Antihepatoma effect of alpha-fetoprotein antisense phosphorothioate oligodeoxyribonucleotides in vitro and in mice

AIM: To evaluate antihepatoma effect of antisense phosphorothioate oligodeoxyribonucleotides (S-ODNs) targeted to alpha-fetoprotein (AFP) genes in vitro and in nude mice. METHODS: AFP gene expression was examined by immunocytochemical method or enzyme-linked immunosorbent assay. Effect of S-ODNs on SMMC-7721 human hepatoma cell growth in vitro was determined using microculture tetrazolium assay. In vitro antitumor activities of S-ODNs were monitored by measuring tumor weight differences in treated and control mice bearing SMMC-7721 xenografts. Induction of cell apoptosis was evaluated by fluorescence-activated cell sorter (FACS) analysis. RESULTS: Antisense S-ODN treatment led to reduced AFP gene expression. Specific antisense S-ODNs, but not control S-ODNs, inhibited the growth of hepatoma cells in vitro. In vitro, only antisense S-ODNs exhibited obvious antitumor activities. FACS analysis revealed that the growth inhibition by antisense S-ODNs was associated with their cell apoptosis induction. CONCLUSION: Antisense S-ODNs targeted to AFP genes inhibit the growth of human hepatoma cells and solid hepatoma, which is related to their cell apoptosis induction.

Long Non-coding RNA PCED1B Antisense RNA 1 Promotes Cell Proliferation and Invasion in Hepatocellular Carcinoma by Regulating the MicroRNA-34a/CD44 Axis

Objective This study aimed to examine the role of long non-coding RNA PCED1B antisense RNA 1(PCED1B-AS1)in the development of hepatocellular carcinoma(HCC).Methods A total of 62 pairs of HCC tissues and adjacent non-tumor tissues were obtained from 62 HCC patients.The interactions of PCED1B-AS1 and microRNA-34a(miR-34a)were detected by dual luciferase activity assay and RNA pull-down assay.The RNA expression levels of PCED1B-AS1,miR-34a and CD44 were detected by RT-qPCR,and the protein expression level of CD44 was determined by Western blotting.The cell proliferation was detected by cell proliferation assay,and the cell invasion and migration by transwell invasion assay.The HCC tumor growth after PCED1B-AS1 was downregulated was determined by in vivo animal study.Results PCED1B-AS1 was highly expressed in HCC tissues,which was associated with poor survival of HCC patients.Furthermore,PCED1B-AS1 interacted with miR-34a in HCC cells,but they did not regulate the expression of each other.Additionally,PCED1B-AS1 increased the expression level of CD44,which was targeted by miR-34a.The cell proliferation and invasion assay revealed that miR-34a inhibited the proliferation and invasion of HCC in vitro,while CD44 exhibited the opposite effects.Furthermore,PCED1B-AS1 suppressed the role of miR-34a.Moreover,the knockdown of PCED1B-AS1 repressed the HCC tumor growth in nude mice in vivo.Conclusion PCED1B-AS1 may play an oncogenic role by regulating the miR-34a/CD44 axis in HCC.

Alu antisense RNA ameliorates methylglyoxal-induced human lens epithelial cell apoptosis by enhancing antioxidant defense

AIM:To determine whether an antisense RNA corresponding to the human Alu transposable element(Aluas RNA)can protect human lens epithelial cells(HLECs)from methylglyoxal-induced apoptosis.METHODS:Cell counting kit-8(CCK-8)and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assays were used to assess HLEC viability.HLEC viability/death was detected using a Calcein-AM/PI double staining kit;the annexin V-FITC method was used to detect HLEC apoptosis.The cytosolic reactive oxygen species(ROS)levels in HLECs were determined using a reactive species assay kit.The levels of malondialdehyde(MDA)and the antioxidant activities of total-superoxide dismutase(T-SOD)and glutathione peroxidase(GSH-Px)were assessed in HLECs using their respective kits.RT-q PCR and Western blotting were used to measure m RNA and protein expression levels of the genes.RESULTS:Aluas RNA rescued methylglyoxal-induced apoptosis in HLECs and ameliorated both the methylglyoxalinduced decrease in Bcl-2 m RNA and the methylglyoxalinduced increase in Bax m RNA.In addition,Aluas RNA inhibited the methylglyoxal-induced increase in Alu sense RNA expression.Aluas RNA inhibited the production of ROS induced by methylglyoxal,restored T-SOD and GSHPx activity,and moderated the increase in MDA content after treatment with methylglyoxal.Aluas RNA significantly restored the methylglyoxal-induced down-regulation of Nrf2 gene and antioxidant defense genes,including glutathione peroxidase,heme oxygenase 1,γ-glutamylcysteine synthetase and quinone oxidoreductase 1.Aluas RNA ameliorated methylglyoxal-induced increases of the m RNA and protein expression of Keap1 that is the negative regulator of Nrf2.CONCLUSION:Aluas RNA reduces apoptosis induced by methylglyoxal by enhancing antioxidant defense.

