Ribozyme Gene Research Articles

Hairpin Ribozyme Genes Curtail Alcohol Drinking: from Rational Design to in vivo Effects in the Rat.

Ribozyme genes were designed to reduce voluntary alcohol drinking in a rat model of alcohol dependence. Acetaldehyde generated from alcohol in the liver is metabolized by the mitochondrial aldehyde dehydrogenase (ALDH2) such that diminishing ALDH2 activity leads to the aversive effects of blood acetaldehyde upon alcohol intake. A stepwise approach was followed to design genes encoding ribozymes targeted to the rat ALDH2 mRNA. In vitro studies of accessibility to oligonucleotides identified suitable target sites in the mRNA, one of which fulfilled hammerhead and hairpin ribozyme requirements (CGGUC). Ribozyme genes delivered in plasmid constructs were tested in rat cells in culture. While the hairpin ribozyme reduced ALDH2 activity 56% by cleavage and blockade (P < 0.0001), the hammerhead ribozyme elicited minor effects by blockade. The hairpin ribozyme was tested in vivo by adenoviral gene delivery to UChB alcohol drinker rats. Ethanol intake was curtailed 47% for 34 days (P < 0.0001), while blood acetaldehyde more than doubled upon ethanol administration and ALDH2 activity dropped 25% in liver homogenates, not affecting other ALDH isoforms. Thus, hairpin ribozymes targeted to 16 nt in the ALDH2 mRNA provide durable and specific effects in vivo, representing an improvement on previous work and encouraging development of gene therapy for alcoholism.

GW26-e2354 R65 Ribozyme Gene Attenuates ox-LDL Induced Apoptosis in HASMCs

GW26-e2354 R65 Ribozyme Gene Attenuates ox-LDL Induced Apoptosis in HASMCs

GW25-e2529 Comparative study of vitro transfection of different human vascular cells by Type 9 recombinant adeno-associated virus mediated R65 ribozyme gene

To evaluate the transfection efficiency using recombinant adeno-associatedvirus serotype 9 (rAAV9) mediated anti-nuclear factor-κB (NF-κB) ribozyme and enhanced green fluorescent protein (rAAV9-EGFP-R65) in Human Umbilical Vein Endothelial Cells (HUVECs) and in Human Aortic Smooth Muscle Cells (

GW25-e2527 Inhibition of the NF-κB pathway by the R65 ribozyme gene via adeno-associated virus serotype 9 in human umbilical vein endothelial cells

GW25-e2527 Inhibition of the NF-κB pathway by the R65 ribozyme gene via adeno-associated virus serotype 9 in human umbilical vein endothelial cells

CONSTRUCTION OF RECOMBINANT ADENO-ASSOCIATED VIRUS SEROTYPE 9 WITH RIBOZYME GENE TARGETING NF-κB AND ITS SUPPRESSION OF NF-κB ACTIVITY IN HELA CELLS

ObjectivesTo construct the recombinant adeno-associated virus serotype 9 containing ribozyme gene (R65) targeting nuclear factor-κ B (NF-κB), and investigate the inhibitory effect of rAAV9-eGFP-R65 on activation of NF-κB as well...

High-throughput screening method for promoter activity using bead display and a ligase ribozyme

In this report, we describe the development of a novel in vitro high-throughput system for detecting and screening promoter activity; the method employs emulsified reactions and a ligase ribozyme. In our study, a promoter DNA fragment containing the ribozyme gene was immobilized on a bead by using emulsion PCR, followed by in vitro transcription of the immobilized DNA in water-in-oil emulsions. Owing to the self-ligation activity of the ribozyme, it was co-transcriptionally linked to the active promoter immobilized on the beads. The bead complex containing the active promoter sequence was then labeled by reverse transcription with a fluorescently labeled primer. Employing flow cytometry, the fluorescence intensity corresponding to the strength of each promoter was observed, indicating the applicability of the system for promoter evaluation. Moreover, two rounds of screening with T7 RNA polymerase using a cell sorter enriched the T7 promoter fragment by 70 folds from a 1:100 mixture of T7 promoter and SP6 promoter fragments, suggesting that this system can be used to screen promoters.

