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.

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.

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.

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).

Inhibitory effect of antisense vascular endothelial growth factor RNA on the profile of hepatocellular carcinoma cell line in vitro and in vivo

AIM： To evaluate the effect of antisense vascular endothelial growth factor （VEGF） RNA （PCMV-FGEV） transfection on the profile of hepatocellular carcinoma （HCC） SMMC-7721 cells in vitro and in vivo.METHODS： SMMC-7721 cells were transfected with PCMV-FGEV antisense, PCMV-VEGF sense and empty vector plasmid encapsulated by lipofectamine as antisense group, sense group and control group respectively. The positive cell clones were selected with G418. The stable transfection and expression of VEGF in the cells were determined by RT-PCR and immunohistochemistry. Cell proliferation was observed by Mnassay. FACS analysis was used to determine the effect of PCMV-FGEV transfection on cell apoptosis. The growth of transfected cells in vivo was also observed in nude mice.RESULTS： VEGF expression was reduced in SMMC-7721 transfected with PCMV-FGEV, which was confirmed by RT-PCR and immunohistochemistry. No effect of PCMV- FGEV transfection was found on cell proliferation and cell apoptosis of SMMC-7721 in vitro. The growth of cells transfected with PCMV-FGEV was slow in nude mice and accompanied with obvious apoptosis. The latent time of tumors in the antisense group was 25.0 ：l： 1.8 d, which was longer than that in sense and control groups （F= 19.455, P〈 0.01）. The average tumor weight in antisense group （0.96 g±0.28 g） was the smallest among the three groups （F= 21.501, P〈 0.01）.CONCLUSION： The expression of VEGF can be inhibited by antisense PCMV-FGEV. Antisense PCMV-FGEV has no effect on cell proliferation and apoptosis of SMMC-7721 in vitro but can inhibit tumor growth and induce cell apoptosis in vivo.

Effect of Antisense RNA Targeting Polo-like Kinase 1 on Cell Growth in A549 Lung Cancer Cells

In order to investigate the effect of Polo-like kinase-1 （Plk1） depletion on cell cycle progression and cell growth in lung cancer cells, a recombinant plasmid containing antisense RNA targeting Plk1 （pcDNA3-Plk1） was transfected into A549 cells by lipofectine. RT-PCR and Western-blot were used to detect the Plk1 gene expression. Cell proliferation was evaluated by direct cell counting and bromodeoxyuridine （BrdU） labeling. Cell cycle distribution and apoptosis were examined by flow cytometry, and the inhibition rate （IR） by vinorebline （NVB） was determined by MTF assay. The results showed that after transfection of pcDNA3-Plk1 into A549 cells, the expression levels of Plk1 mRNA and protein were greatly decreased. In pcDNA3-Plk1 transfected groups, abnormal morphological changes of cells and growth inhibition were observed, and the BrdU labeling index was significantly lower than in the control groups （P〈0.05）. Cells in pcDNA3-Plk1 transfected groups were arresed in G2/M phase and apoptosis was detectable 72 h post transfection. IR induced by vinorebline in pcDNA3-Plk1 transfected groups was significantly higher than in other groups. These data suggested that antisense RNA targeting Plk1 could suppress the Plk1 expression, and therefore, significantly inhibit cell proliferation and induce cell cycle arrest and apoptosis. Moreover, it sensitized lung cancer cells to chemotherapy.

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.

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.

EGFR antisense RNA blocks expression of the epidermal growth factor receptor and partially reverse the malignant phenotype of human breast cancer MDA-MB-231 cells

The effects of human EGFR to the malignant phenotype of human breast cancer cell line MDA-MB-231 were investigated experimentally. A retroviral vector containing a 5'1350bp fragment of the human EGFR cDNA in the antisense orientation was transfected into targeted cells by lipofectamine. The effects on cell proliferation, cell cycle and adherent ability to extracellular matrix (ECM) components were studied after the expression of antisense transcripts to EGFR 5'1350bp fragment in target cells. In vitro studies showed that the growth ability of the transfected cells was partialy inhibited in comparison to parental cells and to cells transfected with the plasmid containing the neomycin resistance gene only. It was found that EGF (10ng/ml) had an augmenation effect on the growth of transfected MDA-AS10 cells but not MDA-MB-231 cells.Flow cytometric analysis showed that the cell cycle of the transfected cells was abnormal with a decrease of cells in G2/M and S phases and an increase of cells in G1 phase,indicating a blockage in phase G1. Immunofluorescence of EGFR expression in transfectants stained with an antiEGFR antibody was decreased and their growth in soft agarose was also severely impaired. The transfected cells showed less adherence to laminin (LN) and fibronectin (FN). In short, EGFR antisense RNA decreases the expression of EGFR on MDA-MB-231 cells and partially reverses their malignant phenotype as well.Effects of antisense EGFR on human breast cancer MDA-MB-231 cells

