中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2010年
8期
1127-1130
,共4页
程文%高建平%张征宇%葛京平%景抗震%解鹏%位志峰%徐锋
程文%高建平%張徵宇%葛京平%景抗震%解鵬%位誌峰%徐鋒
정문%고건평%장정우%갈경평%경항진%해붕%위지봉%서봉
端粒酶%siRNA%膀胱移行细胞癌
耑粒酶%siRNA%膀胱移行細胞癌
단립매%siRNA%방광이행세포암
Telomerase%siRNA%Bladder transitional cell carcinoma
目的 观察基因芯片分析联合小型干扰(siRNA)人端粒酶RNA(hTR)及人端粒酶逆转录酶(hTERT)基因对膀胱移行细胞癌BIU-87细胞株端粒酶活性抑制的影响.方法 根据siRNA设计原则和TERT、TR基因mRNA编码序列,分别设计合成3对不同的且能干扰TR和TERT基因表达的siRNA序列并筛选出最高效抑制序列(pRNAT-TERT-Ⅲ:GTACAGGTTTCACGCATGT基因的第3229位、pRNAT-TR-Ⅲ:CGAAGGTGCCTTGGAATAT基因的第306位).构建以hTR、hTERT基因为靶点的联合RNA干扰(RNAi)装置pRNAT-TR-Ⅲ+pRNAT-TERT-Ⅲ(脂质体法)并将其转染膀胱移行细胞癌BIU-87细胞株,采用荧光定量聚合酶链反应(PCR)分析hTR和hTERT mRNA表达(以目的基因与GAPDH拷贝数比值进行相对定量分析),端粒重复序列扩增-酶联免疫吸附试验(TRAP-ELISA)检测端粒酶活性(测其A值),噻唑蓝(MTT)比色法检测BIU-87细胞的生长抑制(按MTT法检测转染细胞的存活率),并对联合RNAi前后进行基因芯片分析,采用RNA抽提、RNA质量检测、cRNA标记与合成、芯片杂交、化学发光检测、图像采集和数据分析,并进一步采用逆转录(RT)-PCR验证部分差异表达基因.结果 联合RNA干扰hTR及hTERT基因后,pRNAT-TERT-Ⅲ+pRNAT-TR-Ⅲ的联合RNAi可以特异性抑制膀胱移行细胞癌BIU-87细胞株中TERT和TR表达(pRNAT-TERT-Ⅲ+pRNAT-TR-ⅢTERT:0.300±0.033,P＜0.05;TR:0.430±0.028,P＜0.05).且联合RNAi后膀胱移行细胞癌BIU-87细胞株的生长和端粒酶活性均明显受到抑制(pRNAT-TERT-Ⅲ+pRNAT-TR-Ⅲ:1.250±0.012,P＜0.05).联合siRNA前后进行基因芯片分析发现21个基因下调(ATM、BAX、BCL2、BCL2L1、BIRC5、CD44、CTNNB1、E2F1、JUN、MCAM、MTA1、MYC、NFKB1、NFKBIA、NME4、PNN、PRKDC、SERPINE1、THBS1、TNFRSF1A、UCC1).结论 联合RNA干扰hTERT及hTR基因能明显抑制膀胱移行细胞癌BIU-87细胞株的生长;联合siRNA抑制膀胱移行细胞癌BIU-87细胞株的生长的原因为21个基因下调所致.
