抑制survivin基因对人卵巢癌SKOV3细胞顺铂药物敏感性的影响

目的：探讨应用RNAi技术下调survivin基因对人卵巢癌SKOV3细胞增殖、凋亡及顺铂敏感性的影响。方法：构建survivin基因shRNA真核表达载体,转染人卵巢癌SKOV3细胞。定量PCR和Western blotting观察SKOV3细胞survivinmRNA和蛋白表达的改变;噻唑蓝（Myr）检测细胞增殖活性和药物敏感性;流式细胞仪检测细胞凋亡。结果：SKOV3-siRNA组细胞survivinmRNA及蛋白表达下降,同时细胞增殖能力明显降低,细胞凋亡率显著增加（P〈0．05）。SKOV3-siRNA组细胞对顺铂的化学敏感性升高,顺铂的IC50值降低（P〈0．05）。结论：下调survivin基因表达能够抑制卵巢癌SKOV3细胞增殖能力,诱导细胞凋亡,增强细胞顺铂药物敏感性。因此,survivin基因可能成为抗肿瘤治疗的潜在靶点。     

siRNA转染肝癌HepG2细胞Survivin基因凋亡机制探讨

Objective  

The aim of this study was to investigate the molecular mechanism of anti-apoptotic action of survivin to the hepatoma-cellular carcinoma cell line HepG2.     

RNA干扰沉默DcR3对人胰腺癌细胞化疗药物敏感性影响的实验研究

背景与目的：大部分胰腺癌具有高表达诱骗受体-3(decoy receptor 3,DcR3)的特征,而后者与FasL凋亡途径相关,可能导致胰腺癌对化疗耐药。该研究旨在探讨RNA干扰沉默DcR3基因对人胰腺癌细胞化疗药物敏感性的影响及其可能机制。方法：构建带有DcR3-siRNA序列的稳定表达质粒,通过Lipofectamine^TM2000转染至人胰腺癌细胞AsPC-1细胞株,筛选出转染后稳定低表达DcR3的胰腺癌细胞,同时设未转染对照组(control组)和转染阴性质粒对照组(mock组)。应用ELISA和蛋白［质］印迹法(Western blot)检测各组AsPC-1细胞中DcR3的蛋白表达;MTT实验检测各组AsPC-1细胞对吉西他滨的敏感性;流式细胞术检测各组AsPC-1细胞凋亡情况;Western blot和实时荧光定量聚合酶链反应(real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR)检测各组AsPC-1细胞中FasL、Caspase-8、Caspase-3蛋白和mRNA的表达。结果：转染DcR3-siRNA后AsPC-1细胞中DcR3蛋白较其他对照组明显降低;转染DcR3-siRNA后AsPC-1细胞对吉西他滨的敏感性显著增加;沉默DcR3基因可以上调FasL、Caspase-8和Caspase-3的表达并促进吉西他滨诱导的细胞凋亡。结论：RNA干扰沉默DcR3基因可激活FasL/Caspase凋亡途径,促进肿瘤细胞凋亡,增加人胰腺癌AsPC-1细胞对化疗药物的敏感性。     

RNA干扰PLCE1基因表达对食管鳞癌细胞周期和凋亡的影响

目的：通过siRNA干扰PLCE1基因表达,检测PLCE1对食管鳞癌细胞增殖周期和凋亡的影响,以探讨PLCE1的致癌机制。方法：采用免疫组织化学方法检测食管鳞癌组织和癌旁正常组织中PLCE1蛋白的表达水平;PLCE1特异性siRNA转染食管鳞癌细胞,荧光显微镜观察转染效率;半定量RT-PCR法检测转染后PLCE1沉默效果;流式细胞术检测干扰后细胞周期变化及凋亡情况。结果：PLCE1在食管鳞癌组织的表达高于正常组织（P=0.000）;siRNA转染后PLCE1表达明显减少（P=0.000）;与对照组相比,PLCE1干扰后可致G0/G1期细胞阻滞（P=0.001）,细胞凋亡增多（P=0.000）。结论：PLCE1在食管鳞癌组织中有较高表达;沉默PLCE1后可抑制癌细胞的增殖,促进其凋亡。     

