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.

RNA interference and antiviral therapy

RNA interference (RNAi) is an evolutionally conserved gene silencing mechanism present in a variety of eukaryotic species. RNAi uses short double-stranded RNA (dsRNA) to trigger degradation or translation repression of homologous RNA targets in a sequence-specific manner. This system can be induced effectively in vitro and in vivo by direct application of small interfering RNAs (siRNAs), or by expression of short hairpin RNA (shRNA) with non-viral and viral vectors. To date, RNAi has been extensively used as a novel and effective tool for functional genomic studies, and has displayed great potential in treating human diseases, including human genetic and acquired disorders such as cancer and viral infections. In the present review, we focus on the recent development in the use of RNAi in the prevention and treatment of viral infections. The mechanisms, strategies, hurdles and prospects of employing RNAi in the pharmaceutical industry are also discussed.

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.

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...

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.

PCR扩增的shRNA表达盒快速筛选PRRSV有效siRNA序列

有效siRNA的筛选是RNAi研究的关键点之一。本研究选取猪繁殖与呼吸综合征病毒(PRRSV)的核衣壳蛋白(N)作为靶基因,使用http://www.ambion.com的靶位点筛选和设计工具,选取4个siRNA序列(siRNA95、siRNA179、siRNA218和siRNA294),利用一步PCR法产生包含U6启动子的短发夹RNA表达盒技术快速筛选高效siRNA,PCR法制备的shRNA表达盒(PCR-shRNA95、PCR-shRNA179、PCR-shRNA218和PCR-shRNA294)分别与表达N-EGFP融合蛋白的重组质粒pEN-ORF7共转染至293T细胞,48 h后荧光显微镜下检测细胞表达EGFP阳性率,筛选有效siRNA片段,将筛选的PCR-shRNA179的PCR产物转染N-EGFP融合蛋白稳定表达293T细胞系和PRRSV感染的Marc-145细胞,结果表明PCR-shRNA179可明显减少N蛋白的表达、有效减轻PRRSV引起的细胞病变及减少感染PRRSV的Marc-145细胞中的N蛋白阳性细胞。本研究证明一步PCR扩增shRNA表达盒法可用于筛选特异性基因表达抑制的siRNA。

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.

Applications of RNA interference highthroughput screening technology in cancer biology and virology

RNA interference （RNAi） is an ancient intra-cellular mechanism that regulates gene expression and cell function. Large-scale gene silencing using RNAi highthroughput screening （HTS） has opened an exciting frontier to systematically study gene function in mammalian cells. This approach enables researchers to identify gene function in a given biological context and will provide considerable novel insight. Here, we review RNAi HTS strategies and applications using case studies in cancer biology and virology.

Inhibition of hepatitis B virus surface antigen expression by small hairpin RNA in vitro

AIM: To explore the anti-hepatitis B virus effect of RNA interference (RNAi) using small hairpin RNA (shRNA)expression vector.METHODS: Hepatitis B virus surface antigen green fluorescent protein (HBs-GFP) fusion vector and shRNA expression vectors were constructed and cotransfected transiently into HepG2 cells. mRNAs extracted from HepG2 cells were detected by real-time PCR. Fluorescence of HBs-GFP protein was detected by fluorescence-activated cell sorting (FACS). The effective shRNA expression vector was transfected into HepG2.2.15 cells. HBsAg and HBeAg in HepG2.2.15 cells were analyzed by radioimmunoassay (RIA) method.RESULTS: FACS revealed that shRNA targeting at HBsAg reduced the GFP signal by 56% compared to the control.Real-time PCR showed that HBs-GFP mRNA extracted from HepG2 cells cotransfected with pAVU6+27 and HBs-GFP expression plasmids decreased by 90% compared to the empty vector control. The expressions of HBsAg and HBeAg were also inhibited by 43% and 64%, respectively.CONCLUSION: RNAi using shRNA expression vector can inhibit the expression of HBsAg, providing a fresh approach to screening the efficient small interfering RNAs (siRNAs).

