The Study Of NOTCH1 Down-regulating Tumor Suppressor Gene ID4 In T-ALL

T-cell acute lymoblastic leukemia(T-ALL)is a kind of hematologic malignancies which threaten human health seriously,the rate of incidence is higher among children and adults.Currently combination chemotherapy was mainly taken in to treat T-ALL,but mortality was still very tall and patients were likely to relapse.So there is an urgent need to develop new therapies to improve survival rates of T-ALL.More than 50% of T-ALL patients have the acquired function mutations of NOTCH1.The abnormal activation of NOTCH1 is important in the pathogenesis of T-ALL.?-secretase inhibitor Compound E(GSI)can effectively suppress NOTCH1 activation,but the effect is not specific.T-ALL patients with GSI treatment show obvious side effects,the treatment is unsatisfactory.Therefore,that we will search the drugs of targeting NOTCH1 downstream genes is an important direction for future treatment of T-ALL.MicroRNA(miRNA)is small RNA with the length of 20~24nt,negatively regulating gene expression by binding to mRNA specificly.Recent studies have found that miRNA was involved in all types of cancers,including the formation and malignant development of T-ALL.Inhibitor of DNA binding 4(ID4)is closely associated with the occur and stem cell differentiation of tumor.That researchers silenced ID4 gene in leukemia cells could promote the occurrence of leukemia in a variety of leukemias,but whether ID4 gene is still a tumor suppressor gene in T-ALL?How is regulatory mechanism?Little research.Objective:To explore the expression level and function of inhibitor of DNA binding 4(ID4)in T-cell acute lymoblastic leukemia(T-ALL),and to determine the correlation and mechanism between NOTCH1 and ID4.Methods:(1)The expression levels of ID4 in T-ALL patients and normal donors were analyzed from the database of Oncomine,the apoptosis of T-ALL cell line Jurkat with constructing ID4 overexpression was assessed by flow cytometry.(2)The correlation of NOTCH1 and ID4,was analyzed from the database of Pieters R.We construct the Jurkat cell of ICN1 overexpression to activate NOTCH1 and treat Jurkat cell with GSI to inhibit NOTCH1.Then ID4 mRNA and protein levels were detected by qRT-PCR and Western blot after NOTCH1 activation or inhibition,which were adopted to verify the correlation.(3)MiRNA was predicted to target ID4 with miRNA target prediction softwares of TargetScan?PicTar and MiRanda,and the dual luciferase reporter system was carried out to verify the prediction.(4)miR-342 mimics/miR-342 inhibitor was transfected into Jurkat cell by electroporation.ID4 mRNA and protein levels were detected by qRT-PCR and Western blot after overexpressing or silencing miR-342.(5)MiR-342 mimics was transfected into Jurkat cell with NOTCH1 inhibition by electroporation.MiR-342 inhibitor was transfected into Jurkat cell with NOTCH1 activation by electroporation.Then miR-342 and ID4 expression levels were was detected by qRT-PCR and Western blot before and after electroporation.Results:(1)ID4 expression level in T-ALL patients was significantly lower than that in normal donors(P<0.01),overexpression of ID4 in Jurkat cell leads to apoptosis.(2)ID4 was negatively regulated by NOTCH1(r=-0.242,P=0.02).(3)The dual luciferase reporter system validated ID4 as a specific target gene of microRNA-342(miR-342).(4)Down-regulation of miR-342 resulted in sharp increase of ID4 mRNA and protein(P<0.05);and up-regulation of miR-342 resulted in sharp decrease of ID4 mRNA and protein(P<0.05).(5)The expression of miR-342 shows higher expression when activating NOTCH1 in Jurkat cell.Once miR-342 inhibitor was transfected into Jurkat cell with NOTCH1 activation by electroporation,ID4 mRNA and protein levels would resume rising;The expression of miR-342 shows lower expression when inhibiting NOTCH1 in Jurkat cell.Once miR-342 mimics was transfected into Jurkat cell with NOTCH1 inhibition by electroporation,ID4 mRNA and protein levels would resume declining.Conclusion:NOTCH1 activates the negatively regulatory role of miR-342 for ID4,then down-regulates tumor suppressor gene ID4 expression,and may contribute to the pathogenesis of T-ALL.