Objective      To determine the effect of angiogenin (ANG) down-regulation on the prolifera

tion and apoptosis in human bladder cancer T24 cells.       Methods      An ANG-targeting siRNA vector and a non-homologous negative control were designed to transfect human bladder cancer T24 cells with Lipofectamine 2000

and the positive clones were screened by G418 and then named as T24-siANG and T24-NC

respectively. T24 cells without transfection were named as control (T24 group). The expression of ANG  at mRNA and protein levels was detected by RT-PCR and Western blotting. Cell proliferative activity was determined by MTT assay. Cell apoptosis was evaluated by TUNEL kit

Hochest (33342) and flow cytometry. The expression of caspase-3

Bax and Bcl-2 was determined by Western blotting. The tumor cells of the 3 groups of cells at 2×10

6

were respectively injected into the back of BALB/c nude mice (4 to 6 weekes

20±3 g) to establish the xenograft models. The changes of micro-blood vessels in tumor tissue and the expression of CD31 were detected by immunofluorescence and HE staining. Immunohistochemical assay was used to detect the expression of ANG

Bax

Bcl-2 and caspase-3 in the tumors.       Results      Enzyme digestion and DNA sequencing verified the vectors was correctly constructed. The ANG expression at mRNA and protein levels was significantly decreased in the T24-siANG group (

P

0.05)

and the cell proliferative activity reduced markedly (

P

0.05). Flow cytometry showed that the apoptotic rate was obviously higher in the T24-siANG cells than in the T24-NC cells and the T24 cells (

P

0.01).  The number of TUNEL-positive cells was significantly higher in the T24-siANG cells than in the T24 cells and the T24-NC cells. The results of Hochest demonstrated that T24-siANG cells appeared typical apoptotic morphology features such as chromatin condensation

nuclear fragmentation and brighter blue fluorescence. However

the control cells did not have such apoptotic characteristics. BALB/c nude mice injected with the T24-siANG cells showed lower density of tumor microvessel and lower

CD31 expression than those with the control cells (

P

0.01). Immunohistochemical assay results proved that the expression of Bcl-2 was significantly decreased in the T24-siANG cells as compared to that in the T24 and T24-NC cells (

P

0.01)

but the expression of caspase-3 and Bax was stably increased (

P

0.05).       Conclusion      The plasmid expressing ANG-targeting siRNA down-regulates ANG  expression and inhibits the proliferation of T24 cells significantly

and thus induces apoptosis in bladder cancer T24 cells by regulating apoptosis-related proteins caspase-3

Bax and Bcl-2.