靶向PRRSV 5′ UTR的shRNA体外抑制试验

为了明确靶向PRRSV基因组5’非翻译区（UTR）的shRNA能否抑制PRRSV在Marci45细胞中的复制，以5’UTR87～106位序列为靶点构建能转录小发夹RNA（shRNA）的质粒pSUPER-UTR，同时构建融合表达质粒pU—TR—EGFP，将二者共转染HEK293A细胞，观察荧光；将pSUPER—UTR转染Marc145细胞，感染PRRSV后观察细胞病变，进行间接免疫荧光和实时PCR检测。结果shRNA表达质粒能使UTR—EGFP融合蛋白的表达受到抑制，抑制率约为50％；表达的shRNA在PRRsV感染后使病毒的致细胞病变作用不明显，病毒的mRNA转录水平明显降低，间接免疫荧光结果表明shRNA表达质粒转染孔的荧光细胞数显著减少。表明5’UTR与PPRSv的复制有关，5’UTR的87～106位可能是PPRSV复制的关键性位点，靶向该位点的siRNA可以作为控制PRRSV传播的候选分子。

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.

猪繁殖与呼吸综合征病毒ORF2～4特异siRNA的筛选及其抑制病毒复制效果的研究

RNA干扰（RNAi）技术是基因功能研究的有效工具,为了了解猪繁殖与呼吸综合征病毒（PRRSV）次要结构蛋白GP2、GP3、GP4在病毒复制中的作用,针对各自的编码基因ORF2、ORF3、ORF4分别选取4个小干扰RNA（siRNA）位点（共12个）,构建相应的短发夹RNA（shRNA）表达载体,转染MARC-145细胞后,通过荧光定量PCR和病毒滴度检测干扰效果。筛选了可以减少GP2、GP3、GP4相应基因mRNA含量的ORF2、ORF3、ORF4特异shRNA表达载体,病毒效价滴定表明shRNA表达载体处理细胞可以减少GP2、GP3、GP4相应基因mRNA含量,细胞培养上清中的病毒滴度比对照低184~4.65倍。

Effect of vector-expressed shRNAs on hTERT expression

AIM: To study the effect of short hairpin RNAs (shRNAs)expressed from DNA vector on hTERT expression.METHODS: Oligonucleotides coding for four shRNAs against hTERT were cloned into a mammalian shRNA expressionvector pUC18U6 to form pUC18U6ht1-4, which were thenintroduced into HepG2 cells by using liposome-mediated transfection. HepG2 cells transfected by pUC18U6 and pUC18U6GFPsir, which expressed shRNA against green fluorescent protein (GFP), were used as controls. hTERT mRNA in the transfected cells were quantified by using real-time fluorescent RT-PCR.RESULTS: Among the four shRNAs against hTERT, two decreased the hTERT mRNA level. Compared with the controls, pUC18U6ht which expressed the two shRNAs reduced hTERT mRNA by 39% and 49% (P＜0.05).CONCLUSION: hTERT expression is inhibited by the shRNAs expressed from the DNA vector.

Investigation of transcriptional gene silencing and mechanism induced by shRNAs targeted to RUNX3 in vitro

AIM:To investigate transcriptional gene silencing induced by short hairpin RNAs (shRNAs) that target gene prompter regions of RUNX3 gene, and whether shRNAs homologous to DNA sequences may serve as initiators for methylation. METHODS:According to the principle of RNAi design, pSilencer3.1-H1-shRNA/RUNX3 expression vector was constructed, The recombinant plasmid shRNA was transfected into human stomach carcinoma cell line SGC7901 with Lipofectamine 2000. Then, the positive cell clones were screened by G418. The mRNA and protein expression level of RUNX3 in the stable transfected cell line SGC7901 were determined by RT-PCR, Western blotting and immunocytochemistry. Characteristics of the cell lines including SGC7901, pSilencer3.1-H1/SGC7901 and pSilencer3.1-H1-shRNA/RUNX3/SGC7901 were analyzed with growth curves, clone formation rate and cell-cycle distribution. The activated level of RUNX3 was examined after treatment with the different density of 5’-aza-2’-deoxycytidine (5-Aza-CdR) by using semi- quantitative RT-PCR and Western blotting. RESULTS:In the cell line SGC7901 transfected with pSilencer3.1-H1-shRNA/RUNX3, mRNA and proteinexpression of the RUNX3 gene was lost identified by RT- PCR, Western blotting and immunocytochemistry assay. The growth of pSilencer3.1-H1-shRNA/ RUNX3/SGC7901 cells without expression of RUNX3 was the fastest (P < 0.05), its rate of clone formation was the highest (P < 0.01), and the cell distribution in G0/G1 and S/M phases was lowest and highest, respectively (P < 0.05), compared with that of the transfected pSilencer3.1-H1 and non-transfected cells. Through RT-PCR and Western blot assay, inactivated RUNX3 could not be reactivated by 5-Aza-CdR. CONCLUSION:We found that, although shRNAs targeted to gene prompter regions of RUNX3 could effectively induce transcriptional repression with chromatic changes characteristic of inaction promoters, this was independent of DNA methylation, and the presence of RNA-dependent transcriptional silencing showed that RNA-directed DNA methylation might be an existing gene regulatory mechanism relative to the methylated in humans.

