Regulation Of NHE1in Different Cell Lines After DNA Damage Exerts Diverse Cell Function

Background:Apoptosis induced by DNA damage plays an important role in many disease. As a regulater of intracellular pH, NHE1may play a role in pathway of apoptosis and affects the sensitivity responsed to the DNA damage in some hemopoietic malignant disease. BCR/ABL fusion kinase may affect the expression of NHE1.The constitutive activated activity of BCR/ABL affects many signaling pathway to form a complex network which is vital in development in hemopoietic malignant disease.Recently, BCR/ABL has been shown to inhibit apoptosis via NHE1, but the precise molecular mechanisms regulating NHE1expression,remain unclear.Objective:This study aimed to investigate the underlying molecular mechanisms governing NHE1expression in BCR/ABL-positive leukemia cells and breast cancer cells.Meanwhile, trying to expore possible therapeutic target.Methods:We first compared NHE1expression of different cell lines after DNA damage using realtime PCR and western blot.Intercellular pH was measured by method based on high K+buffer. Apoptosis of these cell lines was detected by AnnexinV-FITC/PI method. Then the luciferase reporter plasmids containing human SLC9A1promoter as well as its deletion mutation were constructed to identify the essential responsive elements for activation of SLC9A1promoter responsible to DNA damage. We also constructed a luciferase report vector containing human NHE1mRNA3’untranslated region to determine if there were possible regulating miRNA affecting NHE1mRNA stability.Additionally, selective chemical inhibitors of c-Myc, miR-19,BCR/ABL signaling pathway were employed to determine the signals are responsible for inhibition of NHE1expression. At last, transwell assay was used to detect the matastasis and invasion of breast cancer cell lines affects by NHE1Resuls:the expression level of NHE1mRNA and protein increased in accordance with apoptosis ratio and pHi in BCR/ABL negative cells after treatment with etoposide. But the change was inhibited in k562cells. Relative luciferase activity increased in K562cells after Etoposide treatment.The responsible elements was located in-1259～1029region containing possible cis-acting element in coincidence with OCT1matrices.In K562cells, inhibition of NHE1expression was in the level of post-transcription which depends on miR-19targets on seed region in3’untranslated region of NHE1. Series of inhibitors uncovered BCR/ABL,c-MYC,and miR-19can affecting NHE1expression and pHi as well as the sensitivity to DNA damage.Finally,the change of NHE1expression caused by DNA damage can regulate the matastasis and invasion of breast cancer cells.Conclusion:DNA damage enhance NHE1expression through transcription level,BCR/ABL pathway affects NHE1expression responsible to DNA damage. NHE1expression responsible to DNA damage can regulates different cell behaviors.