Silencing ubiquitin specific peptidase 22 inhibits the cell proliferation of human bladder cancer EJ cells via down-regulated Myc

Abstract

Objective To investigate the role of silencing USP22 gene by aiRNA on the cell proliferation in human bladder cancer EJ cells.Methods USP22 aiRNA and NC-aiRNA were designed and synthesized.The structure of USP22 aiRNA had 15 bp sense sequence and 21 bp antisense sequence and was named USP22 aiRNA (15/21).The EJ cells were transfected with USP22 aiRNA (15/21) or NC-aiRNA for 48 h.The mRNA and protein levels of USP22,Myc,cyclin D1 and cyclin E1 were measured by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot,respectively.The proliferation rate of EJ cells was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-Diphenyl tetrazolium bromide (MTT) assay.The transcriptional activity of Myc gene was determined by luciferase reporter gene assay.The ability of Sp1 binding to Myc promoter was tested by a ChIP assay.Results Compared with control group,the mRNA and protein levels of USP22,Myc,cyclin D1 and cyclin E1 in USP22 aiRNA (15/21) group were reduced by (87.4±5.2)％ and (91.2±7.3)％,(69.2±4.3)％ and (61.0±6.6)％,(78.5±4.1)％ and (67.4±5.5) ％,(81.0±5.5)％ and (78.3 4.0)％ (P＜0.05).MTT assay demonstrated that the cell viability at 24,48,72,96 h in the USP22 aiRNA (15/21) group was (85.4±5.7)％,(71.3±8.4)％,(52.5±6.7)％ and (45.8±6.4) ％ (P＜0.05) and luc,iferase reporter gene assay showed that the transcriptional activity of Myc in USP22 aiRNA (15/21) group was decreased by (65.5±4.2) ％ (P＜0.05).Moreover,ChIP assay confirmed that the ability of Sp1 binding to the Myc promoter in USP22 aiRNA (15/21) group was inhibited by (48.0±3.2) ％ in comparison with control group (P＜0.05).Conclusions These results suggested that silencing USP22 inhibited the EJ cells proliferation by blocking Sp1 binding to the Myc promoter. Key words: Ubiquitin specific peptidase 22; Myc gene; Transcription factor Sp1; Transcription regulation; Bladder cancer