Anti-aging Effects of Alu Antisense RNA on Human Fibroblast Senescence Through the MEK-ERK Pathway Mediated by KIF15

Objective:To investigate whether human short interspersed nuclear element antisense RNA(Alu antisense RNA;Alu asRNA)could delay human fibroblast senescence and explore the underlying mechanisms.Methods:We transfected Alu asRNA into senescent human fibroblasts and used cell counting kit-8(CCK-8),reactive oxygen species(ROS),and senescence-associated beta-galactosidase(SA-β-gal)staining methods to analyze the anti-aging effects of Alu asRNA on the fibroblasts.We also used an RNA-sequencing(RNA-seq)method to investigate the Alu asRNA-specific mechanisms of anti-aging.We examined the effects of KIF15 on the anti-aging role induced by Alu asRNA.We also investigated the mechanisms underlying a KIF15-induced proliferation of senescent human fibroblasts.Results:The CCK-8,ROS and SA-β-gal results showed that Alu asRNA could delay fibroblast aging.RNA-seq showed 183 differentially expressed genes(DEGs)in Alu asRNA transfected fibroblasts compared with fibroblasts transfected with the calcium phosphate transfection(CPT)reagent.The KEGG analysis showed that the cell cycle pathway was significantly enriched in the DEGs in fibroblasts transfected with Alu asRNA compared with fibroblasts transfected with the CPT reagent.Notably,Alu asRNA promoted the KIF15 expression and activated the MEK-ERK signaling pathway.Conclusion:Our results suggest that Alu asRNA could promote senescent fibroblast proliferation via activation of the KIF15-mediated MEK-ERK signaling pathway.

Targeting LncRNA LLNLR-299G3.1 with antisense oligonucleotide inhibits malignancy of esophageal squamous cell carcinoma cells in vitro and in vivo

Accumulating evidence has indicated that long non-coding RNAs(lncRNAs)play critical roles in the development and progression of cancers,including esophageal squamous cell carcinoma(ESCC).However,the mechanisms of lncRNAs in ESCC are still incompletely understood and therapeutic attempts for in vivo targeting cancer-associated lncRNA remain a challenge.By RNA-sequencing analysis,we identified that LLNLR-299G3.1 was a novel ESCC-associated lncRNA.LLNLR-299G3.1 was up-regulated in ESCC tissues and cells and promoted ESCC cell proliferation and invasion.Silencing of LLNLR-299G3.1 with ASO(antisense oligonucleotide)resulted in opposite effects.Mechanistically,LLNLR-299G3.1 bound to cancerassociated RNA binding proteins and regulated the expression of cancer-related genes,including OSM,TNFRSF4,HRH3,and SSTR3.ChIRP-seq(chromatin isolation by RNA purification and sequencing)revealed that these genes contained enriched chromatin binding sites for LLNLR-299G3.1.Rescue experiments confirmed that the effects of LLNLR-299G3.1 on ESCC cell proliferation were dependent on interaction with HRH3 and TNFRSF4.Therapeutically,intravenous delivery of placental chondroitin sulfate A binding peptide-coated nanoparticles containing antisense oligonucleotide(pICSA-BP-ANPs)strongly inhibited ESCC tumor growth and significantly improved animal survival in vivo.Overall,our results suggest that LLNLR-299G3.1 promotes ESCC malignancy through regulating gene-chromatin interactions and targeting ESCC by pICSA-BP-ANPs may be an effective strategy for the treatment of lncRNA-associated ESCC.

