中华实验外科杂志
中華實驗外科雜誌
중화실험외과잡지
CHINESE JOURNAL OF EXPERIMENTAL SURGERY
2012年
6期
1000-1002
,共3页
胡嘏%陈忠%龚化%吴嘉%张勇%王骥%徐华%李龙承%杨为民%叶章群
鬍嘏%陳忠%龔化%吳嘉%張勇%王驥%徐華%李龍承%楊為民%葉章群
호하%진충%공화%오가%장용%왕기%서화%리룡승%양위민%협장군
RNA%真核表达质粒%p21%泌尿系肿瘤
RNA%真覈錶達質粒%p21%泌尿繫腫瘤
RNA%진핵표체질립%p21%비뇨계종류
RNA%Eukaryotic expression plasmid%p21%Urinary cancer
目的 构建可表达具有RNA激活功能的小分子双链RNA(dsRNA)真核表达质粒,并探讨其调控前列腺癌细胞株PC-3和膀胱癌细胞株T-24中抑癌基因p21 WAF1/CIP1表达的功能.方法 构建真核表达质粒dsRNAP21-pGenesil-1,并分别转染至PC-3和T-24细胞株,通过实时定量聚合酶链反应( Real-time qPCR)和Western blot检测p21mRNA转录水平和蛋白表达的变化.结果 测序结果证实目的表达质粒dsRNAP21-pGenesil-1构建成功,将其转染入PC-3细胞和T-24细胞中,Real-time qPCR检测p21基因分别被上调4.35倍和2.83倍,Western blot进一步证明在两种细胞株中p21蛋白表达水平的增加与p21mRNA水平的上调一致,且与对照组比较,差异均有统计学意义(P＜0.05).结论 构建的dsRNAP21-pGenesil-1质粒具备在泌尿系肿瘤中上调抑癌基因p21表达的能力.
目的 構建可錶達具有RNA激活功能的小分子雙鏈RNA(dsRNA)真覈錶達質粒,併探討其調控前列腺癌細胞株PC-3和膀胱癌細胞株T-24中抑癌基因p21 WAF1/CIP1錶達的功能.方法 構建真覈錶達質粒dsRNAP21-pGenesil-1,併分彆轉染至PC-3和T-24細胞株,通過實時定量聚閤酶鏈反應( Real-time qPCR)和Western blot檢測p21mRNA轉錄水平和蛋白錶達的變化.結果 測序結果證實目的錶達質粒dsRNAP21-pGenesil-1構建成功,將其轉染入PC-3細胞和T-24細胞中,Real-time qPCR檢測p21基因分彆被上調4.35倍和2.83倍,Western blot進一步證明在兩種細胞株中p21蛋白錶達水平的增加與p21mRNA水平的上調一緻,且與對照組比較,差異均有統計學意義(P＜0.05).結論 構建的dsRNAP21-pGenesil-1質粒具備在泌尿繫腫瘤中上調抑癌基因p21錶達的能力.
목적 구건가표체구유RNA격활공능적소분자쌍련RNA(dsRNA)진핵표체질립,병탐토기조공전렬선암세포주PC-3화방광암세포주T-24중억암기인p21 WAF1/CIP1표체적공능.방법 구건진핵표체질립dsRNAP21-pGenesil-1,병분별전염지PC-3화T-24세포주,통과실시정량취합매련반응( Real-time qPCR)화Western blot검측p21mRNA전록수평화단백표체적변화.결과 측서결과증실목적표체질립dsRNAP21-pGenesil-1구건성공,장기전염입PC-3세포화T-24세포중,Real-time qPCR검측p21기인분별피상조4.35배화2.83배,Western blot진일보증명재량충세포주중p21단백표체수평적증가여p21mRNA수평적상조일치,차여대조조비교,차이균유통계학의의(P＜0.05).결론 구건적dsRNAP21-pGenesil-1질립구비재비뇨계종류중상조억암기인p21표체적능력.
Objective To construct small double stand RNA (dsRNA) eukaryotic expression plasmid possessing the ability of RNA activation,and investigate its capability to regulate the tumor suppressor gene p21 WAF1/CIP1 expression in human prostate adenocarcinoma cell line PC-3 and human bladder cancer cell line T-24.Methods The dsRNA sequences targeting the p21 promoter at position-322 relative to the transcription start site were synthesized and inserted into dsRNA eukaryotic expression plasmid pGenesil-1,and the positive clone was named dsRNAP21-pGenesil-1.The eukaryotic expression plasmid dsR-NACon-pGenesil-1 was also constructed and used as a negative control.PC-3 and T-24 cells were cultured in vitro and transfected with dsRNAP21-pGenesil-1 or dsRNACon-pGenesil-1 by Lipofectamine 2000.Realtime quantitative polymerase chain reaction (Real-time qPCR) and Western blotting were applied to detect the expression levels of p21 mRNA and protein respectively.Results The eukaryotic expression plasmids dsRNAP21-pGenesil-1 or dsRNACon-pGenesil-1 were confirmed by DNA sequencing.Seventy-two h after transfection,Real-time qPCR showed that dsRNAP21-pGenesil-1 caused a significant induction in p21 mR-NA expression in two cell lines.Compared with mock transfections,induction of p21mRNA was 4.35- and 2.83-fold in PC-3 and T-24 cells,respectively.Western blotting analysis further verified that the elevated levels of p21 protein were strongly correlated to the increase of p21 mRNA expression in above cell lines.The p21 protein expression level in dsRNAP21-pGenesil-1 transfections was significantly higher than in dsCon-pGenesil-1 transfections and mock transfections ( P ＜ 0.05 ).Conclusion The constructed dsRNAP21-pGenesil-1 could have the ability to increase the expression of tumor suppressor gene p21 in urinary cancer.