ＭＭＰ-２特异性ＲＮＡ干扰对人胰腺癌细胞粘附和侵袭的作用

探讨靶向基质金属蛋白酶-２（ＭＭＰ-２）的ＲＮＡ干扰对人胰腺癌细胞粘附和侵袭的作用。方法　设计和构建靶向ＭＭＰ-２的特异性小干扰ＲＮＡ（ｓｉＲＮＡ）表达质粒,将质粒转染入人胰腺癌ＢｘＰＣ-３细胞系中,根据转染质粒靶序列的不同分为５组,即ｐＧＰＵ６-１、ｐＧＰＵ６-２、ｐＧＰＵ６-３、ｐＧＰＵ６-４和阴性对照组ｐＧＰＵ６（－）,并设仅加入转染剂的空白对照组。应用ＲＴ-ＰＣＲ和Ｗｅｓｔｅｒｎｂｌｏｔ法检测各组ＢｘＰＣ-３细胞转染后ＭＭＰ-２蛋白及ｍＲＮＡ表达水平,应用ＭＴＴ法及流式细胞术检测各组细胞增殖和凋亡水平,以平板粘附模型和Ｔｒａｎｓｗｅｌｌ小室模型检测各组转染后肿瘤细胞粘附和侵袭能力,比较各组间差异。结果　与空白对照组和阴性对照组相比,ｐＧＰＵ６-１、ｐＧＰＵ６-２、ｐＧＰＵ６-３组ＭＭＰ-２ｍＲＮＡ抑制率分别为７５.３％、６４.５％、５１.６％和７４.７％、６３.７％、５０.５％,差异均有统计学意义;ＭＭＰ-２蛋白表达抑制率分别为７９.１％、６４％、５２.３％和７８％、６２.２％、５０.５％,差异均有统计学意义;粘附抑制率分别为６３.１％、４２.９％、２５.６％和６２.２％、４１.６％、２３.９％,且穿膜细胞数量明显降低,差异均有统计学意义。对照组与ｐＧＰＵ６-４组的ＭＭＰ-２蛋白及ｍＲＮＡ表达水平无明显差异,在肿瘤细胞粘附和侵袭能力方面也未见明显差异。ＭＴＴ法及流式细胞术的结果并没有提示各组间肿瘤细胞增殖和凋亡水平的差异。结论　靶向ＭＭＰ-２的ＲＮＡ干扰能够明显抑制胰腺癌ＢｘＰＣ-３细胞蛋白水平及ｍＲＮＡ水平的表达,从而抑制肿瘤细胞粘附和侵袭的能力,但并没有导致干扰后肿瘤细胞增殖和凋亡能力的改变。     

Lycorine induces apoptosis and down-regulation of Mcl-1 in human leukemia cells

Lycorine is an alkaloid isolated from the bulb of the Amaryllidaceae Lycoris. Here, we report that treatment with lycorine resulted in survival inhibition and apoptosis induction in human leukemia cell lines. Lycorine induced apoptosis in human leukemia cells via intrinsic mitochondria pathway and caused a rapid-turnover of protein level of Mcl-1 which occurred before caspases activation. Furthermore, pronounced apoptosis accompanied by the down-regulation of Mcl-1 was also observed in blasts from patients with acute myeloid leukemia. Our findings suggest that lycorine may be a good candidate therapeutic agent against leukemia in worth of further evaluation.     

RNA干扰技术靶向hTERT基因治疗肝癌的实验研究

背景与目的：RNA干扰(RNA interference,RNAi)是由双链RNA介导的、在转录后mRNA水平关闭相应基因表达的新基因阻断技术,在基因功能研究、基因治疗方面已显示出巨大的前景。目前,利用RNAi已抑制了包括cyclophilin、GAPDH、p53、c-myc在内的多个内源基因的表达。同时在艾滋病、病毒性肝炎等的治疗研究中也已取得一定进展。但对肝癌等恶性肿瘤中高表达的hTERT基因,国内外还未见相关研究报道。本研究利用RNAi技术,在体内外抑制hTERT基因表达,探讨RNAi对肝癌治疗的可行性。方法：设计干扰hTERT基因的小片段RNA,构建重组表达质粒pTzu6 1-shRNA-hTERT并导入肝癌SMMC7721细胞株和裸鼠移植瘤,在体内外诱导RNAi,采用流式细胞检测技术、RT—PCR法、免疫组化等同时检测RNAi治疗组和对照组hTERT基因表达及细胞增殖变化,结果：体外细胞实验显示,重组质粒pTZU6 1—shRNA—hTERT导入肝癌SMMC7721细胞株3～7天后,肝癌细胞生长抑制率达37．5％;细胞周期相分布发生显著变化,S期细胞明显减少,G1／G0期细胞显著增加;hTERT的mRNA表达由99．4％下调到53．1％,hTERT蛋白表达由86．3％下调到46．6％。裸鼠体内实验结果显示,质粒pTZu6 1-shRNA-hTERT注射裸鼠皮下移植瘤7天后,瘤体积明显缩小,hTERT的mRNA表达由99．1％下调到76．2％,hT．ERT蛋白表达由87．2％下调到61．8％。体内、外对照组各指标均无变化。结论：RNAi明显抑制了靶基因hTERT的表达及肝癌细胞增殖,是潜在的肿瘤治疗新方法。     

