| BackgroundSo far, vascular endothelial growth factor (VEGF) is known as the most importantpositive angiogenesis factor, which can stimulate vascular endothelial cell proliferation,strengthen blood vessel’s permeability, and promote the occurrence and metastasis of tumorcells. In1971, Folkman first put forward the viewpoint of angiogenesis in tumor. Soonafter, VEGF signal pathway had been identified as one of the anti-tumor therapeutic targets.In solid tumors, VEGF signal pathway had been thoroughly researched, such as theextracellular signal regulating kinase Raf/MEK/ERK, PI3K/AKT signal pathway,p38MAPK, JNK/STAT signal pathway, NF-κB, DLL4-Notch signal pathway and so on.And in leukemia, the similar situation happens, there are significant increased bloodvessels, it performances as increased bone marrow microvessel density, and accompaniedby the increased expression level of VEGF. But there are still many questions about thesecretion and expression of VEGF in leukemia cells, we still have no ideas about whichfactor can target-regulate VEGF in the proliferation and differentiation of leukemia cells.ObjectiveBased on the theoretical background talked above, this project first established cellmodel of ATRA induced HL-60cell differentiation, and at the same time VEGF level wasdecreased. And we proceeded on to screeded from several tumor related transcriptionfactor, and find the upscream key factor that can regulate the expression VEGF. We meantto explore the key molecular mechanism of VEGF expression, which can break a path forleukemia treatment.MethodsTake logarithmic phase HL-60cell as experimental subject by adding1μM ATRA for 96hours, incubating in37℃,5%CO2saturated humidity, morphology was observed byWright-Giemsa staining, cell differentiation was detected by NBT reduction experiment,CD11b expression and the cell cycle were measured by flow cytometry, cell proliferationwas determined by MTT colorimetry, immunocytochemical technique was to detect theexpression of VEGF and c-myc in HL-60cell. CD11b、VEGF、HIF-1α、STAT3、c-myc、P53、VHL、COX-2、SP-1and AP-1mRNA were measured by Reverse Transcription-PCR,VEGF、STAT3and c-myc proteins were determined by Western blot. RNA interferenceexperiment checked the relationship between VEGF and c-myc or STAT3. Furthermore,Chromatin Immunoprecipitation confirmed that c-myc is binding onto the promoter ofVEGF gene. Dates are expressed as mean and standard deviations. Comparisons betweenthe treatments and control were performed using the t-test and one-way anova. AP-value<0.05was considered to be statistically significant, A P-value<0.01was consideredto be significant difference.Results1μM ATRA treated for96hours can obviously restrain HL-60cell proliferation, andin addition, the cells appeared apparent differentiation tendency under the invertedmicroscope, which growed like mature granulocyte; The expression of VEGF and c-myc incytoplasm of HL-60cell was remarkable decreased; In cell cycle the rate was as high as77.31%located in G0/G1phase; The expression level of cell surface marker CD11bsignificantly increased which was detected by Flow cytometry (P<0.01); NBT positive ratewas82.59%(P<0.01); VHL mRNA didn’t have obviously change (P>0.05), HIF-1α、P53、COX-2、SP-1and AP-1mRNA levels were increased (P<0.05); VEGF、STAT3、c-myc mRNA and protein expression levels were obviously decreased (P<0.05); RNAiresults showed that when c-myc gene expression was blocked, VEGF gene expressionlevel decreased obviously; ChIP experiment furtherly verify c-myc is binding onto thepromoter of VEGF gene.ConclusionsIn the model of ATRA induced HL-60cell differentiation, VEGF expression level wasreduced with the procession of HL-60cell differentiation, which may be largely correlatedwith the down regulation of c-myc and STAT3. And additional, c-myc is binding onto thepromoter of VEGF gene. Maybe HIF-1α、P53、VHL、COX-2、SP-1and AP-1gene actas transcription repressors in the activity of VEGF regulation, and maybe they are largely relevant to cell differentiation. |
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