Abstract 5131: Identification of the genes that chemosensitize hepatocellular carcinoma cells to interferon-α and 5-fluorouracil therapy

Abstract The increasing numbers of patients with hepatocellular carcinoma (HCC), which is characterized by an extremely poor prognosis, are causing serious problems worldwide. To improve their prognosis, the development of a more effective therapy is required. Combination therapy with interferon-α (IFN-α) and 5-fluorouracil (5-FU) was reported to provide benefits to patients with advanced HCC, although its efficacy is still unsatisfactory. In this study, taking advantage of functional screening, three chemosensitizing genes for this therapy against HCC cells were identified. Identification of chemosensitizing genes was performed using ribozyme-based functional screening. We constructed a plasmid DNA library expressing random ribozyme genes with ∼6.0 × 106 target recognition sequences. We performed a positive-selection strategy based on the ability of 5-FU to eliminate cells expressing a ribozyme that targets chemosensitivity-unrelated genes. The ribozyme library was transfected into HepG2 cells, which were subsequently treated with the IC50 dose of 5-FU. This cycle was repeated 10 times to fully concentrate the ribozymes targeting those genes involved in the sensitivity of 5-FU. Consequently, 100 target ribozyme recognition sequences that were recovered before screening and after 10 cycles of screening were subjected to a BLAST search to retrieve genes with homologous sequences. This analysis indicated 3 genes (PRKAG2, TGFBR2, and EXT1) as candidate genes. The chemosensitizing effects of these genes on IFN-α/5-FU as well as 5-FU treatment were confirmed by adenovirus-mediated gene transfer. In addition, the mechanisms of effects of these genes on enhancing effects on combination therapy with IFN-α/5-FU were also evaluated. Furthermore, to evaluate the relationship between their expression levels in tumor tissues and the clinical outcome of IFN-α/5-FU combination therapy in advanced HCC patients, mRNA expression was measured using real-time reverse transcription-polymerase chain reaction. As a result, PRKAG2, TGFBR2, and EXT1 were identified as novel chemosensitizing genes for the combination therapy against HCC. TGFBR2 and EXT1 induced transforming growth factor-β signal activation and endoplasmic reticulum stress, respectively. Moreover, PRKAG2 and TGFBR2 were suggested to be promising predictive markers for the clinical outcome of this therapy. In conclusion, the identified genes are promising candidates as targets to enhance the therapeutic effect of IFN-α/5-FU combination therapy in advanced HCC and could be used as predictive markers for response to this therapy. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5131. doi:1538-7445.AM2012-5131

E0229 Inhibition of NF-κB attenuates post-infarct left ventricular rupture and remodelling in aged mice by ribozyme gene transfer with adeno-associated virus serotypes 9

ObjectiveUsing intravenous injection of adeno-associated virus serotypes 9 carring ribozyme gene (AAV9-R65), we examined whether inhibition NF-κB would prevent post-infarct left ventricular rupture and remodelling in aged mice.Methods and resultsOld...

Enhanced sensitivity to DNA damage induced by cooking oil fumes in human OGG1 deficient cells

Cooking oil fumes (COFs) have been implicated as an important nonsmoking risk factor of lung cancer in Chinese women. However, the molecular mechanism of COFs-induced carcinogenicity remains unknown. To understand the molecular basis underlying COFs-induced cytotoxicity and genotoxicity as well as the roles of hOGG1 in the repair of COFs-induced DNA damage, a human lung cancer cell line with hOGG1 deficiency, A549-R was established by using a ribozyme gene targeting technique that specifically knockdowned hOGG1 in A549 lung adenocarcinoma cells. MTT and comet assays were employed to examine cell viability and DNA damage/repair, respectively, in A549-R and A549 cell lines treated with COF condensate (COFC). RT-PCR and Western blot results showed that the expression of hOGG1 in A549-R cell line was significantly decreased compared with that in A549 cell line. The concentration of COFC that inhibited cell growth by 50% (the IC50) in the A549-R cell line was much lower than that in the A549 cell line, and more COFC-induced DNA damage was detected in the A549-R cell line. The time course study of DNA repair demonstrated delayed repair kinetics in the A549-R cell line, suggesting a decreased cellular damage repair capacity. Our results showed that hOGG1 deficiency enhanced cellular sensitivity to DNA damage caused by COFC. The results further indicate that hOGG1 plays an important role in repairing COF-induced DNA damage. Our study suggests that COFs may lead to DNA damage that is subjected to hOGG1-mediated repair pathways, and oxidative DNA damage may be involved in COF-induced carcinogenesis.

