Growth inhibition induced by short hairpin RNA to silence survivin gene in human pancreatic cancer cells

BACKGROUND: 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. METHODS: 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 lipfectamine (TM) 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. RESULTS: 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. CONCLUSIONS: 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.

Influence of Osteopontin Short Hairpin RNA on the Proliferation and Activity of Rat Vascular Smooth Muscle Cells

To investigate the influence of osteopontin （OPN） short hairpin RNA （shRNA） on the proliferation and activity of rat vascular smooth muscle cells （VSMCs）, the expressing vector of shRNA targeting OPN was constructed and transferred into the rat VSMCs. After amplification and purification, pGenesil-1/OPNshRNA1 （PG1）, pGenesil-1/OPNshRNA2 （PG2） and pGenesil-1/OPNshRNAHK （PGH） were transfected into the cultured rat VSMC by LipofectamineTM 2000. Transfected cells were visualized by using an inverted fluorescent microscope. VSMCs transfected by optimal recombined plasmid was selected by culturing in G418 48 h later. Nude cells and cells transfected by PGH were used as control. The expression levels of OPN mRNA and protein were assayed by RT-PCR and Western blotting. The OPN of VSMCs was suppressed by transfection of optimal recombined plasmid, and the changes in cell proliferation, adhesion and motility were evaluated by MTT, adhesion test and transwell chamber test. Levels of type I and Ⅲ collagen were measured with ELISA kit. Our results showed that VSMCs stably transfected by OPN shRNA accounted for over 50% of total cells. OPN mRNA and protein were reduced by 81% and 67% （P〈0.01） by PG1, 73% and 52% （P〈0.01） by PG2, respectively while no change was found in PGH and non-treated VSMCs. PG1 significantly suppressed the proliferation, adhesion, mobility of VSMCs and reduced the amount of type Ⅰ and Ⅲ collagen. It is concluded that recombinant plasmid can be success-fully transfected into VSMCs by LipofectamineTM 2000 and inhibit the expression of OPN. The proliferation, adhesion and mobility of VSMCs can be inhibited by knocking down OPN expression. Moreover, the transferring capability of cells is attenuated, and the secretion of type Ⅰ and Ⅲ collagen is inhibited aftter knocking-down of OPN expression. The study provides experimental evidence for clinical prevention of restenosis after percutaneous coronary intervention （PCI） by RNA interference （RNAi） technology.

Short Hairpin RNA-mediated MDR1 Gene Silencing Increases Apoptosis of Human Ovarian Cancer Cell Line A2780/Taxol

Objective： Recurrent ovarian cancer is often resistant to drugs such as paclitaxel. Short hairpin RNA （shRNA） targeting MDRI, a gene involved in the process of drug resistance, may be a promising strategy to overcome drug resistance. Methods： Construction and identification of eukaryotic expression plasmid was transiently transfected into human ovarian cancer ce plasmid of shRNA targeting on MDR1 gene. The ne A2780/Taxol. Apoptosis was determined by flow cytometry using annexin V-FITC/PI double labeling. Expression of MDRI mRNA was detected by quantitative polymerase chain reaction （qPCR） and P-glycoprotein expression was detected using Western blot. Results The IC50 of paclitaxel in MDR1 shRNA-transfected group was significantly reduced （1.986±0.153）μmol/ml as compared with that in negative control （5.246±0.107）μmol/ml and empty vector-transfected group （5.212±0.075）μmol/ml （P〈0.05）. The percent of the relative reverse sensitivity to paclitaxel on A2780/Taxol cells was 67.1%, and the apoptotic rate was significantly increased [（6.977±0.333）%] compared with control [（1.637±0.111）%] and empty vector-transfected group [（1.663±0.114）%] （P〈0.05）. Expressions of MDR1 mRNA and P-glycoprotein were significantly reduced compared with control （P〈0.05）. Conclusion： The present study demonstrated that the eukaryotic expression plasmid of shRNA targeting on MDRI inhibited the expression of MDRI effectively, thus enhance the sensitivity of A2780/Taxol cells to paclitaxel.

