNAi infected group was(28.35±5.91)%, which is lower than the uninfected group(47.58±6.63)%( P<0.01).The transwell experinet showed that the transmembrane cells in miR RNAi infected group was lower than the uninfected group( P<0.01).Conclusion:The AL BMSCs have high expression of HIF-1α.The miR RNAi Lentiviral Vector target HIF-1αcan efficiently silence the HIF-1αexpression of AL BMSCs.Blocking the HIF-1αexpression of AL BMSCs can inhibit the proliferation,adhesion and migration abilities of cocultured Jurkat cells and induce apoptosis.The signal pathway P53/PTEN→HIF-1α→VEGF/SDF-1 may be exist in the AL BMSCs.