Suppression of a DNA base excision repair gene, hOGG1, increases bleomycin sensitivity of human lung cancer cell line

Bleomycin (BLM) has been found to induce 8-oxoguanine and DNA strand breaks through producing oxidative free radicals, thereby leading to cell cycle arrest, apoptosis and cell death. Cellular DNA damage repair mechanisms such as single strand DNA break repair/base excision repair (BER) are responsible for removing bleomycin-induced DNA damage, therefore confer chemotherapeutic resistance to bleomycin. In this study, we have investigated if down-regulation of human 8-oxoguanine DNA glycosylase (hOGG1), an important BER enzyme, could alter cellular sensitivity to bleomycin, thereby reducing chemotherapeutic resistance in human tumor cell. A human lung cancer cell line with hOGG1 deficiency (A549-R) was created by ribozyme gene knockdown technique. Bleomycin cellular sensitivity and DNA/chromosomal damages were examined using MTT, colony forming assay, comet assay as well as micronucleus assay. We demonstrated that hOGG1 gene knockdown enhanced bleomycin cytotoxicity and reduced the ability of colony formation of the lung cancer cell lines. We further demonstrated that bleomycin-induced DNA strand breaks resulted in an increase of micronucleus rate. hOGG1 deficiency significantly reduced DNA damage repair capacity of the lung cancer cell lines. Our results indicated that hOGG1 deficiency allowed the accumulation of bleomycin-induced DNA damage and chromosomal breaks by compromising DNA damage repair capacity, thereby increasing cellular sensitivity to bleomycin.

SOD2 Knockdown Mouse Model of Early AMD

To test the hypothesis that oxidative injury to the retinal pigment epithelium (RPE) may lead to retinal damage similar to that associated with the early stages of age-related macular degeneration (AMD). A ribozyme that targets the protective enzyme manganese superoxide dismutase (MnSOD) was expressed in RPE-J cells, and adeno-associated virus (AAV) expressing the ribozyme gene was injected beneath the retinas of adult C57BL/6 mice. The RPE/choroid complex was examined for SOD2 protein levels and protein markers of oxidative damage using immunoblot analysis and LC MS/MS-identification of proteins and nitration sites. Lipids were extracted from retinal tissue and analyzed for the bis-retinoid compounds A2E and iso-A2E. The mice were analyzed by full-field electroretinography (ERG) for light response. Light and electron microscopy were used to measure cytological changes in the retinas. The treatment of RPE-J cells with Rz432 resulted in decreased MnSOD mRNA and protein as well as increased levels of superoxide anion and apoptotic cell death. When delivered by AAV, Rz432 reduced MnSOD protein and increased markers of oxidative damage, including nitrated and carboxyethylpyrrole-modified proteins in the RPE-choroid of mice. Ribozyme delivery caused a progressive loss of electroretinograph response, vacuolization, degeneration of the RPE, thickening of Bruch's membrane, and shortening and disorganization of the photoreceptor outer and inner segments. Progressive thinning of the photoreceptor outer nuclear layer resulted from apoptotic cell death. Similar to the eyes of patients with AMD, ribozyme-treated eyes exhibited increased autofluorescence and elevated levels of A2E and iso-A2E, major bis-retinoid pigments of lipofuscin. These results support the hypothesis that oxidative damage to the RPE may play a role in some of the key features of AMD.

688. Aiming at Alcoholism with Ribozyme Genes: Silencing the mRNA for an Alcohol Detoxifying Enzyme

688. Aiming at Alcoholism with Ribozyme Genes: Silencing the mRNA for an Alcohol Detoxifying Enzyme

Ultrasensitive monitoring of ribozyme cleavage product using molecular-beacon-ligation system

This paper reports a new approach to detect ribozyme cleavage product based on the molecular-beacon-ligation system. The molecular beacon, designed in such a way that one-half of its loop is complementary to ribozyme cleavage product, is used to monitor ligation process of RNA/DNA complex in a homogeneous solution and to convert directly cleavage product information into fluorescence signal. The method need not label ribozyme and ribozyme substrate, which is fast, simple and ultrasensitive for detection of cleavage product. Detection limit of the assay is 0.05 nmol/L. The cleavage product of hammerhead ribozyme against hepatitis C virus RNA (HCV-RNA) was detected perfectly based on this assay. Owing to its ultrasensitivity, excellent specificity, convenience and fidelity, this method might hold out great promise in ribozyme reaction and ribozyme gene therapy.

