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Objective:To investigate the effects of miR-5011-5p on apoptosis and migration of bladder cancer cell line J82 and the underlying mechanism.Methods:J82 cells were transfected with random sequence molecules (NC group) and miR-5011-5p sequence molecules (miR-5011-5p group). Flow cytometry and scratch experiment were performed to analyze the effects of miR-5011-5p on apoptosis and migration of J82 cells. The target gene of miR-5011-5p was predicted by bioinformatics. Real-time fluorescent quantitative polymerase chain reaction and western blot assay were performed to investigate the effects of miR-5011-5p on target gene expression.Results:The relative expression of miR-5011-5p in J82 cells in the miR-5011-5p group was significantly higher than that in the NC group (10.73 ± 1.67 vs. 1.04 ± 0.16, t = 5.81, P < 0.01). There was significant difference in the apoptosis rate of J82 cells between NC and miR-5011-5p groups [(8.83 ± 1.67)% vs. (34.96 ± 3.80)%, t = 6.30, P < 0.01]. The migration rate of J82 cells differed significantly between NC and miR-5011-5p groups [(71.31 ± 7.69)% vs. (37.43 ± 5.01)%, t = 3.69, P < 0.05]. The target gene of miR-5011-5p may be Yes-related protein 1 (YAP1). Compared with the NC group, miR-5011-5p exhibited an obvious inhibitory effect on the YAP1 expression in J82 cells ( P < 0.01). Conclusion:miR-5011-5p may promote the apoptosis of J82 cells and inhibit their migration in bladder cancer through targeted inhibition of YAP1 gene expression.
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Objective:To observe the expression of long-chain noncoding RNA (lncRNA) SCAMP1-AS1 in esophageal cancer tissues, and explore the effect of SCAMP1-AS1 on the proliferation and migration of esophageal cancer cells and the possible molecular mechanism.Methods:Real-time fluorescent quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression level of SCAMP1-AS1 in 37 cases of esophageal cancer tissues and adjacent tissues surgically resected in Huangshi Central Hospital of Edong Medical Group from March 2017 to August 2020. RT-qPCR was also used to detect the expression level of SCAMP1-AS1 in 4 types of esophageal cancer cells (EC9706, TE-13, KYSE30, Eca109) and normal esophageal epithelial cells HET-1A. The cells with the lowest expression were selected, the negative control lentivirus (LV-NC) infection was used as the control group, and the recombinant lentivirus carrying SCAMP1-AS1 sequence (LV-SCAMP1-AS1) infection was used as the experimental group. RT-qPCR was used to detect the expression of SCAMP1-AS1 in esophageal cancer cells after infection. Cell counting kit 8 (CCK-8) and Transwell chamber method were used to detect the proliferation and migration ability of esophageal cancer cells. Bioinformatics methods predicted the target genes of SCAMP1-AS1, and dual luciferase reporter experiments verified the interaction of SCAMP1-AS1 with target gene. RT-qPCR detected the expression of target genes. Western blotting detected the expression of cell proliferation and migration phenotype proteins.Results:The relative expression level of SCAMP1-AS1 in esophageal cancer tissue was significantly lower than that in adjacent tissues (1.26 ± 0.48 vs. 8.03 ± 1.17, P<0.01). The relative expression levels of SCAMP1-AS1 in esophageal cancer cells EC9706, TE-13, KYSE30, Eca109 were all lower than that in normal esophageal epithelial cells (0.54 ± 0.05, 0.14 ± 0.02, 0.46 ± 0.07, 0.77 ± 0.05 vs.1.00 ± 0.06, P<0.05), and the expression of SCAMP1-AS1 in TE-13 cells was the lowest ( P<0.01). Compared with the control group, the expression of SCAMP1-AS1 in TE-13 cells in the experimental group was up-regulated ( P<0.01), the proliferation ability of the cells was reduced ( P<0.01), and the migration ability of the cells was reduced ( P<0.01). miR-483-5p was the direct target of SCAMP1-AS1. Compared with the control group, the expression of miR-483-5p was down-regulated in TE-13 cells in the experimental group ( P<0.01), and the expression of cell proliferation and migration phenotype proteins was down-regulated. Conclusions:The expression of lncRNA SCAMP1-AS1 is down-regulated in esophageal cancer. SCAMP1-AS1 can inhibit the proliferation and migration of esophageal cancer TE-13 cells by targeting the expression of miR-483-5p. SCAMP1-AS1 is expected to become a potential molecular therapeutic target for esophageal cancer.