Targeting Mcl-1 for multiple myeloma (MM) therapy: Drug-induced generation of Mcl-1 fragment Mcl-1 triggers MM cell death via c-Jun upregulation

Myeloid cell leukemia-1 (Mcl-1, HGNC: 6943), a pro-survival member of the Bcl-2 family, plays a crucial role in Multiple Myeloma (MM) pathogenesis and drug resistance, thus representing a promising therapeutic target in MM. A novel strategy to inhibit Mcl-1 activity is the induction of ubiquitin-independent Mcl-1 degradation. Our own and other previous studies have demonstrated caspase-dependent generation of a 28 kDa Mcl-1 fragment, Mcl-1^128–350, which inhibits MM cell proliferation and survival. Here, we show that similar to bortezomib, the novel proteasome inhibitors carfilzomib and ixazomib, as well as staurosporine and adaphostin, induce the generation of Mcl-1^128–350 in MM cells. Next, the molecular sequelae downstream of Mcl-1^128–350, which mediate its pro-apoptotic activity, were delineated. Surprisingly, we observed nuclear accumulation of drug-induced or exogenously overexpressed Mcl-1^128–350, followed by elevated mRNA and protein levels of c-Jun, as well as enhanced AP-1 reporter activity. Moreover, drug-induced AP-1 activity was blocked after introducing a point mutation into the highly conserved Mcl-1 caspase-cleavage site Asp127, but not Asp157. Consequently, drug-triggered cell death was significantly decreased in MM cells transfected with Mcl-1 D127A, but not with Mcl-1 D157A. Consistent with these data, treatment with bortezomib triggered c-Jun upregulation followed by apoptosis in Mcl-1^wt/wt, but not Mcl-1^Δ/null murine embryonic fibroblasts (MEFs). Transfection of a plasmid carrying Mcl-1^wt into Mcl-1^Δ/null MEFs restored bortezomib-induced Mcl-1 fragmentation, c-Jun upregulation and AP-1 reporter activity. Finally, our data indicate that drug-induced generation of a pro-apoptotic Mcl-1 fragment followed by c-Jun upregulation may also be a novel therapeutic approach in other tumor entities.     

小干扰RNA靶向MIF对胃癌细胞SGC 7901增殖和凋亡的影响#br#

目的探讨靶向巨噬细胞移动抑制因子（MIF）的小干扰RNA（siRNA）对胃癌细胞SGC 7901增殖和凋亡的影响。 方法取对数生长期的SGC 7901细胞,采用脂质体法分别转染靶向人MIF的siRNA（siMIF组）或阴性对照序列（NC组）,48 h后采用Western blotting检测MIF蛋白的表达情况,MTT法观察转染后24、48、72 h的细胞增殖情况,流式细胞仪检测转染后72 h的细胞凋亡率,Western blotting检测凋亡相关蛋白Bcl 2和Bax的表达变化。 结果siMIF组转染48 h后的MIF相对表达量为0321±0104,低于NC组的1078±0212,差异有统计学意义（P＜005）。siMIF组转染48～72 h后的细胞增殖率低于NC组,差异有统计学意义（P＜005）。转染MIF siRNA 72 h后,siMIF组的细胞凋亡率为（235±36）％,高于NC组的（47±17）％,差异有统计学意义（P＜005）。siMIF组Bcl 2的相对表达量为0663±0209,低于NC组的1129±0178,而Bax相对表达量为0981±0225,高于NC组的0587±0254,以上差异均有统计学意义（P＜005）。 结论siRNA靶向沉默MIF能够降低SGC 7901细胞中MIF蛋白表达,抑制SGC 7901细胞的增殖并促进凋亡,在胃癌的靶向治疗中有一定前景。     