Investigation on small molecule-aptamer dissociation equilibria based on antisense displacement probe

Food safety is a major issue to public health and have attracted global attention.Fast,sensitive,and reliable detection methods for food hazardous substances is highly desirable.Aptamers which can bind to the target molecules with high affinity and specificity represent an attractive tool for the recognition of food hazardous substances,which play an important role in the development and application of new food safety detection technology.But current assays for characterizing small molecule-aptamer binding are limited by either the mass sensitivity or the size differentiation ability.Herein,we proposed a comprehensive method for assessing the dissociation equilibria of small molecule-aptamer,which is immobilized-free under ambient conditions.The design employs the Le Chatelier’s principle and could be used to effectively measure small molecule-aptamer interactions.ATP binding aptamer and anti-aflatoxin B1 aptamer were used as the model system to determine their affinity,in which their dissociation equilibria measurements are in excellent close to their previous work.Due to the simplicity and sensitivity of this new method,we believe that it could be recommended as an effective tool for characterizing small molecule-aptamer interactions and promote the further application of small molecular aptamer in food safety.

EXPRESSION OF TOMATO ANTISENSE ACC SYNTHASE GENE IN TRANSGENIC TOBACCO AND ITS ROLE IN SHOOT FORMATION

An ACC synthase cDNA isolated from tomato (Lycopersicum esculentum Mill.) fruit was constructed in antisense orientation under the transcriptional control of CaMV 35S promoter and then introduced into tobacco (Nicotiana tabacum L.) . PCR amplification demonstrated the integration of this antisense gene in tobacco genomes. Northern hybridization and reverse transcription-PCR analyses indicated the expression of this heterologous antisense gene in the transgenic tobacco tissues, which caused a decrease in the ethylene production, particularly when shoot regeneration exhibited. The ability of shoot regeneration of the transgenic plant during the culture process was enhanced remarkably as compared with that of the control. These results indicate at the molecular level that ethylene may play a regulatory role in shoot formation.

Cloning of cDNA Encoding CCoAOMT from Populus tomentosa and Down-regulation of Lignin Content in Transgenic Plant Expressing Antisense Gene

cDNA encoding caffeoyl CoA O-methyltransferase (CCoAOMT) from Chinese white poplar ( Populus tomentosa Carr.) was cloned by RT-PCR and sequenced. Northern analysis displayed that the CCoAOMT was expressed specifically in the developing secondary xylem and its expression was coincident with lignification. The antisense CCoAOMT cDNA was transformed into P. tremula x P. alba mediated by Agrobacterium tumefaciens ( Smith et Townsend) Conn. Transgenic plants were identified with PCR, PCR-Southern and Southern analysis. Lignin content in 5- to 6-month-old transgenic plants was measured. One of the transgenic lines had significant reduction of 17.9% in Klason lignin content as compared with that of untransformed poplar. The results demonstrate that antisense repression of CCoAOMT is an efficient way to reduce lignin content for improving pulping property in engineered trees.

Inhibition of HSP70 Gene Expression by Modified Antisense and Its Effects on Embryonic Sensitivity to Heat Shock

Experiments were performed to evaluate the efficiency of inhibition of HSP70 gene expressionby antisense oligonucleotides complementary to the mRNA of HSP70 and to test the effects ofinhibition of HSP70 gene expression on subsequent embryonic sensitivity to heat shock. Theresults showed that transfection of pre-implantation embryos at 4-cell stage with 5 Mantisense oligo had no effect on in vitro blastocyst development. However, transfection with10 to 40 M antisense oligo had reduced in vitro blastocyst development to 15, 10% and 0; Forthe embryos which exposed to 40 M As arrested at the 16-cell stage, there was no blastocystformation within the heat shock groups. In contrast, transfection had no effect on embryonicsensitivity to heat shock, above 25% of embryos developed to blastocyst stage in controlgroups.

