Advances and Applications in Gene Silencing / 昆明医科大学学报

Gene silencing is a phenomenon in which the specific genes in organisms are not expressed or reduced for various reasons. It is an important mechanism to control gene expression by epigenetic control. At present, scientists intend to explore the treatment of difficult diseases through gene silencing technology,and the research has become a hot spot. With the rapid development of gene engineering, gene silencing technology has been replaced by new technologies. This paper aims to introduce the ribozyme technology, antisense oligonucleic acid technology, gene knockout,RNA interference technology and their applications in gene silencing technology.

Engineering of Thiamin Pyrophosphate Fluorescent Biosensors Based on Ribozyme Switches in Mammalian Cells / 分析化学

Thiamin pyrophosphate (TPP) is a thiamine (vitamin B1) derivative and an essential cofactor in oxidative metabolism of the sugars,fatty acids and amino acids in living cells.By now,numerous TPP-dependent artificial riboswitch systems have been developed to regulate target gene expression but limited in bacteria,fungi or plant cells.Herein,the activating (switch-on) and inhibiting (switch-off) TPP-depended hammerhead ribozyme switches,which are from previous reported structures of prokaryotes screening,were investigated in mammalian cells.These ribozyme switches were inserted into the 3'UTR of the enhanced green fluorescence protein (EGFP) gene to construct the efficient ribozyme-based artificial switches through overlap extension PCR cloning.The HEK293 cells were transfected with the engineered ribozyme switches at increasing concentration of TPP.The EGFP gene-regulatory ability was analyzed with fluorescent microscope and flow cytometry.These TPP-inducible gene regulation devices showed the obvious ligand dose-dependency and excellent specificity.Two switch-on and one switch-off constructs demonstrated 3.1-fold or 1.9-fold increment and 2.3-fold reduction of EGFP level respectively with 150 μ mol/L TPP.The ligand-responsive ribozyme switches,by tuning the change of TPP concentration into the visual reporter genetic expression in cells,enable an efficient development of label-free,noninvasive and high-specific biosensors in living mammalian cells.

Interactions of aminoglycosides with RNAs and proteins via carbohydrate microarray / 生物工程学报

Aminoglycosides are broad-spectrum antibacterials to treat bacterial infections, especially gram-negative bacteria infections. However, aminoglycosides are losing efficacy because of the increase in antibiotic resistance and their inherent toxicity, attracting more interests in developing new aminoglycosides. Several clinically used aminoglycosides are mainly exerted by inhibition of protein synthesis through binding to bacterial rRNA. The bacterial ribosome RNA is the most currently exploited RNA drug target. Identification of new compounds that target RNAs is indispensable to fight with the growing threat that bacteria pose to human safety. In this work, we used carbohydrate microarrays to probe interactions of low molecular weight ligands with RNAs and proteins. Carbohydrate microarrays, comprising hundreds to thousands of different glycan structures on surfaces in a spatially discrete pattern, are sensitive and versatile tools to study the interactions between biological macromolecules. Herein, aminoglycosides have been immobilized onto the modified glass microscope slides and their interactions with RNAs and proteins are then measured through the labeled fluorescence. The results displayed that microarray can be used to detect the binding of aminoglycosides with three types of target molecules, including the small RNA oligonucleotide mimics of aminoglycoside binding sites in the ribosome (rRNA A-site mimics), the large group I ribozyme RNA (approximately 400 nucleotide) and certain proteins (toxicity-causing enzymes, such as DNA polymerase and phospholipase C). For rRNA A-site mimics, the fluorescence intensities of 16S rRNA is stronger than that of 18S rRNA, illustrating that as a screen technique, the microarray method can not only determine the binding affinity to RNA but also detect the specific binding to bacterial rRNA mimic. The ability to screen group I ribozyme RNA can be helpful to the discovery of new RNA therapeutic targets. Binding of immobilized aminoglycosides to toxicity-causing proteins (DNA polymerase and phospholipase C) is a new method to study of aminoglycoside toxicity. These studies lay the foundation for rapid identification of new RNA-binding ligands with strong and specific binding affinity for their desired targets.

