The Study On The Effect And Molecular Mechanisms Of Schlafen2 Gene Expression Regulation Induced By Pleiotrophin

Objective:To analyze the effect of Pleiotrophin(Ptn)on expression of Schlafen2(Slfn2) in the Ptn knock down mouse embryo fibroblast cells line,to investigate what kinds of cytokine and signal pathway are implicated in Ptn-silence induced up-regulation of Slfn2.Methods:Previous study suggest Schlafen2(Slfn2)is up-regulated in mouse embryo fibroblast cells(MEF241)knock down Pleiotrophin(Ptn).At present study,to investigate the possible growth factor involved in expression regulation of Slfn2 in the Ptn knock down mouse embryo fibroblast cells line,mRNA expression of Slfn2 gene was detected in the control cells that incubated with the supernatant of Ptn-siRNA B/MEF241 cells. Then the expression change of IL-1a and IL-1f6 was detected in the Ptn-siRNA B/MEF241 cells that treated by 50ng/μl recombinant human PTN(rh PTN).Northern blotting was used to detect the change of Slfn2 gene expression in the presence of rhIL-1ra and IL-1a affinity purified polyclonal antibody.The further study was made to investigate the possible signal pathway involved in Ptn-silence induced up-regulation of Slfn2.Phosphor-JNK were tested by Western blotting on Ptn-siRNA B/MEF241 system with or without exogenous PTN(50ng/μl),and changes of Slfn2 transcription were tested by Northern blotting on Ptn-siRNA B/MEF241 system in presence of SP600125(JNK inhibitor,20μmol/L)or SB203580(p38 inhibitor,20μmol/L),respectively.At last,to investigate if IL-1 also involved in the regulation of Phospho-JNK,changes.of Phosphor-JNK were also tested in the presence of rhIL-1ra and IL-1a affinity purified polyclonal antibody,respectively.Results:(1)Slfn2 high expression in Ptn-siRNA B/MEF241 cells,and the expression of Slfn2 in the control cells can be induced by incubation with the culture medium of Ptn-siRNA B/MEF241 cells;IL-1a and IL-1f6 over-expression in Ptn-siRNA B/MEF241 cells,and this effect was inhibited by presence of rhPTN;The expression of Slfn2 in the Ptn-siRNA B/MEF241 cells can be inhibited in the presence of rhIL-1ra and IL-1a affinity purified polyclonal antibody.(2)Phospho-JNK is over-expression in Ptn-siRNA B/MEF241 system,and this effect was inhibited by presence of PTN;Slfn2 transcription was inhibited by SP600128,but not SB203508,in Ptn-siRNA B/MEF241 system.(3) The expression of Phospho-JNK in the Ptn-siRNA B/MEF241 cells can be inhibited in the presence of rhIL-1ra and IL-1a affinity purified polyclonal antibody.Conclusions:(1)IL-1 may play an important role in Ptn negative regulation of Slfn2 in Ptn-siRNA B/MEF241 cell.(2)Ptn negativelY regulate Slfn2 transcription probably through JNK signal pathway.(3)IL-1 also involved in the regulation of Phospho-JNK in Ptn-siRNA B/MEF241 cell.