Influence of Osteopontin Short Hairpin RNA on the Proliferation and Invasion of Human Renal Cancer Cells

The influence of short hairpin RNA(shRNA)-mediated osteopontin(OPN)gene silencing on the proliferation and invasion of human renal cancer ACHN cells was investigated.Four types of OPN shRNA recombinant plasmids were constructed and RT-PCR assays were used to screen the most highly functional shRNA recombinant plasmids,which were transferred into the cultured ACHN cells by LipofectamineTM 2000.The cells transfected by shRNA expression vectors(ACHN/OPN)were visualized under an inverted microscope and screened...

Lentivirus-mediated short hairpin RNA interference of CENPK inhibits growth of colorectal cancer cells with overexpression of Cullin 4A

BACKGROUND Colorectal cancer(CRC)is one of the most common malignant tumors worldwide.The identification of novel diagnostic and prognostic biomarkers for CRC is a key research imperative.Immunohistochemical analysis has revealed high expression of centromere protein K(CENPK)in CRC.However,the role of CENPK in the progression of CRC is not well characterized.AIM To evaluate the effects of knockdown of CENPK and overexpression of Cullin 4A(CUL4A)in RKO and HCT116 cells.METHODS Human colon cancer samples were collected and tested using a human gene expression chip.We identified CENPK as a potential oncogene for CRC based on bioinformatics analysis.In vitro experiments verified the function of this gene.We investigated the expression of CENPK in RKO and HCT116 cells using quantitative polymerase chain reaction(qPCR),western blot,and flow cytometry.The effect of short hairpin RNA(shRNA)virus-infected RKO cells on tumor growth was evaluated in vivo using quantitative analysis of fluorescence imaging.To evaluate the effects of knockdown of CENPK and overexpression of CUL4A in RKO and HCT116 cells,we performed a series of in vitro experiments,using qPCR,western blot,MTT assay,and flow cytometry.RESULTS We demonstrated overexpression of CENPK in human colon cancer samples.CENPK was an independent risk factor in patients with CRC.The downstream genes FBX32,CUL4A,and Yesassociated protein isoform 1 were examined to evaluate the regulatory action of CENPK in RKO cells.Significantly delayed xenograft tumor emergence,slower growth rate,and lower final tumor weight and volume were observed in the CENPK short hairpin RNA virus infected group compared with the CENPK negative control group.The CENPK gene interference inhibited the proliferation of RKO cells in vitro and in vivo.The lentivirus-mediated shRNA interference of CENPK inhibited the proliferation of RKO and HCT116 colon cancer cells,with overexpression of the CUL4A.CONCLUSION We indicated a potential role of CENPK in promoting tumor proliferation,and it may be a novel diagnostic and prognostic biomarker for CRC.

Construction of Short Hairpin RNA Vector with σNS & σC Genes of Avian Reovirus and Determination of Interference Effect

[ Objective] The aim was to explore novel method for treatment of Avian Reovirus. [ Method] According to the design principle of siRNA target sequences, siRNA templates were designed and synthesized and then cloned into the shRNA expression vector, namely, pSilencer-CMV 4.1 neo. Short hairpin RNA vector C1, C2, C3, which contain σC gene, and shRNA vector NS1, NS2, NS3, which contain aNS gene, were constructed separately. The constructed shRNA vectors and negative control were co-transfected into DF-1 cells with the eukaryotic expression vector pEG- FP-σC and pEGFP-σNS, respectively. [ Result] Observation through fluorescence microscope indicated that the constructed 6 shRNA could inhibit the expression of fusion protein to different degrees. In addition, results of Real-time PCR suggested that C3 and NS1 have the best interference effect to the viral duplication in vitro. [ Conclusionl Construction and selection of specific shRNA expression vectors inhibiting Avian Reovirus are significant for researching effects of σC and oNS proteins in infection and duplication of ARV, providing new idea for ARV antiviral therapy.