肝癌细胞系BEL-7402中siRNA表达载体介导靶向c-myc基因的RNA干涉实验研究

Objective： To explore the inhibition of the expression of c-Myc in human hepatocellular carcinoma via vector-based RNA interference （RNAi）. Methods： Two RNA interference DNA templates targeting c-Myc oncogene were designed via online software and synthesized. By ligation, the fragments were inserted into pSilencer 1.0-U6 to construct the recombinant siRNA expressing plasmids. The identified recombinants were introduced into BEL-7402 cells with Lipofactamine. The inhibition of c-Myc expression, together with the expression of CDK4, hTERT and Gadd45β in c-Myc down-regulated BEL-7402 cells, were analyzed by semi-quantitative RT-PCR. Results： Two recombinant plasmids pSic-myc-1 and pSic-myc-2, which direct the yields of siRNAs targeting c-Myc in cells, were constructed. Among which, pSic-myc-2 was shown to trigger a RNAi-mediated inhibition of expression of c-Myc in BEL-7402 by up to 90%. In c-Myc knockdown BEL-7402 cells, the expression of CDK4 and hTERT were down-regulated with a ratio of 85% and 57%, respectively, while the expression of Gadd45β was up-regulated by up to 110%. Conclusion： The expression of c-Myc in BEL-7402 could be suppressed by vector-based RNA interference successfully. The knockdown of c-Myc in turn resulted in the changes of expression of genes related to cell proliferation and apoptosis. Thus, our study provided a preliminary data in searching of a c-Myc-targeted RNAi therapy of human hepatocellular carcinoma.     

RNA干扰技术在以survivin为靶点的肿瘤基因治疗中的应用

survivin选择性地在大部分肿瘤中表达,越来越多的研究者将其作为肿瘤基因治疗的理想靶点.RNA干扰技术以其高效、特异性强的特点成为在消化系统肿瘤、乳腺癌、膀胱癌、肺癌、妇科肿瘤等各种恶性肿瘤中研究survivin基因沉默的理想手段.     

Over-expression of the Beclin1 gene upregulates chemosensitivity to anti-cancer drugs by enhancing therapy-induced apoptosis in cervix squamous carcinoma CaSki cells

The purpose of this study was to investigate whether the autophagy-related gene, Beclin1, plays a role in the regulation of chemosensitivity to anti-cancer drugs in cervical cancer CaSki cells. Expression of the Beclin1 protein was up-regulated in pcDNA3.1-Bec transfectants and led to cell arrest in the G₀/G₁ phase of the cell cycle. The MTT assay indicated that over-expression of Beclin1 sensitized CaSki cells to chemotherapeutic drugs (cisplatin, paclitaxel, 5-fluorouracil, and epirubicin) and induced greater degrees of cytotoxicity than vector-only controls. After treatment with anti-cancer drugs, flow cytometric analysis indicated that the Beclin1-transfected group showed a greater increase in apoptosis than did the non-transfected group. Furthermore, pSUPER-Bec transfectants did not lead to a significant increase of resistance to each of these anti-cancer drugs. These results suggest that Beclin1 plays an important role in the regulation of potent anti-tumor activity, and over-expression of Beclin1 in CaSki cells may enhance apoptosis signaling induced by anti-cancer drugs.     

Adenoviral‐mediated RNA interference targeting URG11 inhibits growth of human hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is the second most common malignancy in Asia, with a 5‐year survival rate of less than 5% due to high recurrence after surgery and resistance to chemotherapy. A variety of therapeutic interventions to treat HCC, particularly gene therapy, have recently been investigated in tumor model systems to provide a more complete understanding of hepatocarcinogenesis and effectively design therapeutic strategies to treat this disease. In our study, we constructed an adenoviral vector expressing small interfering RNA (siRNA) targeting a newly discovered gene named upregulated gene 11 (URG11). We introduced this vector into HCC cells to investigate the role of URG11 in HCC carcinogenesis. We observed that upon URG11 knockdown, HCC cell proliferation was inhibited through downregulation of several G1‐S phase related molecules including cyclin D1 and apoptosis was induced as a result of Bcl‐2 downregulation. Besides decreased expression of cyclin D1, CDK4, pRb and Bcl‐2, URG11 also suppressed several other proteins including CAPN9, which was identified by cDNA microarray and 2D gel electrophoresis. Moreover, Ad‐URG11‐siRNA significantly suppressed HCC tumor growth in nude mice. In conclusion, Ad‐URG11‐siRNA can significantly suppress HCC tumor growth in vitro and in vivo by silencing the URG11 gene, and the use of this vector for gene therapy may represent a novel strategy to treat human HCC.     