Research Progresses in Single Molecule Enzymology / 分析化学

The single molecule imaging and technologies that developed in 1990 s have successfully probed the dynamics of single molecule enzyme catalysis in real time in vitro. Ever since then, single molecule enzymology has entered the golden age of rapid developing. Individual features of each enzyme hidden in the overall average have been discovered, and many new catalytic mechanisms have been proposed. Single molecule enzymology sheds light on the dynamic interactions between enzymes and substrates or products, deepening the understanding of biochemical reactions. This review described the recent research progresses of single molecule protease and ribozyme.

Recombinant adeno-associated virus serotype 9 with p65 ribozyme protects H9c2 cells from oxidative stress through inhibiting NF-κB signaling pathway / 老年心脏病学杂志（英文版）

Background Oxidative stress is a major mechanism underlying the pathogenesis of cardiovascular disease. It can trigger inflammatory cascades which are primarily mediated via nuclear factor-κB (NF-κB). The NF-κB transcription factor family includes several subunits (p50, p52, p65, c-Rel, and Rel B) that respond to myocardial ischemia. It has been proved that persistent myocyte NF-κB p65 activation in heart failure exacerbates cardiac remodeling. Mechods A recombinant adeno-associated virus serotype 9 carrying enhanced green fluorescent protein and anti-NF-κB p65 ribozyme (AAV9-R65-CMV-eGFP) was constructed. The cells were assessed by MTT assay, Annexin V–propidium iodide dual staining to study apoptosis. The expression of P65 and P50 were assessed by Western blot to investigate the under-lying molecular mechanisms. Results After stimulation with H2O2 for 6 h, H9c2 cells viability decreased significantly, a large fraction of cells underwent apoptosis. We observed a rescue of H9c2 cells from H2O2-induced apoptosis in pretreatment with AAV9-R65-CMV-eGFP. Moreover, AAV9-R65-CMV-eGFP decreased H2O2-induced P65 expression. Conclusions AAV9-R65-CMV-eGFP protects H9c2 cells from oxidative stress induced apoptosis through down-regulation of P65 expression. These observations indicate that AAV9-R65-CMV-eGFP has the potential to exert cardioprotective effects against oxidative stress, which might be of great importance to clinical efficacy for cardiovascular disease.

Comparative study of in vitro transfection of R65 ribozyme gene to different human vascular cells mediated by recombinant adeno-associated virus 9 / 中华微生物学和免疫学杂志

Objective To evaluate in vitro transfection of anti-nuclear factor-κB ( NF-κB) ribozyme gene to human umbilical vein endothelial cells (HUVECs) and human aortic smooth muscle cells (HASMCs) mediated by recombinant adeno-associated virus 9 carrying enhanced green fluorescent protein gene ( rAAV9-EGFP-R65 ) and to study their effects on cell proliferation and NF-κB P65 expression.Methods HUVECs and HASMCs were respectively transfected with rAAV9-EGFP-R65 at different multiplicity of infection ( MOI=1 ×105 , 1 ×106 and 1×107).The expression of EGFP was observed with fluorescence microscopy .Flow cytometry was performed to evaluate the transfection efficiency .Alamar Blue assay was used to measure the proliferation of the transfected cells.Western blot was used to detect NF-κB P65 expression .Results The fluorescence intensity was enhanced along with an increased MOI and an extended time of transfection .HUVECs and HASMCs transfected with rAAV 9-EGFP-R65 began to express EGFP at 24 h after transfection .The expression peak appeared on the sixth day in HUVECs, and the fifth day in HASMCs.The efficiencies of transfection in HUVECs at MOI of 1×105, 1×106 and 1×107 on the sixth day were (1.40±1.20)%, (12.30±1.35)%and (52.80±2.05)%, respectively.The trans-fection efficiencies of HASMCs on the fifth day were (5.30±1.04)%, (18.30±2.24)% and (52.40±3.21)%at MOI of 1×105 , 1×106 and 1×107 .Cell growth and morphology were not affected by transfection .Alamar Blue assay confirmed that there was no significant difference in the absorbance value between the transfected cells and two types of control cells .Western blot assay showed that the expression of NF-κB P65 was decreased by the trans-fection of rAAV9-EGFP-R65 in HUVECs and HASMCs .Conclusion rAAV9-EGFP-R65 can be efficiently trans-fected into two types of human vascular cells .It shows no inhibitory effects on cell proliferation , but can repress NF-κB P65 expression.

