Combining siRN A-mediated hTR and hTERT expression in bladder uroepithelium cancer BIU-87 cell lines

Objective To perform a DNA microarray analysis of combining small interfering RNA (siRNA) of human telomerase RNA (hTR) and human telomerase reverse transcriptase (hTERT) gene in bladder transitional cell carcinoma cell line BIU-87 telomere Inhibition of enzyme activity. Methods According to the siRNA design principles and TERT.TR gene mRNA coding sequence,respectively,were designed and synthesized three pairs of different and can interfere with TR and TERT gene expression in siRNA sequence and filter out the most efficient suppression sequence ( pRNAT-TERT-Ⅲ: GTACAG-GTTTCACGCATGT gene No. 3229, pRNAT-TR- Ⅲ: CGAAGGTGCCTTGGAATAT gene No. 306). Constructing the hTR,hTERT gene as a target of the joint RNA interference ( RNAi) device pRNAT-TR-Ⅲ+ pRNAT-TERT-Ⅲ(liposome) and transfected into bladder transitional cell carcinoma of BIU-87 cell line, using fluorescent quantitative polymerase chain reaction (PCR) analysis of hTR and hTERT mRNA expression (in the target gene and GAPDH copy number ratios for the relative quantitative analysis) , TRAP-ELJSA detection of telomerase activity (measured A value) ,MTT assay BIU-87 cell growth inhibition (by MTT method Detection of the survival rate of transfected cells) ,and before and after co-RNAi gene chip a-nalysis,using RNA extraction,RNA quality control,cRNA marking and synthesis,chip hybridization, chemi-luminescence detection,image acquisition and data analysis,and further use of part of the reverse transcription (RT)-PCR validation of differentially expressed genes. Results The combined RNA interference hTR and hTERT gene, pRNAT-TERT-Ⅲ+ pRNAT-TR-Ⅲcombined RNAi can specifically inhibit the bladder transitional cell carcinoma cell line BIU-87 in TERT and TR expression (pRNAT-TERT- Ⅲ+ pRNAT-TR-I TERT:0.300 ±0.033 ,P <0.05;TR:0.430 ±0.028,P <0.05). After the combined RNAi and bladder transitional cell carcinoma of BIU-87 cell line growth and telomerase activity were significantly inhibited (pRNAT-TERT-Ⅲ+ pRNAT-TR- Ⅲ: 1.250 ± 0.012, P < 0.05 ). Before and after the joint siRNA gene chip analysis found 21 genes down ( ATM, BAX, BCL2, BCL2L1, BIRC5, CD44, CTNNB1, E2F1, JUN, MCAM, MTA1, MYC, NFKB1, NFKBIA, NME4, PNN, PRKDC, SERP1NE1, THBS1, TNFRSF1A, UCCl). Conclusion The combined RNA interference with hTERT and hTR gene can significantly inhibit bladder cancer BIU-87 cell line growth;joint siRNA inhibited bladder transitional cell carcinoma of BIU-87 cell line causes growth of 21 genes were cut. Key words: Telomerase;  siRNA;  Bladder transitional cell carcinoma