RNA干扰技术失活Rad51基因增加肿瘤细胞放射敏感性的研究

目的 研究RNA干扰技术（RNAi）能否失活Rad51基因，从而使肿瘤细胞的放射敏感性增加。方法 应用Psilencer U6-1．0质粒为载体在人纤维肉瘤HTl080细胞内表达短链干扰RNA（siRNA）。RNAi效应在蛋白水平用Westernblot方法评价；在细胞水平用成克隆分析及放射诱导的凋亡来评价。结果 所选3个靶序列均起到失活Rad51的作用，放射诱导的Rad51高表达也能被RNAi抑制。成克隆分析表明实验组存活分数在照射2、4、6、8Gy后，分别是对照组的80．5％、78．6％、69．0％、57．7％（P=0．020）；实验组放射诱导的凋亡在放射后0、6、18、24、48h分别是对照组的1．03、1．43、1．19、1．29、1．33倍（P=0．017）。结论 RNAi技术能够使内源性Rad51基因及放射诱导的Rad51高表达失活，从而使人纤维肉瘤HT1080细胞放射敏感性增加；RNAi技术是一种新的研究基因功能和放射增敏的方法。     

RNA干扰 Rac1基因表达对结直肠癌LoVo细胞骨架和细胞周期的影响

目的:观察Rac1蛋白在结直肠癌细胞中的表达,并分析其与结直肠癌LoVo细胞骨架、细胞周期和细胞凋亡的相关性.方法:Western blotting测定5种结直肠癌细胞株(LoVo,SW480,SW620,SW1116,HT29)中Rac1蛋白的表达;Rac1-shRNA质粒转染LoVo细胞后,激光共聚焦显微镜观察LoVo细胞骨架的变化,流式细胞仪检测细胞周期和细胞凋亡的变化.结果:5种结直肠癌细胞株中均有Rac1蛋白的高表达.Rac1基因沉默后,LoVo细胞中交联状态的F-actin网明显减少且紊乱,G0/G1期细胞比例较对照组显著增加[(74.63 ±4.40)％ vs(56.46 ±3.09)％,P＜0.05],而S期细胞比例较对照组显著减少[(12.87±1.77)％vs(29.66±1.92)％,P＜0.05];Rac1-shRNA转染组LoVo细胞凋亡率较对照组显著增加[(25.31±2.05)％vs(9.38±1.16)％,P＜0.05].结论:RNA沉默Rac1基因的表达影响了LoVo细胞骨架的形成和细胞周期,为以Rac1为靶点治疗结直肠癌提供了实验依据.     

RNA干扰沉默Sp1基因抑制大肠癌SW620细胞的恶性增生

目的采用RNA干扰技术沉默大肠癌SW620细胞转录因子特化蛋白1（Sp1）的表达,观察其对细胞增生的影响。方法构建靶向人Sp1基因的干扰载体（pGenesil-1-Sp1）,脂质体介导转染SW620细胞,荧光倒置显微镜下观察转染效率,应用实时荧光定量PCR和Western blotting检测其对Sp1 mRNA及蛋白质水平的影响,MTT法检测SW620细胞增生活性的改变。结果成功构建针对Sp1的干扰载体,转染后能够有效抑制大肠癌SW620细胞中Sp1 mRNA和蛋白的表达,且在转染48h抑制作用最强,其对Sp1 mRNA和蛋白质抑制率分别为68．47％和73．82％,与对照组相比,差异有统计学意义（P〈0．05）。MTT实验显示,下调Sp1表达可显著抑制大肠癌SW620细胞的体外增生能力。结论Sp1基因沉默能够抑制人大肠癌SW620细胞的增生,为以Sp1为靶点的大肠癌基因治疗提供新的思路和手段。     

Knockdown of radixin by RNA interference suppresses the growth of human pancreatic cancer cells in vitro and in vivo

Radixin, encoded by a gene on chromosome 11, plays important roles in cell motility, invasion and tumor progression. However, its function in pancreatic cancer remains elusive. In this study, radixin gene expression was suppressed with a lentivirus-mediated short-hairpin RNA (shRNA) method. We found that radixin shRNA caused down-regulation of radixin in PANC-1 cells, associated with inhibition of pancreatic cancer cell proliferation, survival, adhesion and invasive potential in vitro. When radixin-silenced cells were implanted in nude mice, tumor growth and microvessel density were significantly inhibited as compared to blank control cells or nonsense shRNA control cells. Thrombospondin-1 (TSP-1) and E-cadherin were up-regulated in radixin-silenced PANC-1 cells. Our results suggest that radixin might play a critical role in pancreatic cancer progression, possibly through involvement of down-regulation of TSP-1 and E-cadherin expression.     