目的 觀察基因芯片分析聯閤小型榦擾(siRNA)人耑粒酶RNA(hTR)及人耑粒酶逆轉錄酶(hTERT)基因對膀胱移行細胞癌BIU-87細胞株耑粒酶活性抑製的影響.方法 根據siRNA設計原則和TERT、TR基因mRNA編碼序列,分彆設計閤成3對不同的且能榦擾TR和TERT基因錶達的siRNA序列併篩選齣最高效抑製序列(pRNAT-TERT-Ⅲ:GTACAGGTTTCACGCATGT基因的第3229位、pRNAT-TR-Ⅲ:CGAAGGTGCCTTGGAATAT基因的第306位).構建以hTR、hTERT基因為靶點的聯閤RNA榦擾(RNAi)裝置pRNAT-TR-Ⅲ+pRNAT-TERT-Ⅲ(脂質體法)併將其轉染膀胱移行細胞癌BIU-87細胞株,採用熒光定量聚閤酶鏈反應(PCR)分析hTR和hTERT mRNA錶達(以目的基因與GAPDH拷貝數比值進行相對定量分析),耑粒重複序列擴增-酶聯免疫吸附試驗(TRAP-ELISA)檢測耑粒酶活性(測其A值),噻唑藍(MTT)比色法檢測BIU-87細胞的生長抑製(按MTT法檢測轉染細胞的存活率),併對聯閤RNAi前後進行基因芯片分析,採用RNA抽提、RNA質量檢測、cRNA標記與閤成、芯片雜交、化學髮光檢測、圖像採集和數據分析,併進一步採用逆轉錄(RT)-PCR驗證部分差異錶達基因.結果 聯閤RNA榦擾hTR及hTERT基因後,pRNAT-TERT-Ⅲ+pRNAT-TR-Ⅲ的聯閤RNAi可以特異性抑製膀胱移行細胞癌BIU-87細胞株中TERT和TR錶達(pRNAT-TERT-Ⅲ+pRNAT-TR-ⅢTERT:0.300±0.033,P＜0.05;TR:0.430±0.028,P＜0.05).且聯閤RNAi後膀胱移行細胞癌BIU-87細胞株的生長和耑粒酶活性均明顯受到抑製(pRNAT-TERT-Ⅲ+pRNAT-TR-Ⅲ:1.250±0.012,P＜0.05).聯閤siRNA前後進行基因芯片分析髮現21箇基因下調(ATM、BAX、BCL2、BCL2L1、BIRC5、CD44、CTNNB1、E2F1、JUN、MCAM、MTA1、MYC、NFKB1、NFKBIA、NME4、PNN、PRKDC、SERPINE1、THBS1、TNFRSF1A、UCC1).結論 聯閤RNA榦擾hTERT及hTR基因能明顯抑製膀胱移行細胞癌BIU-87細胞株的生長;聯閤siRNA抑製膀胱移行細胞癌BIU-87細胞株的生長的原因為21箇基因下調所緻.
목적 관찰기인심편분석연합소형간우(siRNA)인단립매RNA(hTR)급인단립매역전록매(hTERT)기인대방광이행세포암BIU-87세포주단립매활성억제적영향.방법 근거siRNA설계원칙화TERT、TR기인mRNA편마서렬,분별설계합성3대불동적차능간우TR화TERT기인표체적siRNA서렬병사선출최고효억제서렬(pRNAT-TERT-Ⅲ:GTACAGGTTTCACGCATGT기인적제3229위、pRNAT-TR-Ⅲ:CGAAGGTGCCTTGGAATAT기인적제306위).구건이hTR、hTERT기인위파점적연합RNA간우(RNAi)장치pRNAT-TR-Ⅲ+pRNAT-TERT-Ⅲ(지질체법)병장기전염방광이행세포암BIU-87세포주,채용형광정량취합매련반응(PCR)분석hTR화hTERT mRNA표체(이목적기인여GAPDH고패수비치진행상대정량분석),단립중복서렬확증-매련면역흡부시험(TRAP-ELISA)검측단립매활성(측기A치),새서람(MTT)비색법검측BIU-87세포적생장억제(안MTT법검측전염세포적존활솔),병대연합RNAi전후진행기인심편분석,채용RNA추제、RNA질량검측、cRNA표기여합성、심편잡교、화학발광검측、도상채집화수거분석,병진일보채용역전록(RT)-PCR험증부분차이표체기인.결과 연합RNA간우hTR급hTERT기인후,pRNAT-TERT-Ⅲ+pRNAT-TR-Ⅲ적연합RNAi가이특이성억제방광이행세포암BIU-87세포주중TERT화TR표체(pRNAT-TERT-Ⅲ+pRNAT-TR-ⅢTERT:0.300±0.033,P＜0.05;TR:0.430±0.028,P＜0.05).차연합RNAi후방광이행세포암BIU-87세포주적생장화단립매활성균명현수도억제(pRNAT-TERT-Ⅲ+pRNAT-TR-Ⅲ:1.250±0.012,P＜0.05).연합siRNA전후진행기인심편분석발현21개기인하조(ATM、BAX、BCL2、BCL2L1、BIRC5、CD44、CTNNB1、E2F1、JUN、MCAM、MTA1、MYC、NFKB1、NFKBIA、NME4、PNN、PRKDC、SERPINE1、THBS1、TNFRSF1A、UCC1).결론 연합RNA간우hTERT급hTR기인능명현억제방광이행세포암BIU-87세포주적생장;연합siRNA억제방광이행세포암BIU-87세포주적생장적원인위21개기인하조소치.
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.