Antitumor and antiangiogenic activities of anti-vascular endothelial growth factor hairpin ribozyme in human hepatocellular carcinoma cell cultures and xenografts

To study the effectiveness and mechanisms of anti- human vascular endothelial growth factor (hVEGF) hairpin ribozyme on angiogenesis, oncogenicity and tumor growth in a hepatocarcinoma cell line and a xenografted model. The artificial anti-hVEGF hairpin ribozyme was transfected into hepatocarcinoma cell line SMMC-7,721 and, subsequently, polymerase chain reaction (PCR) and reverse transcription polymerase chain reaction (RT-PCR) were performed to confirm the ribozyme gene integration and transcription. To determine the effects of ribozyme ,VEGF expression was detected by semiquantitative RT-PCR and enzyme liked immunosorbent assay (ELISA). MTT assay was carried out to measure the cell proliferation. Furthermore,the transfected and control cells were inoculated into nude mice respectively, the growth of cells in nude mice and angiogenesis were observed. VEGF expression was down-regulated sharply by ribozyme in transfected SMMC-7,721 cells and xenografted tumor. Compared to the control group, the transfected cells grew slower in cell cultures and xenografts, and the xenograft formation was delayed as well. In addition, the microvessel density of the xenografted tumor was obviously declined in the transfected group. As demonstrated by microscopy,reduction of VEGF production induced by ribozyme resulted in a significantly higher cell differentiation and less proliferation vigor in xenografted tumor. Anti-hVEGF hairpin ribozyme can effectively inhibit VEGF expression and growth of hepatocarcinoma in vitro and in vivo. VEGF is functionally related to cell proliferation, differentiation and tumori-genesis in hepatocarcinoma.

Construction of retroviral vector of p125FAKspecific ribozyme genes and its effects on BGC-823 cells

To construct the retroviral vector of p(125FAK) specific ribozyme genes and to explore the feasibility of ribozyme in BGC-823 gene therapy in vitro. A hammerhead ribozyme DNA targeting p(125FAK) mRNA from nt 1010 to nt 1032 was synthesized and recombined into the retroviral vector pLXSN forming pLRZXSN recon. Using the lipofectin-mediated DNA transfection technique, pLRZXSN was introduced into BGC-823 cells. The effects of ribozyme on the growth of BGC-823 cells and apoptosis were studied by cell colony assay, flow cytometry (FCM), reverse transcriptase-polymerase chain reaction (RT-PCR), detection of DNA fragmentation and electron microscopy. The number of BGC-823 cell colonies was inhibited by 56% after the cells were treated for 48 h. The cell proliferation was inhibited effectively by p(125FAK) ribozyme and the inhibitory effect depended on the concentration and the time of incubation. The expression of p(125FAK) mRNA and protein P(125FAK) decreased sharply in BGC-823 cells treated with p(125FAK) ribozyme. The characteristics of apoptosis, namely sub-G1 peak, DNA fragmentation and morphological changes, were revealed in BGC-823 cells treated with p(125FAK) ribozyme. p(125FAK) ribozyme decreases p(125FAK) gene expression and induces apoptosis of human gastric cancer cells in vitro.

Effect of proto-oncogene protein c-met ribozyme transfection on biological behavior of gastric carcinoma cells

To explore the effect of c-met ribozyme transfection on biological behavior of gastric carcinoma cells. U1/met292 plasmid containing c-met ribozyme gene was transfected into L2 subline of SGC-7901 gastric cell line, and the proliferative ability, distribution of cell cycle, protein expression of VEGF and c-met, as well as the potential of liver metastasis of the transfected subline were determined. There were no significant difference in proliferative ability, distribution of cell cycle between the transfected cells and the control cells. The protein expression of VEGF and c-met, as well as the liver metastatic potential significantly decreased in the transfected cells than those in the control cells (P< 0.05). The liver metastatic potential of c-met positive gastric cancer cells may be prevented by inhibiting c-met expression.

Preparation of anti-mouse caspase-12 mRNA hammerhead ribozyme and identification of its activityin vitro