RNA Mapping of Mutant Myotonic Dystrophy Protein Kinase 3'-Untranslated Region Transcripts

Myotonic dystrophy type 1 (DM1), which is a dominantly inherited neurodegenerative disorder, results from a CTG trinucleotide repeat expansion in the 3'-untranslated region (3'-UTR) of the myotonic dystrophy protein kinase (DMPK) gene. Retention of mutant DMPK (mDMPK) transcripts in the nuclei of affected cells has been known to be the main cause of pathogenesis of the disease. Thus, reducing the RNA toxicity through elimination of the mutant RNA has been suggested as one therapeutic strategy against DM1. In this study, we suggested RNA replacement with a trans -splicing ribozyme as an alternate genetic therapeutic approach for amelioration of DM1. To this end, we identified the regions of mDMPK 3'-UTR RNA that were accessible to ribozymes by using an RNA mapping strategy based on a trans - splicing ribozyme library. We found that particularly accessible sites were present not only upstream but also downstream of the expanded repeat sequence. Repair or replacement of the mDMPK transcript with the specific ribozyme will be useful for DM1 treatment through reduction of toxic mutant transcripts and simultaneously restore wild-type DMPK or release nucleus-entrapped mDMPK transcripts to the cytoplasm.

Chronotherapy by Ribozyme Targeted to Telomerase in Nude Mice with a Transplanted Tumor: Model for Human Hepatic Cancer / 航天医学与医学工程

Objective To investigate therapeutic autcomes of using telomerase inhibitors to treat cancer at the presumably most and least opportune circadian stages basing on our earlier study. Methods Twenty-four BALB/C nude mice were synchronized to a regimen of LD12:12 for 4 wk. Hepatic cancer cells (SMMC-7721) were implanted into both flanks of each mouse.Two weeks after transplantation,the hTERT-5'RZ was used to treat the hepatic cancer transplanted into the nude mice daily for two weeks,the injection times being either 9 or 21 HALO.Results The tumorinhibition ratio of mice treated at 21 HALO (65%) was statistically significantly higher than that of mice treated at 9 HALO (48%). Telomerase activity was also reduced to a greater extent in mice treated withhTERT-5'RZ at 21 than at 9 HALO, that was at the time of maximal circadian telomerase activity. Conclusion Injection of ribozyme targeted to telomerase during the tumor's DNA synthesis is associated with a betterinhibition of tumor growth and a better therapeutic outcome in hepaticcancer.

Hairpin ribozyme targeted vascular endothelial growth factor (VEGF) inhibited the expression of VEGF and the growth of xenografted tumors / 肿瘤

Objective: Hepatocarcinoma cell line was transfected with anti-vascular endothelial growth factor (VEGF) hairpin ribozyme gene to observe the effect of hairpin ribozyme on VEGF expression and the growth of the xenografted tumors. Methods: The artificial anti-VEGF hairpin ribozyme gene was transfected into hepatocarcinoma SMMC-7721 cells via lipofectin mediation. The blank vector and the cell controls were prepared simultaneously. Then, positive clones were screened by genticin (G418). The transcription of ribozyme was confirmed by RT-PCR. The effects of the ribozyme on VEGF expression of SMMC-7721 cells were detected by semi-quantitative RT-PCR and immunohistochemical method. Cells in each group were inoculated into nude mice. The tumor volume and weight were recorded. The change in microvessel density and expression of VEGF was determined by immunohistochemistry. Results: Ribozyme gene was successfully transferred into tumor cells. The proliferation rate of ribozyme-transfected SMMC-7721 cells was significantly slower (P < 0.01). The expression of VEGF significantly decreased in ribozyme-transfected SMMC-7721 cells. After rebozyme transfection, the tumor formation rate significantly decreased and the growth speed of xenografted hepatocarcinoma markedly slowed down. The microvessel density and angiogenesis of the xenografted hepatocarcinoma were obviously reduced. Conclusion: Anti-VEGF hairpin ribozyme gene significantly inhibited the VEGF expression of hepatocarcinoma in vitro and in vivo by inhibiting angiogenesis of tumor cells. This study provided an experimental evidence for anti-angiogenesis gene therapy for hepatocarcinoma.

