MicroRNA-34a inhibits the proliferation and promotes the apoptosis of non-small cell lung cancer H1299 cell line by targeting TGFßR2.

MicroRNAs (MiRNAs) are small non-coding RNA molecules which act as important regulators of post-transcriptional gene expression by binding 3'-untranslated region (3'-UTR) of target messenger RNA (mRNA). In this study, we analyzed miRNA-34a (miR-34a) as a tumor suppressor in non-small cell lung cancer (NSCLC) H1299 cell line. The expression level of miR-34a in four different NSCLC cell lines, H1299, A549, SPCA-1, and HCC827, was significantly lower than that in the non-tumorigenic bronchial epithelium cell line BEAS-2B. In human NSCLC tissues, miR-34a expression level was also significantly decreased in pT2-4 compared with the pT1 group. Moreover, miR-34a mimic could inhibit the proliferation and triggered apoptosis in H1299 cells. Luciferase assays revealed that miR-34a inhibited TGFßR2 expression by targeting one binding site in the 3'-UTR of TGFßR2 mRNA. Quantitative real-time PCR (qRT-PCR) and Western blot assays verified that miR-34a reduced TGFßR2 expression at both mRNA and protein levels. Furthermore, downregulation of TGFßR2 by siRNA showed the same effects on the proliferation and apoptosis as miR-34a mimic in H1299 cells. Our results demonstrated that miR-34a could inhibit the proliferation and promote the apoptosis of H1299 cells partially through the downregulation of its target gene TGFßR2.

Human tryptophanyl-tRNA synthetase is switched to a tRNA-dependent mode for tryptophan activation by mutations at V85 and I311.

For most aminoacyl-tRNA synthetases (aaRS), their cognate tRNA is not obligatory to catalyze amino acid activation, with the exception of four class I (aaRS): arginyl-tRNA synthetase, glutamyl-tRNA synthetase, glutaminyl-tRNA synthetase and class I lysyl-tRNA synthetase. Furthermore, for arginyl-, glutamyl- and glutaminyl-tRNA synthetase, the integrated 3' end of the tRNA is necessary to activate the ATP-PPi exchange reaction. Tryptophanyl-tRNA synthetase is a class I aaRS that catalyzes tryptophan activation in the absence of its cognate tRNA. Here we describe mutations located at the appended beta1-beta2 hairpin and the AIDQ sequence of human tryptophanyl-tRNA synthetase that switch this enzyme to a tRNA-dependent mode in the tryptophan activation step. For some mutant enzymes, ATP-PPi exchange activity was completely lacking in the absence of tRNA(Trp), which could be partially rescued by adding tRNA(Trp), even if it had been oxidized by sodium periodate. Therefore, these mutant enzymes have strong similarity to arginyl-tRNA synthetase, glutaminyl-tRNA synthetase and glutamyl-tRNA synthetase in their mode of amino acid activation. The results suggest that an aaRS that does not normally require tRNA for amino acid activation can be switched to a tRNA-dependent mode.

[Effect of silencing connective tissue growth factor on rat liver fibrosis and the accumulation of extracellular matrix].

OBJECTIVE: To investigate the anti-fibrogenesis property of intraportal vein injection of small interfering RNA targeting connective tissue growth factor (CTGF) in a rat model of liver fibrosis and its effect on the accumulation of extracellular matrix (ECM). METHODS: Thirty male rats were randomly divided into five groups. Some rats received CCl4 subcutaneously every three days for 6 consecutive weeks, and in the meantime they also received either siRNA targeting CTGF (preventive group), saline (model group) or siRNA (siRNA control group) by intraportal vein injections. Other rats received CCl4 by subcutaneous injection for 2 weeks, followed by CCl4 and CTGF siRNA intraportal vein injection for 4 more weeks (as treatment group). The expressions of CTGF and type I and III collagen genes were detected by means of reverse transcription-polymerase chain reaction (RT-PCR) and/or Western blot respectively. Hepatic histology was evaluated by HE and Sirius red stained sections. The collagen staining areas were measured quantitatively using a computer-aided manipulator with slight modifications. Serum procollagen type III and hyaluronic acid were determined by radioimmunoassay. RESULTS: Six weeks after CCl4 injection, prominent upregulation of gene expressions of CTGF, type I and III collagen, and laminin in saline or siRNA-treated rat livers were observed. The expressions of CTGF at mRNA and protein level and type I and III collagen at mRNA level were markedly reduced in rats with CTGF siRNA treated for four or six weeks. Expressions of CTGF at mRNA and protein levels decreased by 76%+/-8%, 80%+/-3% (F = 68.630) and 95%+/-2%, 93%+/-3% (F = 21.234, P < 0.01); type I and III collagen and laminin at mRNA levels decreased by 74%+/-8%, 78%+/-8%, 31%+/-7% and 57%+/-6%, 59%+/-10%, 43%+/-9% (F = 24.219, 16.315, 9.716, P < 0.01) compared with rats in the model group at 72 h. The CTGF siRNA treatment markedly reduced serum levels of procollagen type III and hyaluronic acid and the degrees of liver fibrosis. CONCLUSION: Intraportal vein siRNA injection targeting CTGF could significantly inhibit CTGF gene expression in rats, thereby attenuating liver fibrosis by reducing ECM accumulation.