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Objective:To investigate the expression of microRNA (miR)-206 in chronic obstructive pulmonary disease (COPD) and its effect on the proliferation of human airway smooth muscle cells (HASMCs) and to explore its mechanism.Methods:Lung tissue samples of 15 patients with COPD (COPD group) who underwent lung volume reduction surgery in the General Hospital of Ningxia Medical University from September 2017 to September 2018 and of 15 patients with benign lung tumors without a history of COPD were collected. Microarray technology was used to analyze the miR and RNA omics in lung tissues of 4 COPD patients and normal controls, and reverse transcriptase polymerase chain reaction(RT-PCR) was used to verify the results. Bioinformatics and double luciferase gene reporting assay were used to detect the target genes of miR-206 in HASMCs. The miR-206 mimic/inhibitor was transfected into HASMCs by liposome transfection technology, and the expression level of miR-206 was detected by RT-PCR. Methyl thiazolyl tetrazolium (MTT), flow cytometry and apoptosis assay were used to detect the effects of miR-206 on the proliferation, cell cycle and apoptosis of HASMCs. The expression of PTEN, cell cycle and apoptotic protein in HASMCs was detected by Western blot.Results:The results of miR and mRNA omics analysis showed that the expressions of miR-206, miR-3187-5p and miR-124 in COPD group were significantly up-regulated (0.09 ± 0.01 vs. 2.17 ± 0.57, 0.60 ± 0.04 vs. 1.32 ± 0.15, 0.22 ± 0.08 vs. 1.09 ± 0.23) ( P<0.05), while the expressions of miR-574 and miR-337-3p decreased significantly (0.79 ± 0.03 vs. 0.15 ± 0.02, 0.95 ± 0.02 vs. 0.17 ± 0.01) ( P<0.05). RT-PCR was used to detect the expression of these five miRNAs in 15 COPD lung tissues, and the results showed that their expression was consistent with that in microarray. The prediction results of miRNA target genes showed that miR-206 could directly inhibit the expression of PTEN. RT-PCR results showed that the expression of miR-206 in miR-206 transfected HASMCs was significantly higher than that in miR-NC transfected group(7.44 ± 0.51 vs. 4.02 ± 0.19), and miR-206 inhibitor could significantly inhibit the expression of miR-206 in cells (1.86 ± 0.32), the difference was statistically significant ( P<0.05); MTT and apoptosis experiments showed that miR-206 mimcs could significantly promote the proliferation rate of cells compared with normal HASMCs or miR-NC transfected cells (0.62 ± 0.14 or 0.57 ± 0.09 vs. 0.83 ± 0.05), inhibit cell apoptosis (9.13 ± 1.71 or 10.02 ± 1.15 vs. 3.06 ± 0.82), the differences were statistically significant ( P<0.05), while miR-206 inhibitor could significantly inhibit cell proliferation and promote cell apoptosis ( P<0.05) The results of cell cycle distribution showed that compared with HASMCs group, the proportion of cells in S phase and G2/M phase in miR-206 mimcs group increased significantly ( P<0.05), while the proportion of cells in S phase and G2/M phase in miR-206 inhibitor group decreased significantly ( P<0.05), and there was no significant difference in miR-NC group ( P>0.05). The results of Western blot showed that compared with normal HASMCs or miR-NC transfected cells, miR-206 mimcs could significantly upregulate the expression of cyclin D1 (0.43 ± 0.07 or 0.41 ± 0.02 vs. 0.63 ± 0.17), and cyclin B1 (0.47 ± 0.13 or 0.50 ± 0.09 vs. 0.79 ± 0.31), and inhibit the expression of PTEN (0.34 ± 0.10 or 0.29 ± 0.05 vs. 0.14 ± 0.02), cyclin p21 (0.34 ± 0.03 or 0.30 ± 0.05 vs. 0.11 ± 0.02), and apoptosis related protein caspase-3 (0.29 ± 0.03 or 0.31 ± 0.05 vs. 0.15 ± 0.03), the differences were statistically significant ( P<0.05). miR-206 inhibitor could significantly inhibit the expression of cyclin D1 and cyclin B1, and promote the expression of PTEN, cyclin p21 and caspase-3 ( P<0.05). Conclusions:In COPD patients, miR-206 could targeted inhibit the expression of PTEN protein in airway smooth muscle cells and regulate the progress of cell cycle, so as to up regulate the proliferation of cells and inhibit their apoptosis.