An RNA Mapping Strategy to Identify Ribozyme-Accessible Sites on the Catalytic Subunit of Mouse Telomerase

Telomerase reverse transcriptase (TERT) is an enzymatic ribonucleoprotein that prolongs the replicative life span of cells by maintaining protective structures at the ends of eukaryotic chromosomes. Telomerase activity is highly up-regulated in 85-90% of human cancers, and is predominately regulated by hTERT expression. In contrast, most normal somatic tissues in humans express low or undetectable levels of telomerase activity. This expression profile identifies TERT as a potential anticancer target. By using an RNA mapping strategy based on a trans-splicing ribozyme library, we identified the regions of mouse TERT (mTERT) RNA that were accessible to ribozymes. We found that particularly accessible sites were present downstream of the AUG start codon. This mTERTspecific ribozyme will be useful for validation of the RNA replacement as cancer gene therapy approach in mouse model with syngeneic tumors.

Construction of an RNase P Ribozyme Library System for Functional Genomics Applications

An RNase P ribozyme library has been developed as a tool for functional genomics studies. Each clone of this library contains a random 18-mer and the sequence of M1 RNA, the catalytic subunit of RNase P. Repression of target gene expression is thus achieved by the complementary binding of mRNA to the random guide sequence and the successive target cleavage via M1 RNA. Cellular expression of the ribozyme expression was confirmed, and EGFP mRNA was used as a model to demonstrate that the RNase P ribozyme expression system can inhibit the target gene expression. The constructed RNase P ribozyme library has a complexity of 1.4x10(7). This novel library system should become a useful in functional genomics, to identify novel gene functions in mammalian cells.

Effects of hammerhad ribozyme targeting connective tissue growth factor on collagen Ⅰ synthesis and cell cycle progression of human hepatic stellate cells / 吉林大学学报(医学版)

Objective To study the inhibitory effect of hammerhead ribozyme targeting connective tissue growth factor(CTGF) on collagen I synthesis and cell cycle progression of human hepatic stellate cell line(LX-2) cells.Methods Hammerhead ribozyme cDNA targeting CTGF mRNA plus two self-cleaving sequences were inserted into pTriEx2 vector to construct a recombinant vector pTriCTGF-Rz.LX-2 cells were transfected with either pTriEx2 or pTriCTGF-Rz and further stimulated with or without TGF-1.There were five groups in the experiment:control group,pTriEx2 group,pTriCTGF-Rz group,pTriEx2 plus TGF-?1 group,and pTrCTGF-Rz plus TGF?1 group.Semi-quantitative RT-PCR was used to detect the levels of CTGF mRNA and collagen Ⅰ mRNA.ELISA and flow cytometry were used to detect the levels of collagen Ⅰ secretion and cell cycle.Results Transfection of pTriCTGF-Rz into LX-2 cells reduced the CTGF mRNA and collagen Ⅰ mRNA levels as well as collagen Ⅰ protein level compared with pTriEx2 group(P

Reversal of HCC Drug Resistance by Using Hammerhead Ribozymes against Multidrug Resistance 1 Gene / 华中科技大学学报（医学）（英德文版）

To reverse multidrug resistance(MDR) of HepG2 by anti-MDR1 hammerhead ribozyme,an anti-MDR1 hammerhead ribozyme was developed and delivered to P-gp-overproducing human hepatocarcinoma cell line HepG2 by a retroviral vector containing RNA polymerase Ⅲ promoter.The expression of mdr1/Pgp and Rz was detected in HepG2, HepG2 multidrug-resistant cell line and HepG2 Rz-transfected cells by semi quantitative RT-PCR and Western blot methods. Moreover, MTT assay was employed to detect the sensitivity of these ribozyme-transfected cells, and Rhodamine123 (Rh123) was used to test the function of Pgp. The Rz- transfected HepG2 cells became doxorubicin-sensitive, which was concomitant with the decreased MDR1 expression. The study showed that the retrovirus vector encoding the anti-MDR1 ribozyme may be applicable to the treatment of MDR cells.