Reduction of bilirubin by targeting human heme oxygenase-1 through siRNA.

Neonatal hyperbilirubinemia is a common clinical condition caused mainly by the increased production and decreased excretion of bilirubin. Current treatment is aimed at reducing the serum levels of bilirubin. Heme oxygenase-1 (HO-1) is a rate-limiting enzyme that generates bilirubin. In this study we intended to suppress HO-1 using the RNA interference technique. Small interfering RNA (siRNA)-A, -B, and -C were designed based on human HO-1 (hHO-1) mRNA sequences. siRNA was transfected into a human hepatic cell line (HL-7702). hHO-1 transcription and protein levels were then determined. In addition, the inhibitory effect of siRNA on hHO-1 was assessed in cells treated with hemin or transfected with an hHO-1 plasmid. siRNA-C showed the most potent suppressive effect on hHO-1. This inhibition is dose and time dependent. Compared with control, both hemin and hHO-1 plasmids up-regulated hHO-1 expression in HL-7702 cells. However, the up-regulation was significantly attenuated by siRNA-C. Furthermore, the decrease in hHO-1 activity was coincident with the suppression of its transcription. Finally, siRNA-C was shown to reduce hHO-1 enzymatic activity and bilirubin levels. Thus, this study provides a novel therapeutic rationale by blocking bilirubin formation via siRNA for preventing and treating neonatal hyperbilirubinemia and bilirubin encephalopathy at an early clinical stage.

An oligonucleotide microarray for microRNA expression analysis based on labeling RNA with quantum dot and nanogold probe.

MicroRNAs (miRNAs) play important regulatory roles in animals and plants by targeting mRNAs for cleavage or translational repression. They have diverse expression patterns and might regulate various developmental and physiological processes. Profiling miRNA expression is very helpful for studying biological functions of miRNAs. We report a novel miRNA profiling microarray, in which miRNAs were directly labeled at the 3' terminus with biotin and hybridized with complementary oligo-DNA probes immobilized on glass slides, and subsequently detected by measuring fluorescence of quantum dots labeled with streptavidin bound to miRNAs through streptavidin-biotin interaction. The detection limit of this microarray for miRNA was approximately 0.4 fmol, and the detection dynamic range spanned about 2 orders of magnitude. We made a model microarray to profile 11 miRNAs from leaf and root of rice (Oryza sativa L. ssp. indica) seedlings. The analysis results of the miRNAs had a good reproducibility and were consistent with the northern blot result. To avoid using high-cost detection equipment, colorimetric detection, a method based on nanogold probe coupled with silver enhancement, was also successfully introduced into miRNA profiling microarray detection.

Direct repression of the oncogene CDK4 by the tumor suppressor miR-486-5p in non-small cell lung cancer.

MicroRNAs are a class of non-coding single-stranded RNA, 20-23 nucleotide in length, which can be involved in the regulation of gene expression. Through binding with 3'-untranslated regions (3'-UTR), microRNAs can cause degradation of target mRNAs or inhibition of translation, and thus regulating the expression of genes at the post-transcriptional level. In this study, we found that miR-486-5p was significantly downregulated in non-small cell lung cancer (NSCLC) tissues and cell lines, suggesting that miR-486-5p might function as a tumor suppressor in lung cancer. Additionally, we showed that CDK4, an oncogene that plays an important role in cell cycle G1/S phase progression, was directly targeted by miR-486-5p. Furthermore, our data reveals that knockdown of CDK4 by siRNA can inhibit cell proliferation, promote apoptosis, and impede cell-cycle progression. In epigenetics, the upstream promoter of miR-486-5p was strongly regulated by methylation in NSCLC. Collectively, our results suggest that miR-486-5p could not only inhibit NSCLC by downregulating the expression of CDK4, but also be as a promising and potent therapy in the near future.