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Objective:To investigate the relationship between serum microRNA-1 (miR-1) and microRNA-155 (miR-155) expression and disease severity and prognosis of acute cerebral infarction (ACI) in patients.Methods:A total of 173 patients with ACI who received treatment in Taizhou Municipal Hospital between April 2018 and August 2019 were included in this study. These patients were divided into mild ( n = 78), moderate ( n = 54) and severe ( n = 41) groups according to the National Institutes of Health Stroke Scale (NIHSS) score. A total of 180 patients who concurrently received physical examination were included in the control group. Serum miR-1 and miR-155 expression was determined in all participants using quantitative reverse transcription-polymerase chain reaction. The included patients were divided into poor prognosis and good prognosis groups according to modified Rankin scale score within 90 days after treatment. The efficacy of serum miR-1 and miR-155 expression in the prediction of ACI prognosis and the risk factors for poor prognosis of ACI were evaluated. Results:The history of hypertension, systolic blood pressure, diastolic blood pressure, total cholesterol, low density lipoprotein cholesterol, serum miR-1 and miR-155 expression in the study group were 56.65% (98/173), (134.02 ± 27.35) mmHg, (88.45 ± 9.52) mmHg, (3.78 ± 0.82) mmol/L, (2.08 ± 0.73) mmol/L, (2.07 ± 0.37) and (1.56 ± 0.32), respectively, which were significantly higher than those in the control group [39.44% (71/180), (119.37 ± 22.14) mmHg, (81.46 ± 14.13) mmHg, (3.59 ± 0.68) mmol/L, (1.74 ± 0.69) mmol/L, (1.01 ± 0.22), (1.02 ± 0.24)], high density lipoprotein cholesterol in the study group was significantly lower than that in the control group [(1.24 ± 0.22) mmol/L vs. (1.31 ± 0.26) mmol/L, χ2 = 10.462, t = 5.542, 5.429, 2.373, 4.498, 32.865, 17.982 and 2.725, all P < 0.05]. Serum miR-1 and miR-155 expression in patients with ACI gradually increased with the increase of disease severity ( t = 10.212, 13.050, 3.092, 7.027, 3.983 and 4.099, all P < 0.05). The proportion of patients having a history of hypertension in the poor prognosis group was significantly higher than that in the good prognosis group [64.47% (49/76) vs. 42.27% (41/97), χ2 = 8.419, P < 0.05]. Systolic blood pressure, diastolic blood pressure, NIHSS score, serum miR-1 and miR-155 expression in the poor prognosis group were (136.51 ± 12.56) mmHg, (89.53 ± 6.65) mmHg, (7.26 ± 0.58) points, (1.32 ± 0.15), (1.21 ± 0.12), respectively, which were significantly higher than those in the good prognosis group [42.27% (41/97), (132.19 ± 9.32) mmHg, (86.34 ± 5.62) mmHg, (6.44 ± 0.62) points, (1.01 ± 0.07) (0.99 ± 0.05), t = 2.597, 3.418, 8.880, 10.695 and 4.633, all P < 0.05]. The receiver operating characteristic curve (ROC curve) analysis results showed the area under the curve of serum miR-1 and miR-155 expression alone in predicting ACI prognosis was 0.814 (95% CI: 0.745-0.884) and 0.839 (95% CI: 0.780-0.897), respectively. The area under the curve of miR-1 and miR-155 expression in combination in predicting ACI prognosis was 0.944 (95% CI: 0.912-0.976). Logistic regression analysis results showed that increases in admission NIHSS score, miR-1 and miR-155 expression were the risk factors for poor prognosis of ACI ( P < 0.05). Conclusion:miR-1 and miR-155 expression levels are related to the severity of ACI and therefore may be the predictors of poor prognosis of ACI.