Short hairpin RNA-mediated knockdown of nuclear factor erythroid 2-like 3 exhibits tumor-suppressing effects in hepatocellular carcinoma cells

BACKGROUND Hepatocellular carcinoma(HCC) is one of the most common malignant tumors with high mortality-to-incidence ratios. Nuclear factor erythroid 2-like 3(NFE2 L3), also known as NRF3, is a member of the cap ‘n' collar basic-region leucine zipper family of transcription factors. NFE2 L3 is involved in the regulation of various biological processes, whereas its role in HCC has not been elucidated.AIM To explore the expression and biological function of NFE2 L3 in HCC.METHODS We analyzed the expression of NFE2 L3 in HCC tissues and its correlation with clinicopathological parameters based on The Cancer Genome Atlas(TCGA) data portal. Short hairpin RNA(shRNA) interference technology was utilized to knock down NFE2 L3 in vitro. Cell apoptosis, clone formation, proliferation, migration,and invasion assays were used to identify the biological effects of NFE2 L3 in BEL-7404 and SMMC-7721 cells. The expression of epithelial-mesenchymal transition(EMT) markers was examined by Western blot analysis.RESULTS TCGA analysis showed that NFE2 L3 expression was significantly positively correlated with tumor grade, T stage, and pathologic stage. The qPCR and Western blot results showed that both the mRNA and protein levels of NFE2 L3 were significantly decreased after shRNA-mediated knockdown in BEL-7404 and SMMC-7721 cells. The shRNA-mediated knockdown of NFE2 L3 could induce apoptosis and inhibit the clone formation and cell proliferation of SMMC-7721 and BEL-7404 cells. NFE2 L3 knockdown also significantly suppressed the migration, invasion, and EMT of the two cell lines.CONCLUSION Our study showed that shRNA-mediated knockdown of NFE2 L3 exhibited tumor-suppressing effects in HCC cells.

Complement factor B knockdown by short hairpin RNA inhibits laser-induced choroidal neovascularization in rats

AIM:To evaluate whether recombinant complement factor B(CFB)short hairpin RNA(sh RNA)reduces laserinduced choroidal neovascularization(CNV)in rats.METHODS:Laser-induced rat CNV model was established,and then the animals underwent fundus fluorescence angiography(FFA)and hematoxylin and eosin(HE)staining.On day 3 and 7 after photocoagulation,the expression of CFB and membrane attack complex(MAC)was detected by immunhischemistry.A recombinant CFBsh RNA plasmid was constructed.CFB and scrambled sh RNA plasmids were intravenous injected into rats via the tail vein on the day of laser treatment,respectively.On day 7,the incidence of CNV was determined by FFA,and the expression of CFB and vascular endothelial growth factor(VEGF)in retinal pigment epithelium(RPE)/choroidal tissues was detected by immunhischemistry,Western blot and/or semi-quantitative reverse transcription-polymerase chain reaction(RT-PCR)in CFB and scrambled sh RNA groups.The possible adverse effects of CFB-sh RNA injection were assessed by transmission electron microscopy and electroretinography.RESULTS:FFA and HE results indicated that a laserinduced rat CNV model was successfully established on day 7 after photocoagulation.The expression of CFB and MAC was extremely weak in normal retina and choroid,and increased on day 3 after photocoagulation.However,it started to reduce on day 7.CFB sh RNA plasmid was successfully constructed and induced CFB knockdown in the retinal and choroidal tissues.FFA showed CFB knockdown significantly inhibited incidence of CNV in rats.Moreover,CFB knockdown significantly inhibited the expression of VEGF in RPE/choroidal tissues.CFB sh RNA caused no obvious side effects in eyes.CONCLUSION:CFB knockdown significantly inhibits the formation and development of CNV in vivo through reducing the expression of VEGF,which is a potential therapy target.The alternative pathway of complement activation plays an important role in CNV formation.

