Midkine Gene Expression Of Acute Leukemia And Leukemia Cell Proliferation Activity Of The Study

Objective:Midkine(MK),a heparin-binding growth factor detected in1988,is also called neurite growth-promoting factor 2.MK is stronglyexpressed in midgestation and plays important role in the developmentand differentiation of epithelial tissues,mesenchymal tissues and nervoustissues.The expression of MK in adults is too weak to be detected and isrestricted to vascular endothelial cells and mucus epithelial cells.Theoverexpression of MK gene was found in many kinds of humanmalignant solid tumors,and its expression level was negatively correlatedwith prognosis.However,the expression of MK gene in acute leukemia isless reported.In this study,we detected the expression of MK gene inacute leukemia patients.To analyze the expression of MK gene in variousacute leukemia(AL) patients and its clinical significance,explore thepossible role of MK in the development of AL,as well as furtherdetermine the role of AML1 and AML1-ETO fusion in MK genetranscription.Methods:1.A real-time quantitive RT-PCR(RQ-RT-PCR) method was applied toassay the MK gene expression levels in bone marrow cells of 181 ALpatients including 158 at new diagnosis and relapse/refractory,and 23 atcomplete remission (CR).31 healthy donors were used as normal controls.2.Sequence of the MDK promoter/enhancer (MKp) with Runt domainbinding sites was amplified from the human genome DNA anddirectionally cloned into pGL3-Basic Luciferase and pGL3-PromoterLuciferase reporter vectors to construct the MKP_{Ⅳ-Ⅴ}- pGL3-Basic andMKP _{Ⅰ-Ⅲ}-pGL3-Promoter expression plasmids.Plasmids of thepCMV5-AML1/AML1-ETO and the MKP_{Ⅰ-Ⅲ}-pGL3-Basic,MKP_{Ⅳ-Ⅴ}-pGL3-Basic or MKP_{Ⅰ-Ⅲ}-pGL3-Promoter were then co-transduced intoCV-1 cells and the luciferase relative value was analyzed.3.A MK expression vector pMK-IRES2-EGFP was transfected intoBA/F3 cells,an IL-3 dependent cell line derived from murine Pro-B cell.By G418 screening,stable subclone expressing full-length transcripts wasobtained.By comparing with BA/F3 cells transfected with IRES2-EGFP,the biological function of MK in the development of AL was studied.Results:1.MK gene was expressed in all AL patients,normal controls andpatients in complete remission (CR).Compared with control groupand CR group,AL patients showed a remarkable increase in MK geneexpression (p＜0.001 and p＜0.05 respectively).No statistic differencewas found between CR group and normal group.The expression ofMK exhibited a notable increase in all B-ALL subtypes (includingpro-B-ALL,common-B-ALL,pre-B-ALL) as well as in adult and children B-ALL patients (p＜0.001).Moreover,there were also markedincrease compared with T-ALL and any other FAB subtypes ofANLL(from p＜0.001 to p=0.008);M2 patients also showed significantincreases of MK expression compared with normal controls(p＜0.001)and with any other FAB subtypes of ANLL(from p=0.004 to 0.03);Median MK expression level of M3 patients was much higher thanthat of normal controls(p=0.015),but there was no statistic differencesbetween M3 and other subtypes of AL except M2 and B-ALL.TheMK expression of CD34 positive patients was significantly higherthan that of CD34 negative(P＜0.01).In M2 patients,MK showed anotable increase in patients with t(8;21) compared with that withoutthe translocation(p＜0.05).Conclusion MK gene expression takes aincrease in B-ALL,M2 and M3 patients with different levels,whichprovides novel insights in the diagnosis of acute leukemia andleukemogenesis study.2.The luciferase expression could be slightly induced by physiologicaldose AML1 in MKP_{Ⅰ-Ⅲ}-pGL3-B plasmid containing AML1 possiblebinding siteⅠ,Ⅱ,Ⅲ.However,physiological dose AML 1-ETO didn'tshow any biological activity on MKP_{Ⅰ-Ⅲ}-pGL3-B.On the contrary,the promoting activity of AML1 on MKP_{Ⅰ-Ⅲ}-pGL3-B was impairedwhen co-transduced with AML1 and AML1-ETO.Physiological doseAML1 and AML1-ETO didn't exhibit remarkable activity on MKP _{Ⅰ-Ⅲ}-pGL3-Promoter and MKP_{Ⅳ-Ⅴ}-pGL3-Basic plasmidcontaining AML 1 possible binding siteⅣⅤ.3.By G418 screening,stable subclone expressing full-length transcriptswas obtained.Under different IL-3,MK expressing clones showedmuch higher viablity than those clones transfected with pIRES2-EGFPin cell counting and colony formation assay.The AnnexinV/7-AADapoptosis assay showed there was a remarkable reduction of earlyapoptosis in MK expression clones.4.Conclusion:MK is up-regulated significantly in B-ALL,M2 and M3.Physiological dose AML1 can slightly induce MK expression,whilethe promoting activity of AML 1 can be impaired by AML 1-ETO.MKcan promote the development of AL by promoting cell proliferationand inhibiting cell apoptosis.