Mechanism of RNA interference targeting at survivin gene on apoptosis of hepatoma-cellular carcinoma cell line HepG2

Abstract

Originally identified as a member of the IAPs, survivin was discovered in 1997, and is one of the most powerful inhibiting factor of anti-apoptosis [1] . Studies suggest it has dual function of inhibiting apoptosis and maintaining cell division is located in chromosome 17q25, 14.5 kb in length, and is a protein of 16.3 KD containing 142 amino acid residues. Survivin specifically expresses in most tumor tissues while show rare exhibition in normal tissues, existing in fast proliferating but not terminally differentiation cells. RNA interference is one kind of methods of inducing gene silence, the mechanism of which is degradation of double-stranded RNA by specific RNase, cleavaged to small interference RNA (siRNA) which complement to targeted RNA degraded by specific enzyme, then inhibit or down-regulate the expression of gene. RNA interference (RNAi) is the induction of dsDNA to the cells leading the degradation of targeting mRNA, since block the expression of gene in eukaryotic cells. Pre-experiments suggest the expression of survivin is related to the results of chemical therapy. But the exact molecular mechanism of survivin mediating the apoptosis of hepG2 cells remains unclear.Compared with other IAPs, survivin is structurally unique. Survivin is the smallest mammalian IAP but no other identifiable protein domain. Structural data suggest that survivin forms a stable homodimer in solution [2] , but definitive evidence about the function of this organization is still lacking [3] . Survivin is expressed in embryonic and fetal tissues, but is almost undetectable in adult tissues. RNAi has emerged as one of the most important discoveries of the last years in the field of molecular biology. Following clarification of this highly conserved endogenous gene silencing mechanism, RNAi has largely been Abstract Objective: The aim of this study was to investigate the molecular mechanism of anti-apoptotic action of survivin to the hepatoma-cellular carcinoma cell line HepG2. Methods: Design and synthesize siRNA gene sequence specifically targeting at HepG2 cell. HepG2 cells cultures were divided into five groups: blank control group, negative control group, low dose group, medium dose group and high dose group. HepG2 cells were treated respectively by pshRNA-survivin-387 of different concentrations. The apoptosis index (AI) was determined by flow cytometry (FCM). Cells were stained with rhodomine-123 (Rh123) to detect changes of mitochondrial membrane potentials. The concentration of cytoplasmic cytochrome C (Cyt.C) was continuously determined by ELISA. Relative activities of caspase-9 and caspase-3 were assessed by colorimetric assay. Results: Compared with the control group, due to the function of short interference RNAs (SiRNAs) that suppresses the survivin gene expression, the apoptotic index of transfected groups were significantly higher than those of control groups (F = 13568.68, q = 110.47-327.16, P < 0.01), the apoptosis index of high concentration of transfected cells was higher than the low concentration tra...