Suppression of Replication of Rabies Virus by Short Hairpin RNAs Expressed by Plasmid

Targeting the N gene of rabies virus (RV), four shRNA expression plasmids were designed and constructed based on the vector pRNATU6.3-Hygro that expresses fusion protein with GFP as a reporter gene. Four cell strains (N1, N2, N3, N4) expressing the short hairpin RNAs (shRNA) were obtained after the plasmids were transfected into BHK-21 cells and screened under the pressure of Hygromycin B (300 μg/mL). These cell strains were infected with 100× the TCID 50 of rabies virus CVS-11 strain, and the viral replication was quantified at 24, 48, 72 and 96 hours by directed immunofluorescence assay (DFA), real-time PCR, and the 50% tissue culture infective dose (TCID 50 ). The results showed variable inhibition of viral replication, with BHK-N2 being the most effective strain (99% inhibition). There was close correspondence between results using the three methods of evaluation. The shRNA-mediated inhibition persisted to at least 96 hours after infection. Effective inhibition of replication of RV in BHK-21 cells was achieved by siRNA targeting the N gene, with N 2 , aimed at the region starting at position 701 of the gene, being the most potent.

AAV-mediated expression of p65shRNA and bone morphogenetic protein 4 synergistically enhances chondrocyte regeneration

BACKGROUND:Adeno-associated virus(AAV)gene therapy has been proven to be reliable and safe for the treatment of osteoarthritis in recent years.However,given the complexity of osteoarthritis pathogenesis,single gene manipulation for the treatment of osteoarthritis may not produce satisfactory results.Previous studies have shown that nuclear factorκB could promote the inflammatory pathway in osteoarthritic chondrocytes,and bone morphogenetic protein 4(BMP4)could promote cartilage regeneration.OBJECTIVE:To test whether combined application of AAV-p65shRNA and AAV-BMP4 will yield the synergistic effect on chondrocytes regeneration and osteoarthritis treatment.METHODS:Viral particles containing AAV-p65-shRNA and AAV-BMP4 were prepared.Their efficacy in inhibiting inflammation in chondrocytes and promoting chondrogenesis was assessed in vitro and in vivo by transfecting AAV-p65-shRNA or AAV-BMP4 into cells.The experiments were divided into five groups:PBS group;osteoarthritis group;AAV-BMP4 group;AAV-p65shRNA group;and BMP4-p65shRNA 1:1 group.Samples were collected at 4,12,and 24 weeks postoperatively.Tissue staining,including safranin O and Alcian blue,was applied after collecting articular tissue.Then,the optimal ratio between the two types of transfected viral particles was further investigated to improve the chondrogenic potential of mixed cells in vivo.RESULTS AND CONCLUSION:The combined application of AAV-p65shRNA and AAV-BMP4 together showed a synergistic effect on cartilage regeneration and osteoarthritis treatment.Mixed cells transfected with AAV-p65shRNA and AAV-BMP4 at a 1:1 ratio produced the most extracellular matrix synthesis(P<0.05).In vivo results also revealed that the combination of the two viruses had the highest regenerative potential for osteoarthritic cartilage(P<0.05).In the present study,we also discovered that the combined therapy had the maximum effect when the two viruses were administered in equal proportions.Decreasing either p65shRNA or BMP4 transfected cells resulted in less collagen II synthesis.This implies that inhibiting inflammation by p65shRNA and promoting regeneration by BMP4 are equally important for osteoarthritis treatment.These findings provide a new strategy for the treatment of early osteoarthritis by simultaneously inhibiting cartilage inflammation and promoting cartilage repair.

Apoptosis induced by short hairpin RNA-mediated STAT6 gene silencing in human colon cancer cells

Background The relationship between signal transduction and tumors has become one of the loci in cancer research. Signal transducer and activator of the transcription 6 （STAT6） signaling pathway is found to be activated in some cancer cells. But the function of the pathway in cancer cells is unknown. This study was undertaken to investigate the effect of the Stat6 signaling pathway on apoptosis in human colon cancer cells （HT-29 cells） and the possible mechanism of Stat6 by RNA interference techniques.