HOX transcript antisense intergenic RNA represses E-cadherin expression by binding to EZH2 in gastric cancer

AIM To clarify the mechanisms of HOX transcript antisense intergenic RNA(HOTAIR) in gastric cancer(GC) migration and invasion.METHODS Quantitative real-time polymerase chain reaction(qp CR) was used to detect the expression level of HOTAIR in GC tissues. The correlation of its expression with clinicopathological features was analyzed. Area under receiver operating characteristic curve(AUCROC) was constructed to evaluate the diagnostic value of HOTAIR. Wound-healing assay and Transwell assay were performed to detect the biological effects of HOTAIR in GC cells. qp CR,western blot and immunohistochemistry were used to evaluate the m RNA and protein expression of E-cadherin. RNAbinding protein immunoprecipitation was used for the analysis of EZH2 interactions with HOTAIR. Chromatin immunoprecipitation assay was performed to investigate direct interactions between EZH2 and E-cadherin.RESULTS The expression of HOTAIR was up-regulated in GC tumorous tissues compared with the para-tumorous tissues(p < 0.001). Its over-expression was correlated with tumor-node-metastasis(TNM) stage(p = 0.024),tumor invasion(p = 0.018),lymph node metastasis(p = 0.023),and poor prognosis(p < 0.001). Multivariate Cox regression analysis confirmed expression of HOTAIR as an independent predictor of overall survival(p = 0.033),together with TNM stage(p = 0.002) and lymph node metastasis(p = 0.002). The AUCROC was up to 0.709(95%CI: 0.623-0.785,p < 0.001). Knockdown of HOTAIR by si RNA in GC cells suppressed the migration and invasion of GC cells. Significantly negative correlation between HOTAIR and E-cadherin was found in GC tissues and cell lines,and HOTAIR contributed to the regulation of E-cadherin through binding to EZH2 with the E-cadherin promoter. CONCLUSION HOTAIR may play a pivotal role in tumor cell migration and invasion. It can be used as a potential diagnostic and prognostic biomarker for GC.

Reduction of tumorigenicity of SMMC-7721 hepatoma cells by vascular endothelial growth factor antisense gene therapy

AIM: To test the hypothesis to block VEGF expression of SMMC-7721 hepatoma cells may inhibit tumor growth using the rat hepatoma model. METHODS: Amplify the 200 VEGF cDNA fragment and insert it into human U6 gene cassette in the reverse orientation transcribing small antisense RNA which could specifically interact with VEGF165, and VEGF121 mRNA. Construct the retroviral vector containing this antisense VEGF U6 cassette and package the replication-deficient recombinant retrovirus. SMMC-7721 cells were transduced with these virus and positive clones were selected with G418. PCR and Southern blot analysis were performed to determine if U6 cassette integrated into the genomic DNA of positive clone. Transfected tumor cells were evaluated for RNA expression by ribonuclease protection assays. The VEGF protein in the supernatant of parental tumor cells and genetically modified tumor cells was determined with ELISA. In vitro and in vivo growth properties of antisense VEGF cell clone in nude mice were analyzed. RESULTS: Restriction enzyme digestion and PCR sequencing verified that the antisense VEGF RNA retroviral vector was successfully constructed.After G418 selection, resistant SMMC-7721 cell clone was picked up. PCR and Southern blot analysis suggested that U6 cassette was integrated into the cell genomic DNA. Stable SMMC-7721 cell clone transduced with U6 antisense RNA cassette could express 200 bp small antisense VEGF RNA and secrete reduced levels of VEGF in culture condition. Production of VEGF by antisense transgene-expressing cells was 65+/-10 ng/L per 10(6) cells, 42045 ng/L per 10(6) cells in sense group and 485+/-30 ng/L per 10(6) cells in the negative control group, (P【 0.05). The antisense-VEGF cell clone appeared phenotypically indistinguishable from SMMC-7721 cells and SMMC-7721 cells transfected sense VEGF. The growth rate of the antisense-VEGF cell clone was the same as the control cells. When S.C. was implanted into nude mice, growth of antisense-VEGF cell lines was greatly inhibited compared with control cells. CONCLUSION: Expression of antisense VEGF RNA in SMMC-7721 cells could decrease the tumorigenicity, and antisense-VEGF gene therapy may be an adjuvant treatment for hepatoma.