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Objective@#To investigate the level and clinical significance of miRNA-106a in non-small cell lung cancer tissues.@*Methods@#Paraffin-embedded specimens of non-small cell lung cancer tissues and adjacent tissues from 80 patients with non-small cell lung cancer from January 2012 to December 2013 in Yiwu Central Hospital were selected in this study.The real-time fluorescence quantitative polymerase chain reaction (QRT-PCR) was used to determine the level of miRNA-106a in lung cancer tissues and adjacent tissues.@*Results@#The level of miRNA-106a in non-small cell lung cancer tissues (2.42±0.23) was higher than that in adjacent tissues (1.00±0.06) (t=53.433, P=0.000). The miRNA-106a levels in lung cancer tissues of stage Ⅲ-Ⅳ, lymph node metastasis and recurrence time<6 months were high than those of the stage Ⅰ-Ⅱ, no lymph node metastasis and recurrence time ≥ 6 months(t=7.641, 11.115, 2.183, P=0.000, 0.000, 0.032). The level of miRNA-106a was not associated with age, gender, pathological type, degree of differentiation and vascular invasion (all P>0.05). The level of miRNA-106a in non-small cell lung cancer tissues was cut by 2.12.The area under the ROC curve for the diagnosis of non-small cell lung cancer was 0.823(95% CI=0.820-0.825, P=0.000), and the sensitivity was 54.37%, the specificity was 89.21%.The cumulative survival rate and progression-free survival rate of patients with low-expression of miRNA-106a in non-small cell lung cancer were higher than those with high expression of miRNA-106a (P=0.027, 0.012).@*Conclusion@#The miRNA-106a level is elevated in non-small cell lung cancer tissues.The miRNA-106a may be involved in the development of non-small cell lung cancer and is of great value in the diagnosis and prognosis evaluation of non-small cell lung cancer.
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The skin pathologic scar is a skin fibrous proliferative disease characterized by abnormal proliferation of fibroblasts and overdeposition of extracellular matrix. Unclarity of genesis and development mechanism is the main reason that restricts its diagnosis and treatment. In recent years, it has been found that microRNAs play important roles in the regulation mechanism of pathological scars. The competing endogenous RNAs (ceRNAs) have microRNA response elements which can be competitively combined with microRNAs through sponge adsorption. Through the mutual regulation of RNAs, ceRNAs regulate the expression of target gene and participate in the development of disease. Based on the ceRNA hypothesis, this paper systematically reviews the biological functions and clinical significance of ceRNAs in pathological scars of skin, and discusses the role of ceRNAs and " RNA-microRNA-RNA" regulation network in pathologic scars. The ceRNA therapy may become a new model therapy for skin scars in the future.
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Objective To investigate the level and clinical significance of miRNA －106a in non－small cell lung cancer tissues.Methods Paraffin－embedded specimens of non －small cell lung cancer tissues and adjacent tissues from 80 patients with non －small cell lung cancer from January 2012 to December 2013 in Yiwu Central Hospital were selected in this study.The real－time fluorescence quantitative polymerase chain reaction (QRT－PCR) was used to determine the level of miRNA －106a in lung cancer tissues and adjacent tissues.Results The level of miRNA－106a in non－small cell lung cancer tissues (2.42 ±0.23) was higher than that in adjacent tissues (1.00 ± 0.06) (t＝53.433,P＝0.000).The miRNA －106a levels in lung cancer tissues of stage Ⅲ －Ⅳ,lymph node metastasis and recurrence time ＜6 months were high than those of the stage Ⅰ－Ⅱ,no lymph node metastasis and recurrence time≥6 months(t＝7.641,11.115,2.183,P＝0.000,0.000,0.032).The level of miRNA－106a was not associated with age ,gender,pathological type,degree of differentiation and vascular invasion (all P＞0.05).The level of miRNA－106a in non－small cell lung cancer tissues was cut by 2.12.The area under the ROC curve for the diagnosis of non －small cell lung cancer was 0.823(95%CI＝0.820－0.825,P＝0.000),and the sensitivity was 54.37%,the specificity was 89.21%.The cumulative survival rate and progression －free survival rate of patients with low－expression of miRNA －106a in non －small cell lung cancer were higher than those with high expression of miRNA－106a (P＝0.027,0.012).Conclusion The miRNA －106a level is elevated in non －small cell lung cancer tissues.The miRNA－106a may be involved in the development of non －small cell lung cancer and is of great value in the diagnosis and prognosis evaluation of non －small cell lung cancer.
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Em laboratório de biologia molecular existem normas para prevenir que nucleases destruam os ácidos nucleicos em análise. Rígida adesão a estas normas é primordial, principalmente em laboratórios de análises clínicas e ao se lidar com amostras com número restrito de cópias do genoma-alvo. Em contraposição, diversas nucleases têm tido importância fundamental, por exemplo, na identificação do ácido nucleico de vírus, investigação de RNA mensageiro, purificação de vírus em abordagem metagenômica, edição de genomas com o sistema CRISPR/Cas e descoberta de enzimas. O conhecimento de como nucleases podem ser tanto vilãs quanto aliadas é essencial na formação de todos que trabalham no campo de biologia molecular.