Reversal of multidrug resistance in renal cell carcinoma by short hairpin RNA targeting MDR1 gene

Background Over-expression of P-glycoprotein （P-gp）, encoded by the MDR1 gene, confers multidrug resistance （MDR） in renal cell carcinoma （RCC） and is a major reason for unsuccessful chemotherapy. This study aimed to determine the effct of RNA interference （RNAi） on the reversal of MDR in human RCC. Methods We designed and selected one short hairpin RNA （shRNA） targeting MDR1 gene, which is stably expressed from integrated plasmid and transfected by lentivirus fluid in human RCC A498 cell. Results The MDRl-targeted RNAi resulted in decreased MDR1 gene mRNA level （P 〈0.001）, almost abolished P-gp expression and reversed MDR to different chemotherapy drugs in the RCC A498 cell line. Conclusion MDR could be reversed by RNAi in human RCC A498 cell line, which may be used for clinical application

Characteristics of short double stranded RNA against hepatitis C virus: a literature-based analysis

Objective： To describe the characteristics of short interfering double stranded RNA （dsRNA） against hepatitis C virus （HCV） and to fred out the determining factors in design for desirable inhibitory efficacy. Methods： The data were collected and analyzed by retrieval of 229 published short dsRNAs designed for degradation ofHCV RNA. Results： Statistical analyses showed that the most frequently involved short dsRNAs were directing against 5＇NTR/core and genotype lb, accounting for 64.2% and 69.9%, respectively. Inhibitory efficacy varied with the structural characteristics of short dsRNAs, of which the most potential were those directed against HCV core region with inhibitory efficacy of 70.2%. Moreover, the mean inhibitory efficacy of short dsRNAs with GC contents from 30% to 52% was higher than that of those with GC contents out of this range. Conclusion： Based on this pooled data in a relatively large sample, the present results provided clues to design for short dsRNAs with more potent inhibitory efficacy.

EIF4A3 shRNA慢病毒载体的构建及其稳定转染细胞系的建立

目的:构建真核细胞翻译起始因子4A3(EIF4A3)-短发夹RNA(shRNA)慢病毒载体,建立Neuro-2a-EIF4A3-shRNA稳定转染细胞系。方法:通过美国国家生物技术信息中心(NCBI)数据库检索EIF4A3基因序列,设计并合成PCR鉴定引物,并将其连接至经EcoRⅠ和AgeⅠ酶切的慢病毒GV493载体,构建GV493-EIF4A3-shRNA慢病毒质粒,PCR筛选阳性克隆并测序鉴定。将GV493空载质粒和GV493-EIF4A3-shRNA重组质粒分别转染至HEK293T细胞中,分别为GV493对照慢病毒和GV493-EIF4A3-shRNA慢病毒,转染48 h后收集慢病毒进行包装并测定病毒滴度。将Neuro-2a细胞分为空白组、GV493对照组和GV493-EIF4A3 shRNA组,空白组不作处理,GV493对照组和GV493-EIF4A3 shRNA组分别采用相应慢病毒感染Neuro-2a细胞,慢病毒感染复数(MOI)为100,使用10 mg·L-1嘌呤霉素筛选成功感染慢病毒的Neuro-2a细胞,荧光显微镜观察各组Neuro-2a细胞的生长状态和绿色荧光表达情况;实时荧光定量PCR(RT-qPCR)法和Western blotting法检测各组Neuro-2a细胞中EIF4A3 mRNA及蛋白表达水平。结果:PCR测序结果显示GV493-EIF4A3-shRNA重组质粒基因序列与设计合成的EIF4A3-shRNA序列一致,成功构建GV493-EIF4A3慢病毒载体。荧光显微镜观察可见HEK293T细胞荧光表达强烈,生长状态良好,慢病毒包装成功。GV493-对照慢病毒和GV493-EIF4A3-shRNA慢病毒的滴度均为2×10~8 TU·mL^(-1),GV493对照组和GV493-EIF4A3 shRNA组Neuro-2a细胞生长状态良好且表达绿色荧光,表明慢病毒感染稳定细胞系构建成功。RT-qPCR法,与空白组和GV493对照组比较,GV493-EIF4A3shRNA组Neuro-2a细胞EIF4A3mRNA表达水平明显降低(P<0.01)。Western blotting法,各组在相对分子质量49000处出现特异性条带,提示Neuro-2a细胞中EIF4A3蛋白表达成功;与空白组和GV493对照组比较,GV493-EIF4A3 shRNA组Neuro-2a细胞中EIF4A3蛋白表达水平明显降低(P<0.01)。结论:成功构建GV493-EIF4A3-shRNA慢病毒载体,建立了Neuro-2a-EIF4A3-shRNA稳定转染细胞系,为EIF4A3在颅内动脉粥样硬化的作用机制研究提供了参考。