Inhibition of Telomerase with hTERT Antisense Increases Susceptibility of Leukemic Cells to CDDP-induced Apoptosis

Objective: To investigated the e?ect of inhibition of telomerase with hTERT antisense on leukemic cells (HL-60 and K562) to CDDP-induced apoptosis. Methods: Antisense phosphorothioate oligodeoxynucleotide (AS PS-ODN) was synthesized and puri?ed. Telomerase activity was detected by Telomerase PCR ELASA kit and cell apoptosis was observed by morphological method and determined by ?owcytometry. Results: AS PS-ODN could signi?cantly inhibit telomerase activity by down regulat- ing the hTERT expression, and increase the susceptibility of leukemic cells to CDDP-induced apoptosis. Conclusion: Inhibition of telomerase with hTERT antisense can increases the susceptibility of leukemic cells to CDDP-induced apoptosis.

Inhibitory Effect of Tissue Transglutaminase (tTG) Antisense Oligodeoxynucleotides on tTG Expression in Cultured Bovine Trabecular Meshwork Cells

To study the effect of tTG fully phosphorothioated antisense oligodeoxynucleotides （tTG-ASDON） on tTG expression in cultured bovine trabecular meshwork cells （BTMCs） in vitro and explore a new treatment alternative for primary open angle glaucoma （POAG）, the ASDON1 and ASDON2 complementary to the protein codogram region of tTG were designed, synthesized and phosphorothioated according to the secondary structure of tTG. The ASDON1 and ASDON2 were embedded in Lipofectamine and transfected into BTMCs. The untreated group served as negative controls. The expression of tTG in the mRNA and protein level were measured by semi-quantitative RT-PCR and immunohistochemical technique-Supervision method respectively. Our results showed that both the mRNA and the protein of tTG with tTG-ASDON1 and tTCr-ASDON2 were significantly decreased as compared with that of the controls （P〈0.05）. On the other hand, no significant difference was found between the ASDON1 group and the ASDON2 group. It is concluded that the expression of tTG mRNA and protein in cultured BTMC are down-regulated by tTG- ASDON. As a result, tTG-ASDON may be used for the treatment of POAG through the inhibitory effect on the expression of tTG.

Inhibition of human telomerase in MKN-45 cell line by antisense hTR expression vector induces cell apoptosis and growth arrest

AIM: To investigate the effects of antisense human telomerase RNA (hTR)on the biologic behavior of human gastric cancer cell line: MKN-45 by gene transfection and its potential role in the gene therapy of gastric cancer. METHODS: The hTR cDNA fragment was cloned from MKN-45 through RT-PCR and subcloned into eukaryotic expression vector (pEF6/V5-His-TOPO) in cis-direction or trans-direction by DNA recombinant methods. The constructed sense, antisense and empty vectors were transfected into MKN-45 cell lines separately by lipofectin-mediated DNA transfection technology. After drug selection, the expression of antisense hTR gene in stable transfectants and normal MKN-45 cells was detected by RT-PCR, the telomerase activity by TRAP, the apoptotic features by PI and Hoechst 33258 staining, the cell cycle distribution by flow cytometry and the population doubling time by cell counting. Comparison among the stable transfectants and normal MKN-45 cells was made. RESULTS: The sense, antisense hTR eukaryotic expression vectors and empty vector were successfully constructed and proved to be the same as original design by restriction endonuclease analysis and sequencing. Then, they were successfully transfected into MKN-45 cell lines separately with lipofectin. The expression of antisense hTR gene was only detected in MKN-45 cells stably transfected with antisense hTR vector (named as MKN-45-ahTR) but not in the control cells. In MKN-45-ahTR, the telomerase activity was inhibited by 75%, the apoptotic rate was increased to 25.3%, the percentage of cells in the G0/G1 phase was increased to 65%, the proliferation index was decreased to 35% and the population doubling time was prolonged to 35.3 hours. However, the telomerase activity, the apoptotic rate, the distribution of cell cycle, the proliferation index and the population doubling time were not different among the control cells. CONCLUSION: Antisense hTR can significantly inhibit telomerase activity and proliferation of MKN-45 cells and induce cell apoptosis. Antisense gene therapy based on telomerase inhibition can be a potential therapeutic approach to the treatment of gastric cancer.