Functional Modification of a Specific RNA with Targeted Trans-Splicing

The self-splicing group I intron from Tetrahymena thermophila has been demonstrated to perform splicing reaction with its substrate RNA in the trans configuration. In this study, we explored the potential use of the trans-splicing group I ribozymes to replace a specific RNA with a new RNA that exerts any new function we want to introduce. We have chosen thymidine phosphorylase (TP) RNA as a target RNA that is known as a valid cancer prognostic factor. Cancer-specific expression of TP RNA was first evaluated with RT-PCR analysis of RNA from patients with gastric cancer. We determined next which regions of the TP RNA are accessible to ribozymes by employing an RNA mapping strategy, and found that the leader sequences upstream of the AUG start codon appeared to be particularly accessible. A specific ribozyme recognizing the most accessible sequence in the TP RNA with firefly luciferase transcript as a 3' exon was then developed. The specific trans-splicing ribozyme transferred an intended 3' exon tag sequence onto the targeted TP transcripts, resulting in a more than two fold induction of the reporter activity in the presence of TP RNA in mammalian cells, compared to the absence of the target RNA. These results suggest that the Tetrahymena ribozyme can be a potent anti-cancer agent to modify TP RNAs in tumors with a new RNA harboring anti-cancer activity.

Down-regulation of the bcl-2 expression level in a human cholangiocarcinoma cell line using ribozyme / 第三军医大学学报

Objective To study the role of down regulation of bcl 2 expression of human cholangiocarcinoma cells with bcl 2 mRNA cleaving ribozyme (RZ). Methods Synthetic gene of bcl 2 mRNA cleaving RZ was recombined with the expression vector of eukaryotic cells. The recombined vector was used to transfect human cholangiocarcinoma cells. The expression of bcl 2 in human cholangiocarcinoma cells was observed by immunocytochemistry and flow cytometry. Results The expression of bcl 2 in human cholangiocarcinoma cells transfected with RZ gene was lower than that of control group. Spontaneous apoptosis was observed in a few of the cells. Conclusion bcl 2 mRNA cleaving RZ can down regulate the expression of bcl 2 of cholangiocarcinoma cells notably and can also induce spontaneous apoptosis.

The suppression of MUC1 expression in cultured cholangiocarcinoma cells using antisense MUC1 ribozyme strategy.

Ribozyme strategy has been employed to suppress the expression of target genes with a better efficacy than that of a regular antisense RNA strategy due to its favorable stoichiometry of the hybridization. A plasmid capable of controlling the expression of a portion of antisense RNA whose sequence is complementary to the 5' region of the coding sequence of MUC1 was constructed to include the requisite sequence of a hammerhead ribozyme. This plasmid is intended to suppress MUC1 expression. This novel plasmid was examined to see whether it functioned inside the harboring cell. Cultured cholangiocarcinoma cells prepared from the sediment of intrahepatic biliary fluid were employed as the transfection target since these cells have been confirmed to have high levels of MUC1 expression. The suppression of MUC1 expression in these cells after stimulation of the plasmid function for 16 h was demonstrated using flow cytometric analysis.

Construction of specific ribozymes against second exon of ?_1 Ⅰ and ?_1 Ⅲ procollagen genes / 中华医学美学美容杂志

Objective To construct two ribozyme genes (gⅠand gⅢ) by using PCR according to the requirement of ribozyme's domains and the nucleotide sequences of ?_1Ⅰand Ⅲ procollagen genes. Methods First we constructed two ribozyme genes (g, gⅢ) by using PCR, and then separately cloned into the downstream of T7 promoter of pGEM-T vector. Results The resulting recombinant plasmids (pT-gⅠ, pT-gⅢ) were verified by restriction enzymes, PCR and sequencing. Conclusion The expected two ribozyme genes are constructed successfully and this work affords the basis for the preparation of large amounts of ribozyme molecules and for the study on interaction between mRNA and ribozyme in vivo.