Survivin shRNA增强人卵巢癌耐药细胞OVCAR3对泰素敏感性的研究

背景与目的:耐药是目前恶性肿瘤治疗急需解决的难题。最近研究显示,卵巢癌组织及细胞中均有Survivin高表达,可能与其抑癌耐药有关。本研究探讨Survivin的短发夹状RNA(shorthairpinRNA,shRNA)对人卵巢癌耐药细胞OVCAR3Survivin基因表达、凋亡及其对泰素、顺铂敏感性的影响。方法:脂质体介导SurvivinshRNA转染OVCAR3。转染空载体或脂质体的细胞及未转染细胞作为对照。逆转录聚合酶链反应(reversetranscription-polymerasechainreaction,RT-PCR)检测SurvivinmRNA的表达,流式细胞仪分析Survivin蛋白的表达及细胞凋亡率。四甲基偶氮唑蓝法(MTT法)测定SurvivinshRNA转染后OVCAR3细胞对泰素的敏感性。结果:与未转染组、空脂质体组、空载体组相比较,SurvivinshRNA处理24h后细胞SurvivinmRNA及蛋白表达水平均明显下调。SurvivinshRNA转染12、24、36、48h后的细胞凋亡率分别为20.7%、31.9%、39.0%、46.7%,呈时间依赖性。MTT结果显示,泰素对未转染组、空脂质体组、空载体组、转染组OVCAR3细胞的IC50分别为(0.305±0.032)μmol/L、(0.157±0.031)μmol/L、(0.175±0.010)μmol/L、(0.019±0.001)μmol/L;顺铂对4组OVCAR3细胞的IC50依次为(9.410±0.796)μmol/L、(6.675±1.739)μmol/L、(6.930±1.273)μmol/L、(7.862±0.081)μmol/L,SurvivinshRNA使OVCAR3对泰素的敏感性提高16倍(P<0.01),但是对顺铂的影响不大(P>0.05)。结论:靶向Survivin的序列特异性shRNA可有效抑制OVCAR3细胞中Survivin基因的表达,同时可以增强OVCAR3对泰素的敏感性,但不增加其对顺铂的敏感性。