MiR-146a-5p inhibits cell proliferation and cell cycle progression in NSCLC cell lines by targeting CCND1 and CCND2.

Previous studies have indicated that miR-146a-5p acts as an oncogene in several types of cancer, yet a tumor suppressor gene in others. In non-small cell lung cancer (NSCLC), one report showed that it was downregulated and played the role of tumor suppressor. However, another study showed that miR-146a-5p was overexpressed in the serum of NSCLC patients compared to healthy controls. Therefore, it is obvious that further study of the function of miR-146a-5p in NSCLC is necessary to fully understand its importance. Herein, we have verified that miR- 146a- 5p acts as a tumor suppressor in NSCLC. Our data revealed that the expression level of miR-146a-5p was significantly decreased in several human NSCLC cell lines, and also less abundant in human NSCLC tissues, when compared with controls. Moreover, we observed that miR-146a-5p could suppress cell proliferation, both in vitro and in vivo. Our results also showed that miR-146a-5p directly targeted the 3'-UTR of CCND1 and CCND2 mRNAs as well as decreased their expression at both mRNA and protein levels, causing cell cycle arrest at the G0/G1 phase. Furthermore, siRNA-mediated downregulation of CCND1 or CCND2 yielded the same effects on proliferation and cell cycle arrest as miR-146a-5p upregulation did in the NSCLC cell lines. We confirmed that the expression of miR-146a-5p had negative relationship with CCND1 or CCND2. Besides, we also found that miR-146a-5p could inhibit tumor growth in xengroft mouse models, and CCND1 and CCND2 were downregulated in miR-146a-5p overexpressed xengroft tumor tissues. In summary, our results demonstrated that miR-146a-5p could suppress the proliferation and cell cycle progression in NSCLC cells by inhibiting the expression of CCND1 and CCND2.

Inhibition of genes expression of SARS coronavirus by synthetic small interfering RNAs.

RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence. dsRNA-mediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression. Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3' overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNA duplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80% at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0 approximately 60 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5'end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.

Preparation of anti-mouse caspase-12 mRNA hammerhead ribozyme and identification of its activity in vitro.

AIM: 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. METHODS: 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). RESULTS: 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%. CONCLUSION: 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.

Effects of ribozyme targeting platelet-derived growth factor receptor beta subunit gene on the proliferation and apoptosis of hepatic stellate cells in vitro.

BACKGROUND: Activation and proliferation of hepatic stellate cells (HSC) is essentially involved in the development and progression of hepatic fibrosis. The most potent growth factor for HSC is platelet-derived growth factor receptor (PDGF) and PDGF receptor beta subunit (PDGFR-beta) is the predominant signal transduction pathway of PDGF which is overexpressed in activated HSC. This study investigated the cleavage activity of hammerhead ribozyme targeting PDGFR-beta mRNA in HSC and the effect on biological characteristics of HSC. METHODS: Expression vector of anti-PDGFR-beta ribozyme was constructed and transfected into rat activated HSC with lipofectamin. The positive cell clones were gained by G418 selection. The expression of PDGFR-beta, alpha-smooth muscle actin, and typeI and type III collagen were detected by using Northern blot, Western blot and immunocytochemical staining, respectively. The cell proliferation was determined with MTT colorimetric assay. The cell apoptosis was analyzed by using flow cytometry, acridine orange fluorescence vital staining and transmission electron microscopy. RESULTS: The expression of PDGFR-beta at mRNA and protein level was markedly reduced in ribozyme-transfected HSC by 49% - 57% (P < 0.05 - 0.01). The proliferation and alpha-smooth muscle actin expression of ribozyme-transfected HSC were significantly decreased (P < 0.05 - 0.01), and the type I and type III collagen synthesis were also reduced (P < 0.01). In addition, the proliferative response of ribozyme-transfected HSC to PDGF BB was significantly inhibited. Otherwise, the apoptotic cells were significantly increased in ribozyme-transfected HSC (P < 0.01), and typical apoptotic cells could be found under transmission electron microscopy. CONCLUSIONS: The anti-PDGFR-beta ribozyme effectively cleaved the target RNA and significantly inhibited its expression, which blocked the signal transduction of PDGF at receptor level, inhibited HSC proliferation and collagen synthesis, and induced HSC apoptosis. These results suggest that inhibiting PDGFR-beta expression of HSC may be a new target for the therapy of liver fibrogenesis, and ribozyme may be a useful tool for inhibiting PDGFR-beta expression.