In a molecular biology laboratory there are standards to prevent nucleases from destroying the nucleic acids under analysis. Strict adherence to these standards is paramount, mainly in clinical analysis laboratories and when dealing with samples with a limited number of copies of the target genome. In contrast, several nucleases have been of fundamental importance, for example, in the identification of the type of viral nucleic acid, investigation of messenger RNA, virus purification in metagenomic approach, genome editing with the CRISPR/Cas system, and enzyme discovery. Knowledge of how nucleases can be both villains and allies is essential in the training of all working in the field of molecular biology.
Subject(s)
Ribonucleases , Viruses , Clinical Laboratory Techniques , Deoxyribonucleases , Molecular BiologyABSTRACT
Em laboratório de biologia molecular existem normas para prevenir que nucleases destruam os ácidos nucleicos em análise. Rígida adesão a estas normas é primordial, principalmente em laboratórios de análises clínicas e ao se lidar com amostras com número restrito de cópias do genoma-alvo. Em contraposição, diversas nucleases têm tido importância fundamental, por exemplo, na identificação do ácido nucleico de vírus, investigação de RNA mensageiro, purificação de vírus em abordagem metagenômica, edição de genomas com o sistema CRISPR/Cas e descoberta de enzimas. O conhecimento de como nucleases podem ser tanto vilãs quanto aliadas é essencial na formação de todos que trabalham no campo de biologia molecular.
In a molecular biology laboratory there are standards to prevent nucleases from destroying the nucleic acids under analysis. Strict adherence to these standards is paramount, mainly in clinical analysis laboratories and when dealing with samples with a limited number of copies of the target genome. In contrast, several nucleases have been of fundamental importance, for example, in the identification of the type of viral nucleic acid, investigation of messenger RNA, virus purification in metagenomic approach, genome editing with the CRISPR/Cas system, and enzyme discovery. Knowledge of how nucleases can be both villains and allies is essential in the training of all working in the field of molecular biology.
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Objective: To observe the expression of long non-coding RNA myocardial infarction associated transcript (LncRNA-MIAT) in tumor necrosis factor-α (TNF-α) induced endothelial cells (ECs) inflammation in vitroand to study the impact of LncRNA-MIAT on inflammatory regulation. Methods: LncRNA-MIAT expression in ECs was induced by TNF-α at different time and concentration. Expressions of intercellular adhesion molecule-1 (ICAM-1) and LncRNA-MIAT in inflammatory ECs were examined by quantitative real time polymerase chain reaction (qRT-PCR) and Western blot analysis. Moreover, ECs was transfected by siRNAMIAT to observe the effect of LncRNA-MIAT knock-down on ICAM-1 expression. Results: LncRNA-MIAT expression showed the increasing trend by elevated time and concentration of TNF-stimulation. Compared with TNF-α stimulation at 0h, 6h and 12h, LncRNA-MIAT expressions were increased at 24h and 48h of TNF-αstimulation respectively, allP<0.05; compared with TNF-α concentration at 0ng/ml and 0.125ng/ml, LncRNA-MIAT expressions were elevated by TNF-α stimulation at 1.000ng/ml and 10.000ng/ml respectively, allP<0.05. With siRNAMIAT knock-down, TNF-α induced ICAM-1 protein expression was significantly reduced in ECs,P<0.05. Conclusion: LncRNA-MIAT might be involved in ECs inflammatory response and it may play a role to promote inflammation.
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In recent years, with the introduction of novel vectors capable of highly efficient transduction ,ribozymes and dexyribozymes have been become an ideal approach to anticancer therapy due to their high efficiency and lack of severe adverse effects. As many high efficiency targets have been found for cancer therapy, targeted therapy using ribozymes and deoxyribozymes may have multiple effects such as induction of tumor apoptosis and inhibition of tumor growth, metastasis, and angiogenesis.