shRNA靶向干扰NHERF1对PC-3M前列腺癌细胞生物学行为的影响

目的 探讨shRNA靶向干扰钠氢交换调节因子(NHERF)1后对前列腺癌细胞PC-3M生物学行为的影响.方法 将pSuper.puro NHERF1 shRNA质粒和阴性对照质粒pSuper.puro luciferase shRNA分别转染人PC-3M前列腺癌细胞,经嘌呤霉素筛选,建立稳定低表达NHERF1的PC-3M细胞株.经Western印迹验证后,采用四甲基偶氮唑蓝(MTT)法检测肿瘤细胞增殖活性,采用划痕实验检测NHERF1-shRNA对前列腺癌PC-3M细胞迁移能力的影响,采用流式细胞仪检测NHERF1-shRNA对PC-3M细胞凋亡的影响,采用Western印迹检测NHERF1-shRNA对凋亡相关蛋白Bax及Bcl-2表达的影响.结果 转染NHERF1-shRNA组比未转染及空载组细胞的增殖能力明显降低(F=62.63,P=0.004);转染NHERF1-shRNA组比未转染及空载组细胞的迁移率明显降低.相比于未转染PC-3M细胞和转染阴性对照质粒细胞,转染NHERF1-shRNA组细胞的凋亡百分比明显增加[(2.55±0.92)％和(11.98±3.28)％],达4倍以上(t=-2.392,P=0.001);Bcl-2蛋白的表达降低,Bax蛋白的表达升高.结论 靶向NHERF1的序列特异性shRNA可抑制PC-3M细胞的增殖和迁移,并促进细胞凋亡.     

慢病毒介导RNA干扰Pin1异构酶的鼻咽癌稳定细胞株的建立和鉴定

目的:应用慢病毒介导的RNA干扰技术,建立稳定表达siPin1的鼻咽癌细胞株。方法:将对照组病毒液lv-3和实验组病毒液siPin1感染CNE2细胞,通过嘌呤霉素和绿色荧光检测筛选siPin1稳定细胞株,在荧光显微镜下观察细胞的荧光表达,通过定量PCR和Western blot鉴定siPin1细胞Pin1 mRNA和蛋白的表达。结果:筛选siPin1稳定表达细胞株嘌呤霉素筛选浓度为1μg/ml,病毒感染后超过90%细胞发出绿色荧光,实验组siPin1与对照组lv-3相比,Pin1 mRNA表达水平下降,Western blotting检测Pin1蛋白表达明显减少。结论:成功构建Pin1干扰的稳定表达鼻咽癌细胞株,为后续研究提供工具细胞。     

慢病毒载体介导RNA干扰体外抑制人胰腺癌细胞VIM基因的表达

Objective: To construct a lentiviral expression vector for RNA interference (RNAi) of human VIM gene; and assess its gene silencing effect in pancreatic cancer cell line Panc-1. Methods: Three pairs of human VIM gene short hairpin RNA (shRNA) sequences were designed using a software available on-line and one pair came from document. After synthesis and annealing, four double-stranded oligonucleotides (dsOligo) were cloned into the pGCL-GFP/U6 plasmid, which were subsequently confirmed by polymerase chain reaction (PCR) and DNA sequencing analysis. Real-time PCR and Western blotting were used to screen the effective pGCL-GFP-shRNA plasmid in 293T cells, then the most effective one was packed into the recombinant lentivirus Lv-VIM-shRNA with lentiviral packing materials pHelper 1.0 and pHelper 2.0 in 293T cells.The titer of lentivirus was determined by hole-by-dilution titer assay. The silencing effect of Lv-VIM-shRNA in Panc-1 cells were validated by real-time PCR and Western-blotting. Results: An effective Lv-VIM-shRNA was successfully constructed.The titer of lentivirus was determined on 2×109TU/mL. The expressions of VIM mRNA and vimentin were down-regluated in the Panc-1 cells infected with Lv-VIM-shRNA. Conclusion: An effective Lv-VIM-shRNA could inhibit the expression of VIM gene in Panc-1 cells in vitro, which provides a tool for investigating the role of VIM gene in the signaling pathway involved in tumorigenesis and progression of pancreatic cancer and searching new therapeutic targets.