[Preparation and identification of hammerhead ribozyme in vitro against caspase-12 mRNA fragments].

OBJECTIVE: To design and synthesize ribozymes targeting 138 and 218 sites of the mRNA nucleotide of mouse caspase-12, a key intermedium of ER stress mediated apoptosis, and to identify their activities through in vitro transcription and cleavage. METHODS: The mouse caspase-12 gene fragment was obtained by RT-PCR and cloned into the PGEM-T vector under the control of T7 RNA polymerase promoter. The transcription product of the target was labeled with a-32P UTP, while ribozymes were not labeled. Ribozyme and target RNA were incubated for 90 min at 37 degree C in a reaction buffer to perform the cleavage reaction. RESULTS: It was found that under a condition of 37 degree C, pH 7.5 and with Mg2+ in a concentration of 10 mmol/L, Rz138 and Rz218 both cleaved targets at predicted sites, and the cleavage efficiency of Rz138 was 100%. CONCLUSION: Rz138 and Rz218 prepared in vitro possess the perfect specific catalytic cleavage activity. Rz138 has excellent cleavage efficiency. It may be a promising tool to prevent ER stress induced apoptosis through catalytic cleavage of caspase-12 mRNA in vivo. It also can be used to verify whether caspase-12 is necessary in ER stress induced apoptosis.

[Inhibition of mutant-type p53 by a chimeric U6 maxizyme in hepatocellular carcinoma cell lines].

OBJECTIVE: To study the inhibition of maxizyme (Mz) directed against the mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG --> AGT) both in cell-free system and in MHCC97 cell lines. METHODS: Maxizyme and control mutant maxizyme (G5 --> A5) were designed by computer and cloned into the eukaryotic expression vector pBSKneoU6 (pU6Mz, pU6asMz). Mz was driven by T7 RNA polymerase promoter in vitro. In the cell lines, U6 promoter was driven by RNA PolIII. The mutant type p53 gene fragment was cloned into the pGEM-T vector under the T7 promoter control. The 32P-labeled mtp53 transcript was the target RNA. Cold maxizyme transcripts were incubated with 32P-labeled target RNA in vitro. pU6Mz was introduced into MHCC97 cells by Lipofectamine2000 and mtp53 expression was analyzed by RT-PCR and Western blot. RESULTS: In vitro cleavage showed that pU6Mz was very active with cleavage efficiency of 42% while pU6asMz was not. The wild type p53 was not cleaved. Partial down-regulation of mtp53 mRNA and mtp53 protein were observed in MHCC97 cells transfected with pU6Mz but not those with pU6asMz. The proliferation of MHCC cells was inhibited by MTT analysis. CONCLUSION: Our findings suggest that the chimeric U6 maxizyme against the mtp53 is a new promising gene therapeutic agent in treating hepatocellular carcinoma.

[Inhibition of mouse hepatocyte apoptosis by anti-caspase-12 small interfering RNA].

OBJECTIVES: To study the inhibition of primary mouse hepatocyte apoptosis by small interfering RNA (siRNAs) against caspase-12. METHODS: The Balb/c mouse primary hepatocytes were isolated in situ with two-step liver perfusion with 0.5 g/L collagenase type IV, and apoptosis were induced with 4 micromol/L thapsigargin (TG). The three kingds of siRNAs targeting different gene sites (130, 214, 521) were synthetized chemically. The single-stranded RNAs were annealed to produce double-stranded siRNAs, then the mouse primary hepatocytes were transfected by oligofectamine package. The inhibition of caspase-12 was analyzed with RT-PCR and Western-blot. The viable hepatocytes following the induction of apoptosis were evaluated with MTT. RESULTS: All the three kinds of siRNAs could obviously inhibit normal mouse hepatocyte caspase-12 mRNA. The siRNA (214) were more effective than the other two when the concentration was 100 nmol/L. The caspase-12 mRNA expression was inhibited by 52.08%, while that of siRNA (521) was 30.73% (t=4.30, P <0.05). However when the concentration was 200 nmol/L, the inhibitions were similar (88.07%, 86.22% and 89.41% respectively). siRNA (214) could downregulate the expression of apoptotic hepatocytes procaspase-12 by 51.43% ( t=4.30, P <0.01). Contrasted with apoptotic hepatocytes, the cell activity, which was analyzed with MTT, increased by 48.76% (t=2.23, P <0.01). CONCLUSION: siRNAs could effectively downregulate the expression of caspase-12 at mRNA and protein levels and prevent mouse primary hepatocytes from apoptosis.