Construction and Characterization of an Antisense RNA Eukaryotic Expression Vector for Survivin

Objective: To inhibit specifically survivin expression and block its function in leukemia cells, an antisense RNA expression plasmid for survivin was constructed and transfected into a leukemia cell line.Methods: A cDNA fragment of survivin obtained by RT-PCR was inserted into a plasmid vector named pcDNA3 in the reverse direction. Antisense RNA of survivin was confirmed by restriction enzyme digestion and DNA sequencing. The recombinant plasmid was transfected into the cell line HL-60 by electroporation. The effect of survivin antisense RNA on survivin mRNA expression in transfected cells was examined by RT-PCR.Results: The correct construction of the recombinant plasmid has been shown by restriction enzyme digestion and DNA sequencing. As compared to controls, the level of survivin mRNA expression in transfected cells decreased significantly.Conclusion: An antisense RNA vector for survivin has been successfully constructed and may be useful as a specific inhibitor in leukemia cells. Thus, antisense therapy on the basis of survivin can be further explored in leukemia. Key words leukemia - survivin - antisense RNA This project was supported by a grant from National Key Basis Research Program of China (No. CB 513109) and the National Natural Sciences Foundation of China (No. 39970693).

Effects of Ghrelin Antisense Inhibition on VEGF and Its Receptor Flt-1 mRNA Expression

[ Objective] This study was to investigate the effect of VEGF and its receptor Fit-1 mRNA expression in Mongolia sheep umbilical vein endothelial cells by ghrelin antisense inhibition. [ Method] Experiments were divided into 4 groups： group Ⅰ （blank control group） ; group Ⅱ （liposome group） ; group Ⅲ （SCON group： 20 μmol/L sense oligonucleotide） ; group Ⅳ （ASCON： 20 μmol/L antisense oligonucleotide）. VEGF and its receptor Fit-1 mRNA expression changes were detected by using real-time fluorescence quantitative detection after 24, 36 and 48 h. [ Result] The expression of VEGF mRNA in group Ⅰ, group Ⅱ were insignificantly different at higher expression levels, and did not change significantly with the time; the expression of VEGF mRNA in group Ⅲ assumed a slight decrease, but there were no significant differences between group I and group Ⅱ （P 〉0.05）, the expression of VEGF mRNA in group Ⅳ（antisense oligonucleotide group ） decreased significantly （P 〈 0.05） ; the expression of VEGF receptor FLT-1 mRNA was similar to that of VEGF. [ Conclusion] Antisense inhibition ghrelin has a downward effect to the expression of VEGF and its receptor Fit-1 the mRNA.

Apoptosis and Lipolysis of White Adipocytes Induced by Neuropeptide Y- Y5 Receptor Antisense Oligodeoxynucleotides

Objective: To investigate the influence of central administration ofneuropeptide Y-Y5 receptor antisense oligodeoxynucleotides (ODNs) on body weight, fat pads of SDrats, and the effects of white adipocytes lipolysis and apoptosis. Methods: Y5 receptor antisense,sense, mismatched ODNs or vehicle was intracerebroventricularly (i. c. v.) injected. Averageadipocyte area was calculated. DNA ladders were measured to evaluate adipocyte apoptosis, and RT-PCRwas used to analyse the expression of Bcl-2 and Bax gene. Results: Central administration of Y5antisense ODNs significantly decreased body weight, and average adipocyte area. DNA fragmentationwas present after electrophoresis at epididymal adipose tissue. The expression of Bcl-2 gene wasdownregulated, while the expression of Bax upregulated. Conclusion: Lipolysis and adipocyteapoptosis may be important mechanisms far 75 antisense therapy.

Inhibiting effect of antisense oligonucleotides phosphorthioate on gene expression of TIMP-1 in rat liver fibrosis