Construction of the recombinant adenovirus vector carrying antisense multidrug resistance gene

BACKGROUND: Multidrug resistance proteins serve as transporters for chemical drugs in human malignancies. The objective of this study was to construct a homologous recombinant adenovirus carrying a reversal fragment of multidrug resistance gene 1 (mdr1) gene cDNA sequence. METHODS: The fragment of the mdr1 gene from the plasmid pHaMDRI-1 carrying the whole human mdr1 cDNA sequence was inserted reversely into the shuttle plasmid pAdTrack-CMV of adenoviral vector system AdEasy. The homologous recombination process was taken place in E. coli BJ5183 with the backbone plasmid pAdEasy-1. After packaging in 293 cells, recombinant adenoviral plasmid was generated. The recombinant adenoviral plasmid was identified by polymerase chain reaction (PCR), restriction endonucleases digest, DNA sequence analysis and fluorescence microscopic photograph, respectively. RESULTS: The recombinant adenovirus pAdEasy-GFPASmdr1 was successfully constructed and identified by PCR, restriction digest, and sequencing with strong green fluorescence expression in fluorescence microscopic photograph. CONCLUSIONS: The recombinant adenoviral mdr1 vector would introduce the antisense mdr1 gene into the human multidrug resistance hepatocellular cell fine effectively, which would provide an experimental basis to study the multidrug resistance in human hepatocellular carcinoma.

Effects of adenovirus-mediated human cyclooxygenase-2 antisense RNA on the growth of hepatocellular carcinoma

AIM: To investigate the relation between the expression of cyclooxygenase-2 (COX-2) and liver cancer, to construct the recombinant adenovirus encoding human COX-2antisense RNA, and to explore its effects on liver cancer cell proliferation.METHODS: We studied the expression of COX-2 in 34cases of hepatocellular carcinoma (HCC) and SMMC7402and SMMC7721 by immunohistochemical technique.Recombinant adenovirus Ad-AShcox-2 was constructed and transfected into human HCC cell lines SMMC7402and SMMC7721, and its effects on COX-2 expression, cell apoptosis and cell cycle were analyzed by flow cytometry.Cell proliferation was determined by colony-forming efficiency.RESULTS: We observed COX-2 expression in 82.4% of HCC and SMMC7402 cells, but no COX-2 expression in SMMC7721 cells. In addition, recombinant adenovirus encoding antisense COX-2 fragment Ad-AShcox-2 was obtained with the titer of 1.06× 1012 PFU/mL. Ad-AShcox-2 could reduce the expression of COX-2 and enhance the percentage of cells in G1/G0 phase in SMMC7402 cell line.The difference of apoptotic index between the Ad-AShcox2 group and control group was statistically significant(tcontrol group= 32.62 and tAd-LacZ= 10.93, P＜0.001) in SMMC7402 but not in SMMC7721. Similarly, colony-forming rates of SMMC7402 and SMMC7721 cell lines, after the transfer of Ad-AShcox-2, were (2.7±0.94)% and(33.6±4.24)%, respectively.CONCLUSION: Reduction in the expression of COX-2 can inhibit COX-2 expressing HCC cells.

Antisense RNA of Survivin Gene Inhibits the Proliferation of Leukemia Cells and Sensitizes Leukemia Cell Line to Taxol-induced Apoptosis

