Radioimmunoassay Center & Clinical Laboratory, Second Affiliated Hospital, Soochow University, Suzhou 215004, China.
Hepatobiliary Pancreat Dis Int. 2010 Feb;9(1):69-77.
Survivin is known to be overexpressed in various human malignancies, including pancreatic cancer, and mediates cancer cell proliferation and tumor growth, so the regulation of this molecule could be a new strategy for treating pancreatic cancer. In this study, short hairpin RNAs (shRNAs) specific to survivin were introduced into human pancreatic cancer Patu8988 cells to investigate the inhibitory effects on survivin expression and cell proliferation in vitro and in vivo.
Three kinds of shRNA specific to the survivin gene were designed and cloned into eukaryotic expression plasmid pGenesil-1 vector. Subsequently the recombinant plasmids were transfected into human pancreatic cancer Patu8988 cells with lipfectamineTM 2000 reagent. The mRNA and protein expressions of survivin in the transiently transfected Patu8988 cells were determined by RT-PCR, flow cytometry, and Western blotting analysis. The proliferation inhibition rates of stably transfected Patu8988 cells were determined by MTT assay. The antitumor activities of the three kinds of survivin-shRNA plasmids were evaluated in BALB/c nude mice inoculated with Patu8988 cells and bearing human pancreatic cancer.
The three survivin-shRNA plasmids named pGenesil-1-survivin-1, pGenesil-1-survivin-2 and pGenesil-1-survivin-1+2 (with double interfering RNA sites) were successfully constructed, and were confirmed by restriction enzyme cutting and sequencing. At 48 hours after transfection, the expression of survivin mRNA and protein was inhibited in Patu8988 cells transfected with pGenesil-1-survivin-1, pGenesil-1-survivin-2, and pGenesil-1-survivin-1+2 when compared with that of either pGenesil-1-NC (with scrambled small interfering RNA) transfected cells or control cells (P<0.05). The MTT results showed that the proliferation rates of Patu8988 cells stably transfected with survivin-shRNA plasmids were reduced when compared with that of either pGenesil-1-NC transfected cells or control cells (P<0.01). Furthermore, when Patu8988 cells stably transfected with survivin-shRNA were injected into BALB/c nude mice, tumor growth was dramatically lower and the tumor was smaller than that of either pGenesil-1-NC transfected cells or control cells (P<0.01). The inhibitory effect of pGenesil-1-survivin-1 was the best among the three kinds of survivin-shRNA plasmids, but no combination of inhibitory effects was found in pGenesil-1-survivin-1+2.
shRNAs specific to survivin have gene silencing effects and inhibit pancreatic cancer cell proliferation. shRNA activity against survivin could be of potential value in gene therapy for pancreatic cancer. However, shRNAs with double combining sites did not significantly enhance the interference compared with single site shRNAs, therefore further studies on this are needed.
Survivin 已知在多种人类恶性肿瘤中过度表达，包括胰腺癌，并介导癌细胞增殖和肿瘤生长，因此该分子的调节可能是治疗胰腺癌的新策略。在这项研究中，将短发夹 RNA（shRNA）特异性引入人胰腺癌细胞 Patu8988 中，以研究体外和体内对 survivin 表达和细胞增殖的抑制作用。
设计并克隆了三种针对 survivin 基因的 shRNA，并将其插入真核表达质粒 pGenesil-1 载体中。随后，使用 lipfectamineTM 2000 试剂将重组质粒转染入人胰腺癌细胞 Patu8988 中。通过 RT-PCR、流式细胞术和 Western blot 分析检测瞬时转染的 Patu8988 细胞中 survivin 的 mRNA 和蛋白表达。通过 MTT 测定法测定稳定转染的 Patu8988 细胞的增殖抑制率。在接种 Patu8988 细胞并携带人胰腺癌的 BALB/c 裸鼠中评估三种 survivin-shRNA 质粒的抗肿瘤活性。
成功构建了三种名为 pGenesil-1-survivin-1、pGenesil-1-survivin-2 和 pGenesil-1-survivin-1+2（具有双干扰 RNA 位点）的 survivin-shRNA 质粒，并通过限制性内切酶切割和测序进行了验证。转染 48 小时后，与 pGenesil-1-NC（带有乱序小干扰 RNA）转染细胞或对照细胞相比，转染 pGenesil-1-survivin-1、pGenesil-1-survivin-2 和 pGenesil-1-survivin-1+2 的 Patu8988 细胞中 survivin mRNA 和蛋白的表达均受到抑制（P<0.05）。MTT 结果表明，与 pGenesil-1-NC 转染细胞或对照细胞相比，稳定转染 survivin-shRNA 质粒的 Patu8988 细胞的增殖率降低（P<0.01）。此外，当稳定转染 survivin-shRNA 的 Patu8988 细胞注入 BALB/c 裸鼠时，肿瘤生长明显降低，肿瘤体积明显小于 pGenesil-1-NC 转染细胞或对照细胞（P<0.01）。三种 survivin-shRNA 质粒中，pGenesil-1-survivin-1 的抑制效果最好，但 pGenesil-1-survivin-1+2 中没有发现联合抑制作用。
特异性针对 survivin 的 shRNA 具有基因沉默作用，并抑制胰腺癌细胞增殖。针对 survivin 的 shRNA 活性在胰腺癌的基因治疗中可能具有潜在价值。然而，具有双结合位点的 shRNA 与单结合位点 shRNA 相比并没有显著增强干扰作用，因此需要进一步研究。