To prepare and identify specific anti-mouse caspase-12 hammerhead ribozymes in vitro, in order to select a more effective ribozyme against mouse caspase-12 as a potential tool to rescue cells from endoplasmic reticulum stress induced apoptosis. Two hammerhead ribozymes directed separately against 138 and 218 site of nucleotide of mouse caspase-12 mRNA were designed by computer software, and their DNA sequences were synthesized. The synthesized ribozymes were cloned into an eukaryotic expression vector-neo(r)pBSKU6 and embedded in U6 SnRNA context for further study. Mouse caspase-12 gene segment was cloned into PGEM-T vector under the control of T7 RNA polymerase promoter (containing gene sequence from positions nt 41 to nt 894) as target. In vitro transcription both the ribozymes and target utilize T7 promoter. The target was labeled with (alpha- (32)P)UTP, while ribozymes were not labeled. After gel purification the RNAs were dissolved in RNase free water. Ribozyme and target were incubated for 90 min at 37 degrees in reaction buffer (40 mmol/L Tris-HCL, pH 7.5, 10 mmol/L Mg(2+)). Molar ratio of ribozyme vs target was 30:1. Samples were analyzed on 6% PAGE(containing 8 mol/L urea). Both caspase-12 and ribozyme gene sequences were successfully cloned into expression vector confirmed by sequencing. Ribozymes and caspase-12 mRNA were obtained by in vitro transcription. Cleavage experiment showed that in a physiological similar condition (37 degrees, pH 7.5), Rz138 and Rz218 both cleaved targets at predicted sites, for Rz138 the cleavage efficiency was about 100%, for Rz218 the value was 36.66%. Rz138 prepared in vitro can site specific cleave mouse caspase-12 mRNA with an excellent efficiency. It shows a potential to suppress the expression of caspase-12 in vivo, thus provided a new way to protect cells from ER stress induced apoptosis.

Inhibition of Fas-mediated apoptosis in Yac-1 cell via Anti-Fas ribozyme.

To detect a new and more effective way against apoptosis mouse lymphomatic cell line Yac-1 in which fas gene was expressed highly was used as a model for studying the effects of anti-Fas ribozyme on Fas-mediated apoptosis. A hammerhead ribozyme gene targeting the fas mRNA was synthesized and its in vitro transcription vector was constructed, which was transfected into Yac-1 cells using electroporation. Rz596 expression was detected using RT-PCR, and Fas expression in Yac-1 cells was detected using RT-PCR, Western blot and flow cytometry. After treated with anti-Fas antibody (JO2), Yac-1 cell viability was measured with MTT assay, caspase-3 proteolytic activity was detected, and cell apoptosis was measured according to annexin V apoptosis detecting kit. Anti-Fas ribozyme could cleave fas mRNA efficiently in vivo and in vitro. Fas expression in Yac-1 cells transfected with anti-Fas ribozyme was decreased remarkably and correlated with resistance to Fas-mediated apoptosis as determined by flow cytometry and caspase-3 proteolytic activity. Anti-Fas ribozyme was detected in cells transfected with pU6-RZ596 and pU6-dRZ596 and could remarkably decrease the Fas expression in Yac-1 cells, which made Yac-1 cells get rid of Fas-mediated apoptosis. Because of wide expression of fas in organs and tissues, our research was very useful for studying the inhibition of apoptosis of many organs and tissues in the future.

Construction of specific ribozymes against second exon of α1 I and α1 III procollagen genes

目的 构建出针对α1Ⅰ型及α1Ⅲ型前胶原基因第2外显子(Exon 2)片段的核酶.方法根据α1Ⅰ型及α1Ⅲ型前胶原基因的碱基序列以及锤头型核酶(ribozyme)的结构域要求,利用聚合酶链反应(PCR)分别构建了针对各自的Exon 2 区域的核酶基因,并克隆到T载体(pGEM-T vector)T7启动子下游.结果分别以引物对PⅠL/PⅠR及PⅢL/PⅢR进行PCR扩增,经琼脂糖凝胶电泳各得到约62bp的DNA片段,与预期的核酶基因大小一致.结论经PCR限制性内切酶酶切及序列测定分析,证实为所设计的插入正确的核酶基因,从而为大量制备核酶及其体外切割实验和胞内与mRNA相互作用的研究打下了基础。

Effect of specific nucleotide G 11 on cleavage activity of hairpin ribozymes in vitro *

Abstract To study the effect of specific G11 nucleotide on cleavage activity of hairpin ribozyme in vitro, the 32P-labeled pKC transcript (267 nt) containing hepatitis B virus core region is used as target RNA. Synthetic hairpin ribozyme genes with G 11 mutational site are cloned into ribozyme vector pl.5 to create pHpRz for preparation of ribozyme. Non-labeled HpRz transcripts are incubated with 32P-labeled target-RNAs under different conditions and autoradiographed after gel electrophoresis. The results show that the cleavage activity of hairpin ribozyme is closely related to the matching of the bases between hairpin ribozyme and the substrate. The G11 site is not essential to cleavage activity of hairpin ribozyme in vitro, so the selection of target sequence matching G11 site of hairpin ribozyme is not limited to this site.