短发夹RNA沉默hTERT基因对人喉癌裸鼠移植瘤的生长抑制作用

背景与目的:RNA干扰(RNAinterference,RNAi)是指双链RNA导入细胞后诱导靶mRNA发生特异性的降解,导致基因转录后沉默的现象。RNAi在基因功能和抗病毒基因治疗等方面均有报道。但对喉癌等头颈恶性肿瘤中高表达的人端粒酶逆转录酶(humantelomerasereversetranscriptase,hTERT)基因,目前尚未见报道。本课题利用RNAi技术,研究短发夹RNA(shorthairpinRNA,shRNA)抑制hTERT基因表达对裸鼠喉鳞状细胞癌皮下移植模型的抑瘤作用。方法:根据hTERTcDNA序列构建表达hTERTmRNA特异的、含荧光素基因的shRNA真核表达质粒pshRNA。建立人喉癌Hep-2细胞株裸鼠皮下接种模型。将pshRNA转染入荷瘤裸鼠瘤体内,观察肿瘤生长情况。以激光共聚焦显微镜观察质粒在瘤组织内的表达;以HE染色法观察质粒治疗后瘤组织的病理改变;以原位末端标记法(TUNEL法)检测肿瘤细胞的凋亡情况;以免疫组化SP法检测hTERT蛋白在肿瘤内的表达;光镜下观察心、肝、肾、脾结构的改变并测量血液学和血液生化指标。结果:pshRNA组(A组)、空质粒载体组(B组)与生理盐水组(C组)之间比较,A组与C组瘤体积有显著性差异(P<0.01),B组与C组之间瘤体积无显著性差异(P>0.05),抑瘤率为76.50%。pshRNA及空质粒载体转染入瘤体后,共聚焦显微镜下见大量的癌细胞表达绿色荧光。病理学检查及TUNEL检测发现:A组肿瘤生长受到抑制,细胞分裂相少见,可见大量肿瘤细胞坏死及凋亡,该组肿瘤细胞的凋亡指数[(26.47±4.25)%]明显高于B组[(3.40±1.41)%]和C组[(2.73±1.35)%]。给予pshRNA后瘤体内hTERT蛋白表达明显下调,而且对心、肝、肾、脾无明显损害,对造血系统无影响。结论:表达shRNA的DNA载体质粒沉默hTERT基因可显著抑制人喉癌裸鼠肿瘤的生长,而且对心、肝、肾、脾及造血系统无明显的毒副作用。

bcl-x_L短发夹状RNA诱导人鼻咽癌细胞株CNE-2Z凋亡(英文)

背景与目的:实验证明bcl-xL 在鼻咽癌细胞株CNE-2Z 中存在高表达,它可能在鼻咽癌的发生发展、转移及治疗过程中产生耐药等方面发挥重要作用。本研究拟探讨bcl-xL 短发夹状RNA (shorthairpin RNA ,shRNA )诱导CNE-2Z 细胞凋亡的作用。方法:把表达bcl-xL shRNA 的重组质粒pm U6-RNAi转染到CNE-2Z细胞中,用荧光染色和流式细胞术等方法检测细胞凋亡;用逆转录聚合酶链反应(reverse transcription-polym erase chain reaction,RT-PCR )检测bcl-xL、bcl-2、survivin和caspase-3的m RNA 表达水平;用W estern blot检测Bcl-xL、Caspase-3和P53的蛋白表达水平。结果:流式细胞术检测发现,pm U6-RNAi重组质粒作用24h 后,细胞出现明显的凋亡峰;H oechst33258单染在荧光显微镜下可见细胞缩小、染色质固缩和核断裂;RT-PCR 分析pm U6-RNAi重组质粒作用细胞后明显下调了bcl-xL、bcl-2和caspase-3的m RNA 表达水平, 对survivin 的m RNA 表达水平无影响;W estern blot分析pm U6-RNAi质粒作用细胞后明显下调了Bcl-xL、Caspase-3的蛋白表达水平,而大幅度上调了P53的蛋白表达水平。结论:bcl-xL shRNA 可诱导CNE-2Z 细胞凋亡;CNE-2Z 细胞的凋亡可能与bcl-2、caspase-3和p53有着密切的关系;质粒表达的bcl-xL shRNA