AIM: To observe the inhibition of antisense oligonucleotides (asON) phosphorthioate to the tissue inhibitors metalloproteinase-1 (TIMP-1) gene and protein expression in the liver tissue of immunologically induced hepatic fibrosis rats. The possibility of reversing hepatic fibrosis through gene therapy was observed. METHODS: Human serum albumin (HSA) was used to attack rats, as hepatic fibrosis model, in which asONs were used to block the gene and protein expressing TIMP-1. According to the analysis of modulator, structure protein, coding series of TIMP-1 genome, we designed four different asONs. These asONs were injected into the hepatic fibrosis models through coccygeal vein. The results was observed by RT-PCR for measuring TIMP-1 mRNA expression, immunohistochemistry and in situ hybridization for collagen I, II, special staining of collagen fiber, and electron microscopic examination. RESULTS: Hepatic fibrosis could last within 363 days in our modified model. The expressing level of TIMP-1 was high during hepatic fibrosis process. It has been proved by the immunohistochemical and the electron microscopic examination that the asON phosphorthioate of TIMP-1 could exactly express in vivo. The effect of colchicine was demonstrated to inhibit the expressing level of mRNA and the content of collagen I, III in the liver of experimental hepatic fibrosis rats. However, the electron microscopy research and the pathologic grading of hepatic fibrosis showed that there was no significant difference between the treatment group and the model group (P】 0.05). CONCLUSION: The experimental rat model of hepatic fibrosis is one of the preferable models to estimate the curative effect of anti-hepatic fibrosis drugs. The asON phosphorthioate of TIMP-1 could block the gene and protein expression of TIMP-1 in the liver of experimental hepatic fibrosis rats at the mRNA level. It is possible to reverse hepatic fibrosis, and it is expected to study a new drug of antihepatic fibrosis on the genetic level. Colchicine has very limited therapeutic effect on hepatic fibrosis, furthermore, its toxicity and side effects are obvious.

Effect of Bcl-2 Antisense Oligodeoxynucleotides on Human Lung Carcinoma Transplanted Subcutaneously in Nude Mice

Objective： To investigate the inhibitory effects of the Bcl-2 antisense oligodeoxynucleotides （ASODN） on tumor formation and growth of human lung carcinoma transplanted subcutaneously in nude mice. Methods： Human NCI-H460 cells treated with Bcl-2 ASODN or nonesense oligodeoxynucleotide （NSODN） and untreated NCI-H460 cells were respectively implanted subcutaneously into nude mice. When the diameters of tumor were above 0.5 cm after untreated NCI-H460 cells injection, the mice bearing tumor were randomly divided into three groups： saline control group, Bcl-2 ASODN group, NSODN group. ODN was directly injected into the tumor body for 3 weeks. The weight and volume of subcutaneous tumors were measured, and the morphology of tumor cells was observed. Results： The tumorigenic ability of the treated NCI-H460 cells by Bcl-2 ASODN was reduced. The mean time at which tumor can be detected was prolonged up to 12.6 days （P〈0.01）. The maximum tumor growth inhibitory rate was 87.5%. In therapeutic efficacy, growth of tumor was significantly inhibited in Bcl-2 ASODN group as compared with that in NSODN group, saline-treated group （P〈0.01）. The NSODN control was ineffective. In comparison with NSODN-treated, saline-treated mice, those treated with Bcl-2 ASODN showed a significant decrease in median weight of subcutaneous tumors （P〈0.01）. The growth inhibitory rate was 71.0% in ASODN group. Conclusion： Bcl-2 ASODN could inhibit tumor formation and tumor growth in nude mice.

Selection of Effective Bcl-2 Antisense Oligodeoxynucleotides with Computer Software and Experimental Assay

Objective： To explore and investigate the selection of effective antisense oligodeoxynuleotides with the help of computer and RNAstructure folding software. Methods： Bcl-2 gene was used as the target gene and five antisense oligodeoxynuleotides were designed to be bound to Bcl-2 mRNA optimal secondary structure regions that were predicted free from intramolecular fold or instability of free energy. The five antisense oligodeoxynucleotides were studied with experimental assay of leukemia cells, including cell grow assay with tropan blue exclusion, expression of Bcl-2 protein detected with immunochemistry and flowcytometry, Bcl-2 mRNA content detected with RT-PCR technique, as well as apoptosis observed and determined with morphonological method, electrophoresis and flowcytometry. Results： The results showed that two of the five antisense oligodeoxynucleotides were effective antisense oligodeoxynucleotides, which were able to inhibit cell growth in leukemia, to decrease the level of Bcl-2 mRNA and protein, to induce apoptosis of leukemia cells significantly. Conclusion： The computational prediction of antisense efficacy is faster than other methods and more efficient, which can potentially speed the development of sequences for both research and clinical applications.