INTRACELLULAR INHIBITION OF VIRAL GENE EXPRESSION BY HAMMERHEAD RIBOZYMES AGAINST HEPATITIS C VIRUS 5′ NCR AND C REGIONS / 解放军医学杂志

To investigate the potential use of synthetic stabilized ribozymes for the treatment of chronic hepatitis C virus (HCV) infection,we designed and synthesized 2 hammerhead ribozymes (Rz213 and Rz498) targeting conserved sites in the 5′noncoding region (NCR) and C gene of HCV RNA.Constructed to the eukaryotic vector pcDNA3, the two ribozymes were respectively or simultaneously transfected with lipofectamine into WISHnc transgenic cells, which could express permanently HCV C luciferase protein under the control of HCV 5′NCR.The expression of C luciferase was measured by luminometer.The results showed that the luciferase activities were significantly down regulated in the WISHnc cells, and the inhibitory rates were 42.94%～67.81% within 7 days after ribozymes transfection. There was no significant differences between Rz213 or Rz498 and co transfection, but adding the target site of ribozymes might prevent host cells from the loss of ribozyme therapeutic effect due to viral gene mutation.

Cleavage activity of anti-transforming growth factor ?_1 RNA by U1 snRNA chimeric ribozyme in vitro / 中国病理生理杂志

AIM: To determine the cleavage activity of anti-transforming growth factor ?1 hammerhead ribozymes which was inserted into U1 small nuclear RNA in cell-free system.METHODS: The hammerhead ribozyme targeting against transforming growth factor ?1 was designed through the analysis of computer software.The ribozyme fragments were synthesized and cloned into the U1 snRNA ribozyme vector pZeoU1EcoSpe,which contained U1 snRNA promoter/enhancer and terminator.TGF ?1 cDNA partial fragment was generated by RT-PCR,and then cloned into the T-vector at the downstream of T7 promoter.The transcripts of ribozyme and target RNA incorporated into isotope were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis.-labeled U1 snRNA chimeric ribozyme transcripts were incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE.RESULTS: U1snRNA chimeric ribozyme(U1Rz803) cleaved TGF?1 mRNA efficiently and specifically at 37 ℃,while the disable ribozyme(U1Rz803m) showed no cleavage activity,so these indicated the design of U1Rz803 was correct.CONCLUSION: U1Rz803 prepared in this study possesses the perfect specific catalytic cleavage activity in cell-free system.These results indicate that U1 snRNA chimeric ribozyme U1Rz803 may suppress the expression of TGF?1 in vivo,therefore it may provide a new means for exploring the role of TGF?1 in hematopoietic regulation in the future.

MDR1 specific ribozyme reverses multidrug resistance (MDR) of human hepatocellular carcinoma, an in vivo experimental study / 中国普通外科杂志

Objective To investigate the reversal of MDR by using anti-MDR1 ribozyme N2A + tRNAimet-iMDR1- sRz (sRz) in nude mice bearing human hepatocellular carcinoma. Methods The nude mice model with implanted human hepatocellular carcinoma was randomly devided into group A ( saline 40?l + Lipofect AMINE?000 10?l), B( N2A + tRNAimet10?g/40?l + Lipofect AMINE?000 10?l) and C(sRz 10?g/40?l + Lipofect AMINE?000 10?l). After one week, mice was peritoneally injected E-ADM 15 mg?kg-1 once a week for 4 weeks. The size of tumors was measured with B-ultrasound and the tumor inhibition rate was calculated. One week after chemotherapy mice was sacrificed and MDR1 mRNA and P-gp were investigated with RT-PCR and Western blot. Results In group C tumor shrank upon each chemotherapy (F = 659. 99, P