应用RNA干扰技术阻断PC-3细胞中survivin基因的表达

Inhibition of Survivin Gene Expression in PC-3 Cells by RNAi

目的：用真核转录载体pSilencer^TM3.1-H1 neo构建针对survivin基因的重组真核转录载体,并转染前列腺癌细胞系PC-3,通过RNA干扰（RNAi）阻断PC-3细胞中survivin基因的表达,并研究survivin基因沉默后对PC-3细胞凋亡产生的影响.方法：将合成的DNA正义链及反义链变性、退火,形成的双链DNA与pSilencer^TM3.1-H1 neo线性质粒用T4DNA连接酶连接,重组质粒经酶切及DNA测序鉴定后,用脂质体法转染PC-3细胞,通过RT-PCR、免疫印迹和免疫组织化学染色实验检测survivin的表达变化,并用流式细胞术检测转染后PC-3细胞的凋亡变化,MTr法检测细胞生长速度的变化.结果：酶切及测序证实设计合成的DNA已正确插入载体.RT-PCR、免疫印迹和免疫组织化学染色实验表明,pSilencer3.1-SVV2和pSilencer3.1-SVV3重组载体有效地阻断了PC-3细胞中survivin基因的表达,survivin基因在mRNA和蛋白水平上的表达明显下调（P＜0.01）,转染后PC-3细胞的凋亡增加了10%～15%,细胞生长速度明显变慢.结论：成功构建了针对survivin基因的真核转录载体pSilencer3.1-SVV1、pSilencer3.1-SVV2和pSilencer3.1-SVV3,后两者可有效地阻断前列腺癌细胞系PC-3细胞中survivin基因的表达,并使转染后PC-3细胞的凋亡增加了10%～15%,细胞生长速度明显变慢.

Objective： To construct eukaryotic expression vectors by using the pSilencer^TM3.1-H1 neo vector for inhibiting human survivin gene by RNA interference, and to detect the effect of the silenced survivin gene on PC-3 cells. Methods ： Three target gene segments were synthesized and cloned into the pSilencer^TM3. 1-H1 neo vector respectively to construct three recombinant eukaryotic expression vectors ： pSilencer3. 1-SVV1, pSilencer3. 1-SVV2 and pSilencer3. 1-SVV3, which were identified by enzyme digestion analysis and DNA sequencing. Then the PC-3 cells were transfected with the recombinant vectors and the interference effect detected by RT- PCR, Western blot and immunohistochemical staining. The apoptosis index of the PC-3 cells was detected by flow cytometry and their proliferation detected by MTT method. Results： Enzyme digestion analysis and DNA sequencing showed that three target segments were cloned into pSilencer^TM3. 1-H1-neo vectors. The results of RT-PCR, Western blot and immunohistochemieal staining indicated that pSilencer3.1-SVV2 and pSilencer3. 1-SVV3 vectors could knock clown the transcription and expression of survivin gene. After transfected with pSilencer3.1-SVV2 and pSilencer3.1-SVV3 vectors, the apoptosis index of the PC-3 cells was increased by 10% - 15% and their growth obviously slowly down. Conclusion ： The transcription and expression of survivin gene were inhibited effectively by the recombinant eukaryotic expression vectors（ pSilencer3.1-SVV2 and pSilencer3.1-SVV3 ） in the prostate cancer cell line PC-3. After transfected with pSilencer3.1-SVV2 and pSilencer3.1-SVV3 vectors, the apoptosis index of the PC-3 cells was increased and their growth inhibited.