The Expression Of Survivin And C-Myc And The Effects On Apoptosis In Retinoblastoma

Objective:To observe the expression of survivin and c-Myc protein and their relationship with apoptosis in 23 retinoblastoma(Rb) samples. And to clarify the induction of cell cycle arrest and apoptosis of human retinoblastoma cell line Hxo-rb44 in vitro induced by trichostatin A(TSA) and the changes of the expression of survivin and c-Myc protein in apoptosis. The plasmid containing short hairpin RNA(shRNA) of survivin protein was costructed in order to be observed the expression of survivin gene in human retinblastoma cell line Hxo-rb44 in vitro. And to explore its effect on the expression of survivin and c-Myc protein and apoptosis.Methods : (1) The cohort consisted of 23 patients diagnosed with primary retinoblastoma from December 1994 to August 2005 in the Union Hospital Affiliated to Tongji Medical College of Huazhong University of Science and Technology. The Sp method (streptavidin-peroxidase conjunct method) of immunohistochemical technique(IHC) was used to detect the expression of survivin and c-Myc protein in all paraffin embedded retinoblastoma samples and evidence of apoptosis cells was examined via the TUNEL (terminal deoxynucleotidyl transferase-mediate deoxyuridinetriphosphate nick end labeling, TdT-mediate d-UTP nick end labeling) technique. (2) The retinoblastoma cell line Hxo-rb44 was chosen in this experiment. The cell cycle arrest effects and apoptosis of Hxo-rb44 cell line were assayed with Flow cytometer(FCM). Expression of survivin and c-Myc protein was detected by Western blot. (3) 64 bp reverse repeated motifs of survivin target sequence were synthesized and inserted into plasmid to construct the plasmid shRNA-survivin and the plasmid was transfected into retinblastoma cell line Hxo-rb44. The exprsession of survivin protein was detected by Western blot. The apoptosis ratio of transfected retinoblastoma cell was evaluated by Hoechst33258 stain. And the growth curve was drawn.Results: (1) Results showed that survivin protein was expressed in nearly 60.9% (14/23) of samples while the c-Myc protein was positively expressed in 17/23 samples (73.9%). Average apoptosis index (AI) is 0.52%. Expression of survivin protein was found to be significantly related with c-Myc protein (p<0.05). Survivin protein expression level was negatively correlated with apoptosis index. (2) Trichostatin A obviously induced cell cycle arrest and apoptosis with dose and time dependent manner. Occurrence of apoptosis was detected by Anexin V-PI staining also in dose dependent manner. The expression survivin and c-Myc protein markedly decreased with increasing incubation time with trichostatin A. (3) The recombinant plasmid shRNA-survivin was successfully constructed and the recombinant plasmid depress the survivin protein expression by 66% and c-Myc by 53% in retinoblastoma cell. After transfection the apoptosis ration was promoted to 36.1±19.66% compared to 3.5±1.29%.Conclusions: (1)Survivin and c-Myc protein might play synergetic role in the carcinogenesis of retinoblastoma by inhibiting tumor cell apoptosis and promoting proliferation. (2)Trichostatin A can effectively induce cell cycle arrest and apoptosis in retinoblastoma cell line Hxo-rb44 and the inhibition of survivin and c-Myc gene expression may play a critical role in the retinoblastoma cell apoptosis induced by trichostatin A. (3)The short hairpin RNA of survivin can efficiently reduce its expression in retinoblastoma cell and induce apoptosis. The inhibition of the expression of survivin and the reduced c-Myc protein may also contribute to the promoted apoptosis ratio.