The Correlation Of MEPE/OF45, SERPINB2 And RB

Since MEPE/OF45 (Matrix Extracellular Phospho-glycoprotein/ Osteoblast/ Osteocyte Factor 45) was first cloned in 2000, however, its role as one member of SIBLING (Small Integrin-Binding Ligand N-linked Glycoprotein) family had been limited to regulation of bone metabolism and phosphate homeostasis.Recently, the previous research in our lab and our collaborative lab has shown a new function of MEPE/OF45. That is, MEPE/OF45 is involved in cellular DNA damage response and affects cellular resistance to DNA damage inducers. By using gene microarray to explore the function of MEPE/OF45, we found that MEPE/OF45 may inhibite apoptosis, and it is found that the cells with high expression of MEPE/OF45 show high expressions of SERPINB2 and RB. Therefore, it is very important to study the correlation of MEPE, SERPINB2 and RB in elucidation the redioresistance and inhibiting apoptosis of MEPE/OF45.Taken together, in this study we identified that (1) Serine (or cysteine) proteinase inhibitor B2(SerpinB2) gene was expressed in E.coli, SERPINB2 and MEPE/OF45 proteins were purified, and the polyclonal antibody against SERPINB2 and MEPE/OF45 were prepared in mice, the titer of these antibodies were determined by ELISA and were about 1:20000,. the specificity was identified by Western blot and SERPINB2 and MEPE/OF45 expressed in prokaryotic and eukaryotic cells can be recognized by these antiserum. (2) SerpinB2-siRNA vectors were constructed, and two SerpinB2-siRNAs which repress SerpinB2 expression in 293T cells were obtained, SerpinB2 expression was repressed by siRNAs. (3) By using overexpression of MEPE/OF45, inhibiting expression of MEPE/OF45 by miRNA and decreasing expression of SERPINB2 by siRNA, the correlation of MEPE/OF45, SERPINB2 and RB in protein levels were studied and the expression levels were accordant, it may be one of the pathways that MEPE/OF45 affectes apoptosis. Our data will not only increase our knowledge on DNA damage response but also give a clue to future research concerned on targets of radiotherapy.