OBJECTIVETo delineate the effects of HBV X gene and of As(2)O(3) on p53 expression and activity in HepG2 cells by shRNA-mediated RNA interference (RNAi).

METHODSHepG2 cells and cells with stable expression of HBV X gene, HepG2-X, were treated with 2 micromol/L As(2)O(3), and the corresponding untreated cells were used as controls. Cell and nuclear lysates were extracted. Total level and the relative activity absorbance of p53 were detected by modified ELISA. HBV X gene sequence-specific shRNA expression vectors, Xi-S1 and Xi-S2, and sequence-unrelated control Xi-S3 were transfected into HepG2-X. The effect of As(2)O(3) on p53 expression and activity were retested.

RESULTSTotal p53 level was up-regulated and its relative activity ratio was enhanced by As(2)O(3) in HepG2 and HepG2-X cells. The total p53 level induced by As(2)O(3) was further up-regulated by HBX expression, while its relative activity was significantly suppressed. The suppression was removed after HBX expression was suppressed by shRNA.

CONCLUSIONAs(2)O(3) could up-regulate p53 expression and enhance its activity. shRNA-mediated RNA interference is conveniently being used in studies on the effect of HBV X gene expression on p53 expression and activity. HBV X expression could up-regulate p53 gene expression level induced by As(2)O(3), while it suppressed the activity of p53.