Tanshinones suppress AURKA through up-regulation of miR-32 expression in non-small cell lung cancer.

Tanshinone is the liposoluble constituent of Salia miltiorrhiza, a root used in traditional herbal medicine which is known to possess certain health benefits. Although it is known that tanshinones, including tanshinone I (T1), tanshinone IIA (T2A), and cryptotanshinone (CT), can inhibit the growth of lung cancer cells in vitro, the mechanism under which they act is still unclear. AURKA, an oncogene, encodes a serine-threonine kinase which regulates mitotic processes in mammalian cells. Here, we reported that tanshinones mediate AURKA suppression partly through up-regulating the expression of miR-32. We found that tanshinones could inhibit cell proliferation, promote apoptosis, and impede cell-cycle progression, thus performing an antineoplastic function in non-small cell lung cancer (NSCLC). Additionally, we demonstrated that tanshinones attained these effects in part by down-regulating AURKA, corroborating previous reports. Our results showed that in NSCLC, similar effects were obtained with knock-down of the AURKA gene by siRNA. We also verified that AURKA was the direct target of miR-32. Collectively, our results demonstrated that tanshinones could inhibit NSCLC by suppressing AURKA via up-regulating the expressions of miR-32 and other related miRNAs.

Expression and in vitro cleavage activity of anti-caspase-7 hammerhead ribozymes.

AIM: To prepare hammerhead ribozymes against mouse caspase-7 and identify their cleavage activity in vitro, in order to select a ribozyme with specific cleavage activity against mouse caspase-7 as a potential gene therapy for apoptosis-related diseases. METHODS: Anti-caspase-7 ribozymes targeting sites 333 and 394 (named Rz333 and Rz394) were designed by computer software, and their DNA sequences encoding ribozymes were synthesized. Caspase-7 DNA sequence was acquired by RT-PCR. Ribozymes and caspase-7 DNA obtained by in vitro transcription were cloned into pBSKneo U6' and pGEM-T vectors, respectively. The cleavage activity of ribozymes against mouse caspase-7 was identified by cleavage experiments in vitro. RESULTS: Rz333 and Rz394 were designed and their DNA sequences were synthesized respectively. The expression vector of caspase-7 and plasmids containing Rz333 and Rz394 were reconstructed successfully. Ribozymes and caspase-7 mRNA were expressed by in vitro transcription. In vitro cleavage experiment showed that 243-nt and 744-nt segments were produced after caspase-7 mRNA was mixed with Rz333 in equivalent, and the cleavage efficiency was 67.98%. No cleaved segment was observed when caspase-7 mRNA was mixed with Rz394. CONCLUSION: Rz333 can site-specific cleave mouse caspase-7 mRNA, and it shows a potential for gene therapy of apoptosis-related diseases by down-regulating gene expression of caspase-7.

Preparation and identification of anti-transforming growth factor beta1 U1 small nuclear RNA chimeric ribozyme in vitro.

AIM: To study the preparation and cleavage activity of anti-transforming growth factor (TGF)beta1 U1 small nuclear (sn) RNA chimeric hammerhead ribozymes in vitro. METHODS: TGFbeta1 partial gene fragment was cloned into T-vector at the downstream of T7 promoter. (32)p-labeled TGFbeta1 partial transcripts as target RNA were transcribed in vitro and purified by denaturing polyacrylamide gel electrophoresis (PAGE). Anti-TGFbeta1 ribozymes were designed by computer, then synthetic ribozyme fragments were cloned into the U1 ribozyme vector pZeoU1EcoSpe containing U1 snRNA promoter/enhancer and terminator. (32)p-labeled U1 snRNA chimeric ribozyme transcripts were gel-purified, incubated with target-RNAs at different conditions and autoradiographed after running denaturing PAGE. RESULTS: Active U1snRNA chimeric ribozyme (U1Rz803) had the best cleavage activity at 50 degrees; at 37 degrees, it was active, K(m)=34.48 nmol/L, K(cat)=0.14 min(-1); while the point mutant ribozyme U1Rz803(m) had no cleavage activity, so these indicated the design of U1Rz803 was correct. CONCLUSION: U1Rz803 prepared in this study possessed the perfect specific catalytic cleavage activity. These results indicate U1 snRNA chimeric ribozyme U1Rz803 may suppress the expression of TGFbeta1 in vivo, therefore it may provide a new avenue for the treatment of liver fibrosis in the future.

