Influence on pertinent index of CREB by CRE decoy oligodeoxynucleotide in SKª²Nª²SH cell line treated by chronic morphine

    ZHANG Guoª²Zhong,MA Chunª²Ling£¬SU Yanª²Jun,ZHANG Jin,ZUO Min,GU Zhenª²Yong,YAO Yuª²Xia,LI Shuª²Jin£¬CONG Bin

    Department of Forensic Medicine,Hebei Medical University,Shijiazhuang 050017,China

    ¡¾Abstract¡¿ AIM: To investigate the influence on pertinent index of cAMP response element binding protein (CREB) by cAMP response element (CRE) decoy oligodeoxynucleotide in SKª²Nª²SH cell line treated by chronic morphine.METHODS: A singleª²stranded phosphorothioate oligodeoxynucleotide,composed of the CRE sequence and control oligonucleotides,was synthesized.DOTAP liposomal was used as transfection reagent and the CRE decoy ODN and control oligonucleotides were separately added to SKª²Nª²SH cells culture.In one group,the cells had been treated by chronic morphine and in another group the morphine treated cells had been precipitated by naloxone.The stability of cellª²incorporated 32Pª²labeled CREª²decoy ODN was examined by nondenaturing polyacrylamide gel electrophoresis and autoradiography.The effect of CREª²decoy ODN on CREB DNAª²binding activity was detected by electrophoresis mobility shift assay(EMSA).The proteins of CREBª²1 and phosphorylated CREBª²1 were detected by western blot.RESULTS: CREª²decoy ODN was successfully transfected to the cells.Compared with that in control groups,chronic morphine and acute naloxone precipitation significantly activated the CREB DNAª²binding activity.Phosphorylated CREBª²1 increased (P<0ª±01),but the protein of CREBª²1 had no obvious change.CREª²decoy ODN specifically downregulated the upper changes(P<0ª±01).CONCLUSION: CREª²decoy ODN can dramatically downregulate the CREB DNA binding activity and phosphorylated CREBª²1 induced by chronic morphine and acute naloxone in SKª²Nª²SH cells. ......