短发夹结构RNA干扰新城疫病毒的增殖

以新城疫病毒(NDV)NP基因为标靶,构建3个细胞内表达发夹样结构小干扰RNA(shRNA)的质粒载体,在鸡胚成纤维细胞(CEF)和鸡胚上进行了RNAi试验,筛选出一个有效抑制病毒复制的小分子ndv1.用阳离子脂质体转染试剂Silent-fect将ndv1转染CEF,以不相关shRNA质粒载体HK为阴性对照,4h后接种NDV,与对照相比,干涉组在病毒感染后3hNP基因的表达量降低2.3倍,6h降低21.1倍,9h降低9.8倍;ndv1能在48h内完全阻断NDV在CEF中的增殖,延缓病变出现时间,减轻病变程度.将Silent-fect-ndv1混合物与NDV同时注入10日龄SPF鸡胚绒毛尿囊腔,能使105ELD50NDV感染后17h鸡胚尿囊液中病毒增殖量减少94.4%,使106ELD50 NDV感染后17h鸡胚尿囊液中病毒增殖量减少62.5%.实验结果证实,在CEF中存在RNAi机制,抑制ND VNP基因的表达能有效阻断该病毒增殖,说明NP基因在NDV复制过程中起重要作用.实验结果为进一步利用RNAi技术在CEF和鸡胚中研究病毒基因组功能及筛选抗病毒小分子奠定了基础.

靶向GPR48的shRNA抑制人宫颈癌HeLa细胞的侵袭转移

背景与目的:GPR48受体偶联Gs蛋白介导细胞内信号转导通路,通过调节微管聚合状态和基质金属蛋白酶活性,影响肿瘤细胞的侵袭转移。本研究探讨靶向GPR48的短发夹RNA(short hairpin RNA,shRNA)对人宫颈癌细胞株HeLa体外侵袭能力及在体转移能力的影响。方法:以人GPR48mRNA编码区中两条不同序列作为RNA干扰靶点,构建靶向GPR48的shRNA真核表达质粒,同时构建不针对任何已知mRNA的阴性shRNA真核表达质粒,分别转染至HeLa细胞,新霉素抗性筛选稳定表达siRNA的转化克隆。采用RT-PCR和Western blot检测GPR48的表达变化。通过Boyden小室实验以穿过人工基底膜的细胞数量评估HeLa细胞体外侵袭能力的变化;分别将转染和未转染GPR48shRNA的HeLa细胞接种于裸鼠皮下,观察肿瘤生长情况及肺部转移情况。结果:RNA干扰使HeLa细胞GPR48表达下调80%;与阴性对照组比较,转染靶向GPR48重组质粒的实验组穿膜细胞数显著减少(28.3±1.5和17.6±1.5vs.94.4±15.7,P<0.01)。裸鼠在体肺转移实验中,与阴性对照组比较,转染靶向GPR48重组质粒的实验组肺转移结节数显著减少(1.3±0.2和1.5±0.4vs.7.8±1.8,P<0.01)。结论:shRNA真核表达载体能明显抑制HeLa细胞的GPR48表达,有效抑制HeLa细胞的体外侵袭和在体转移。

针对Survivin基因的短发卡RNA诱导U251细胞凋亡

目的:观察针对Survivin基因的短发卡RNA(shRNA)对人脑胶质母细胞瘤U251细胞在体外凋亡的影响。方法:对U251细胞,稳定转染Survivin基因shRNA真核表达载体pWH1-SR的U251-SR细胞,以及稳定转染空载体pWH1的U251-P细胞,分别采用相差显微镜、HE染色、Hoechst染色、TUNEL染色以及透射电镜观察各组细胞的形态学特征;采用流式细胞术(FCM)定量测定各组细胞的凋亡细胞数量。结果:相差显微镜、HE染色、Hoechst染色、TUNEL染色以及透射电镜观察显示,U251-SR凋亡细胞明显增多,并呈现典型的细胞凋亡形态学改变;FCM定量分析凋亡细胞数量显示,与U251(2.1%)和U251-P(2.7%)相比,U251-SR凋亡细胞增加约6倍,达14.4%。结论:针对Survivin基因的shRNA能够在体外诱导U251细胞发生大量凋亡。