Recombinant adenovirus-mediated shRNA silencing of midkine gene in BxPC-3 cells

Objective：To investigate the silencing effects of recombinant adenovirus Ad-shRNA-MK on midkine（MK） gene in pancreatic cancer cells. Methods：Ad-shRNA-MK was used to infect pancreatic cancer BxPC-3 cells. Assays were conducted for knockdown of the MK gene on the day of infection and on the 1 ^st, 3^rd, 5^th, 7^th, and 9^th days post-infection by using immunocytochemistry, real-time RT-PCR, and Western blot analysis. Results：The adenoviral Ad-shRNA-PTN was constructed successfully, and infection was confirmed by electron microscopic observation. By using real-time RT-PCR, the inhibition rates of MK mRNA expression in the BxPC-3 cells were 20%, 80%, 55%, and 23% on the 1st, 3^th, 5^th, and 7^th days post-infection. Immunocytochemistry and Western blot analysis confirmed this effect at the gene product level. Conclusion：Efficient and specific knockdown of MK in pancreatic cancer cells by adenoviral Ad-shRNA-PTN is a potentially powerful tool for the study of gene therapy of pancreatic cancer nerve infiltration.

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。

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。

MDR1短发夹RNA表达质粒的构建及其功能的初步研究

目的 构建在哺乳动物细胞中表达MDR1短发夹RNA(shorthairpinRNA ,shRNA)的表达质粒 ,并初步探讨其对耐药肝癌细胞MDR1mRNA的抑制作用及抗肿瘤药物耐药性的影响。方法 根据Genbank中MDR1mRNA设计的两条多聚核苷酸序列 ,退火形成双链DNA ,再与经双酶切后的载体PGE 1连接 ,构建 pshRNA MDR1重组质粒 ,在脂质体的介导下转染肝癌细胞株BEL 74 0 2 /ADM ,RT PCR分析MDR1mRNA的表达 ,MTT法检测阿霉素对细胞的半数抑制浓度 (IC50 )。结果 PCR和DNA测序证实了表达质粒构建成功 ,并能明显地抑制BEL 74 0 2 /ADMMDR1mRNA的表达 ,对阿霉素的耐药指数降低了17.5倍 ( 2 99.2 / 17.1)。结论 构建的 pshRNA MDR1表达质粒能有效地抑制转染细胞MDR1mRNA ,从而提高肿瘤细胞的药物敏感性。

shRNA沉默VEGF基因表达对大肠癌细胞生物学特性的影响

目的观察Pavu6+27VEGFsiRNA重组体在细胞体内表达的VEGF短发夹状RNA(shorthairpinRNA,shRNA)对人大肠癌HCT116细胞株生物学特性的影响。方法将自行构建的表达短发夹状RNA的重组质粒转染到大肠癌HCT116细胞株中,以空质粒Pavu6+27转染为对照,经G418筛选出稳定表达shRNA细胞,行RTPCR检测VEGFmRNA表达的改变,Westernblot检测VEGF蛋白表达,流式细胞技术分析细胞周期分布,MTT比色法检测细胞的生长抑制率。结果成功转染重组体的HCT116细胞中VEGFmRNA表达明显下降,约降低为对照组的61.2%(P<0.05)。VEGF蛋白表达明显下降,光密度分析比较差异有显著性(P<0.05)。G0/G1期细胞比例增高,另一方面S期和G2/M期细胞比例降低,细胞的增殖指数明显降低,实验组细胞增殖指数为(25.63±4.54)%,对照组细胞的增殖指数为(31.90±2.19)%(P<0.05)。结论Pavu6+27VEGFsiRNA重组体在细胞内表达的短发夹状RNA能有效抑制人大肠癌细胞VEGFmRNA和VEGF蛋白表达,细胞增殖能力减弱,为质粒介导的RNAi技术运用于大肠癌的基因治疗提供一定的实验依据。