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Objective To construct and express ribonuclease-resistant virus-like particles containing long chimeric RNA sequence by changing quantity and affinity of ms2 19mer stem-loop.Methods 1.7 kb maturase and coat protein gene of ms2 were amplified.We used a C-variant aptamer of the wild-type stem-loop where uridine-5 has been substituted by cytosine.The 1700 bp PCR-amplified DNA fragments was gel-purified and then digested with BamH Ⅰ and Hind Ⅲ and ligated to linearized pET28b vector to generate recombinant plasmid pET-MC-pac.The five target DNA sequences were spliced by overlapping extension(including 3 fragment of SARS-CoV,one fragment of HCV and one fragment of HSN1).PCR was carried out using primers contained Not Ⅰ site and then PCR were cloned into a pGEM(R) T-easy vector and then excised with the Not Ⅰ restriction enzymes from the resulting recombinant plasmid.simoutaniously the pET-MC-pac plasmid was digested with Not Ⅰ restriction enzymes,fragments were ligated into the linearized pET-MC-pac plasmid to produce a new donor plasmids pET-MC-3V.Simoutaniously,we constructed three recombinant expression vector for control,including N-P3V-pET-P、N-P3V-pET-Cand P-3V-pET-P.N-P3V-pET-P contained one wild type pacsite located behind ms2 sequence:N-P3V-pET-C contained one C-variant pacsite located behind ms2 sequence:P-3V-pET-P contained two wild type pacsites that one is located behind ms2 sequence,another is located between SARS-CoV 3 and HCV sequence.Then these four expression vector were transformed into E.coli strain BL21(DE3),respectively.The 3V Armored RNA was expressed and purified.A260 absorbance value of expression product was determined.Results Four expression plasmids were constructed successfully.The Armored RNA with 1891 bases was snccessfully expressed by the two-paesite expression system of pET-ms2-3V and P-3V-pET-P;for N-P3V-pET-P、N-P3V-pET-C,only 1 200 bp target sequence was packaged into virus-like particles.Expression efficiency of N-P3V-pET-P、N-P3V-pET-C、P-3V-pET-P and N-P3V-pET-P was 0.23,0.35,0.35,0.51 mg/ml,respectively.Armored RNA was shown to have the charaeterization of Ribonuclease-resistant and stable at 4℃,37℃ and 25℃ respectively.Conclusion The expression plasmids containing two pacsites were constructed successfully and prokaryotic expression system can be used as an expressing plasmid platform for preparation of Ribonuclease-resistant virus-like particles containing long chimeric RNA sequence.
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Objective To provide a express plasmid carrier platform that can be in common use for preparation of RNase-resistant RNA standards and controls. Methods A cDNA fragment of MS 2 phage RNA genome, which encodes coat protein and maturase protein, and expression vector pET28b DNA are ligated together with T 4 DNA ligase after digested with HindⅢ and EcoR Ⅰ restriction nucleases. Then, a new expression plasmid carrier pI NCCL is contructed。 The prokaryotic expression was carried out by transform pI NCCL into BL21-DE3 E。Coli. Results A new expression plasmid carrier pI NCCL is contructed successfully. RNase-resistant virus-like particles were obtained after prokaryotic expression of pI NCCL. Conclusion The expression plasmid carrier pI NCCL contructed and prokaryotic expression system can be used as a express plasmid carrier platform that can be in common use for preparation of RNase-resistant RNA standards and controls.
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Objective To study the effect of specific hammerhead ribozyme (Rz) to hepatitis C virus (HCV) in vitro. Methods Rz 1 Rz 2 were designed to cleavage at 5′-NCR nucleotide positions under 136～160 and 313～337, Rz 3 was designed to cleavage at C region nucleotide position under 373～388. As a control, cleavage deficient Rzm that have A→G point mutations in the catalytic loop of the hammerhead domain. 32P-labeled transcript of target HCV RNA was incubated with gel-purified Rz ( Rz 1, Rz 2, Rz 3 and Rzm ) respectively at different concentration based on specified condition and autoradiographed after denaturing gel-electrophoresis. Results Except Rzm, Rz 1 Rz 2 Rz 3 were active at 37 ℃ and more so at higher concentration, and more so with cleavage site nearly to the HCV initial code. Conclusions The HCV specific hammerhead ribozyme can be designed in vitro, further study about cleavage in vitro and in vivo will continue.
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Objective: To design the ribozymes to cleave human TIMP 1 mRNA, and embed them into U 6snRNA to make them stable. Methods: Ribozymes were designed according to the “hammerhead structure” described by Symons.Computer was used to analyze the possible cleavage sites. Results: Three ribozymes targeting the nt123, nt299 and nt353 on TIMP 1 mRNA were designed. Embedding ribozyme in U 6snRNA had little effect on its binding with the substrate. Conclusion: Computer assisted design is indispensable in studying ribozyme. Embedding ribozymes in U 6snRNA may be a good way to solve the problems existing in ribozyme study. [