Maxizyme-mediated specific inhibition on mutant-type p53 in vitro.

AIM: To evaluate the specific inhibition of maxizyme directing against mutant-type p53 gene (mtp53) at codon 249 in exon 7 (AGG-AGT) in vitro. METHODS: Two different monomers of anti-mtp53 maxizyme (maxizyme right MzR, maxizyme left MzL) and control mutant maxizyme (G(5)-A(5)) were designed by computer and cloned into vector pBSKU6 (pBSKU6MzR, pBSKU6MzL). After being sequenced, the restrictive endonuclease site in pBSKU6MzR was changed by PCR and then U6MzR was inserted into pBSKU6MzL, the recombinant vector was named pU6Mz and pU6asMz (mutant maxizyme). Mtp53 and wild-type p53 (wtp53) gene fragments were cloned into pGEM-T vector under the T7 promoter control. The (32)p-labeled mtp53 transcript was the target mRNA. Cold maxizyme transcripts were incubated with (32)p-labeled target RNA in vitro and radioautographed after denaturing polyacrylamide gel electrophoresis. RESULTS: In cell-free systems, pU6Mz showed a specific cleavage activity against target mRNA at 37 degrees and 25 mM MgCL(2). The cleavage efficiency of pU6Mz was 42 %, while pU6asMz had no inhibitory effect. Wtp53 was not cleaved by pU6Mz either. CONCLUSION: pU6Mz had a specific catalytic activity against mtp53 in cell-free system. These lay a good foundation for studying the effects of anti-mtp53 maxizyme in HCC cell lines. The results suggest that maxizyme may be a promising alternative approach for treating hepatocellular carcinoma containing mtp53.

Anti-HBV hairpin ribozyme-mediated cleavage of target RNA in vitro.

AIM: To study the preparation and cleavage activity of HpRz directed against the transcript of HBV core gene in vitro. METHODS: HpRz gene designed by computer targeting the transcript of HBV core gene was cloned into the vector p1.5 between 5'-cis-Rz and 3'-cis-Rz. 32p-labeled HpRz transcript proved whether the vector fit for the preparation of hairpin ribozyme in vitro. 32p-labeled pKC transcript containing HBV core region as target-RNA was transcribed using T7 RNA polymerase and purified by denaturing PAGE. Cold HpRz transcript was incubated with 32p-labeled target-RNAs under different conditions and radio autographed after denaturing polyacrylamide gel electrophoresis. RESULTS: HpRz has the specific ability of cleavage of target RNA at 37 degrees and 12 mM MgCl2. Km=26.31 nmol/L, Kcat=0.18/min. These results revealed that the design of HpRz was correct. CONCLUSION: HpRz prepared in this study possesses specific catalytic activity from the identification of cleavage activity. These results indicate that hairpin ribozyme may intracellularly inhibit the replication of HBV, therefore it may become a novel potent weapon for the treatment of hepatitis B.

Selection with SELEX Method of Small RNA Molecules Specifically Binding to Starch.

A 73-base DNA library with T7 promoter was chemically synthesized according to the sequence specified by the 2.5S RNA component of glycogen branching holoenzyme (EC 2.4.1.18) from rabbit muscle, in which 20 consecutive bases corresponding to a stem-loop structure were completely randomized. The in vitro transcribed RNA library was subjected to 9 successive rounds of selection with SELEX method, resulting in a sharp increase of the percentage of the starch-binding RNA from less than 0.01% for the first round pool to 31% for the ninth-round pool. This demonstrates the existence of small RNA species that can specifically bind to starch. Structural and functional analysis of them is in progress.

A Discriminator Base in tDNA(Trp).

The discriminator base G73 was one of the major identity elements of tRNA(Trp). Aminoacylation assay and CD spectrum of tDNA(Trp)-NCCrA demonstrated that under the same pH, the change of discriminator base affected tRNA conformation at the 3' terminus while tDNA(Trp) with the same discriminator base exhibited different conformation and aminoacylation activity under various pH conditions. Our results showed that it was the conformation of tRNA, but not the base itself, that determined the accurate recognition between tRNA and its cognate synthetase.