The effects of survivin antisense RNA on proliferation of leukemia cell line HL-60 and taxol-induced chemotherapy was explored. A cDNA fragment of survivin obtained by RT-PCR was inserted into a plamid vector named pcDNA3 in the reverse direction. The vector encoding antisense RNA of survivin was confirmed by restriction enzyme digestion and DNA sequencing. The recombinant plasmid was delivered into HL-60 cells by electroporation. Growth curves were plotted based on cell counting. Trypan blue dye exclusion assay and MTT assay were carried out after the cells were incubated with taxol. DNA gel electrophoresis and nuclear staining were performed for cell apoptosis assay. The correct construction of the recombinant plasmid has been identified by restriction enzyme digestion and DNA sequencing. A stable down-regulation has been achieved in HL-60 SVVas cells after G418 selection. Compared to HL-60 cells, the proliferation of HL-60 SVVas cells was significantly inhibited （P〈0.05）. Cytotoxicity assays indicated that IC50 of HL-60 SVVas for taxol was relatively lower than controls （P〈0.01）. Apoptosis assays revealed that taxol-induced apoptosis was detected in HL-60 SVVas cells incubated with 50 ng/ml taxol for 12 h, while in HL-60 cells incubated with 100 ng/ml taxol for 72 h. It was suggested that Survivin antisense RNA could inhibit the proliferation of HL-60 cells and enhance taxol-induced apoptosis in HL-60 cells, which may lay an experimental foundation for further research on gene therapy in leukemia.

REVERSION OF MALIGNANT PHENOTYPES OF HUMAN LUNG SQUAMOUS CARCINOMA CELLS BY ORNITHINE DECARBOXYLASE ANTISENSE RNA

Abnormally elevated activity of ornithine decarboxylase (ODC), and subsequent polyamine accumulation are intimately associated with the genesis.development and metastasis of cancer. In the present study, to control the growth of tumor cells, ODC antisense RNA was used to transfect human lung squamous carcinoma cell line LTEP-78. Compared with the parental cells, growth of the antisense transfected LTEP-78 cells arrested in G0/Gl phase and colony formation in soft agarose and tumorigenicity in nude mice were significantly reduced. Nucleic acid hybridization demonstrated that the transfectants expressed a high level of ODC antisense RNA and a significantly reduced level of endogenous ODC mRNA.The results suggest that the reversion of malignant phenotypes of human lung squamous carcinoma cells transfected with ODC antisense RNA is associated with the inhibition of polyamine biosynthesis.

ANTISENSE RNA OF SURVIVIN ENHANCES THE SENSITIVITY OF PANCREATIC CANCER CELL LINE PANC-1 TO DOXORUBICIN

Objective： This paper attempts to discuss the effects of surviving antisense RNA on doxorubicin-induced apoptosis in pancreatic cancer cell line PANC-1. Methods： A surviving antisense eukaryotic vector pcDNA3-SV Vas prepared in previous study was delivered into PANC-1 by electroperforation. Cell survival fraction and MTT assay were used to investigate the sensibility of transfected cells to doxorubicin. Apoptosis was detected by DNA gel electrophoresis. Results： We obtained two positive cell clone PANC-1/SVVas and PANC-1/neo cells, the growth of PANC-1/SV Vas cells was significantly reduced （P〈0.05）. By MTT assay, the IC50 to doxorubicin of PANC-1/SV Vas, PANC-1/neo and PANC-1 cells were （0.285±0.012） μmol/L, （1.528±0.317） μmol/L and （1.540±0.253） μmol/L respectively, the difference was significant by statistic analysis （P〈0.01）. Agarose gel electrophoresis of genomic DNA from PANC/SV Vas showed typical DNA ladder, but DNA from PANC-1/neo and PANC-1 did not. Conclusion： Survivin antisense RNA could enhance doxorubicin-induced apoptosis in pancreatic cancer cell line PANC-I. This may lay an experimental foundation for further research of gene therapy in pancreatic cancer.

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.

Construction and packaging of pseudotype retrovirus containing human N-ras cDNA antisense sequence and its biological effects on human hepatoma cells

N-ros is one of the transforming genes in human hepatic cancer cells. It has been found that N-ras was overexpressed at the mENA and protein level in hepatoma cells. In order to explore the biological roles of N-ras in human hepatic oaroinogenesis and the potential application in control of cancer cell growth, a pseudotype retrovirus containing antisense sequence of human N-ras was constructed and packaged. A recombinant rebrovirus vector containing antisense or sense sequences of N-ras oDNA was constructed by pZIP-NeoSV(X)1. The pseudotype virus was packaged and rescued by transfeotion and infection in PA317 and ψ2 helper cells. It has been demonstrated that the pseudotype retrovirus containing antisense N-ras sequence did inhibit the growth of human PLC/PEF/5 hepatoma cells accompanied with inhibition of p21 expression, while the retrovirus containing sense sequence had none. The pseudotype virus had no effect on human diploid fibroblasts.

Effect of the Antisense BcMF12 Driven by the BcA9 Promoter on Gene Silencing in Brassica campestris L.ssp.chinensis

The study analyzed the silencing of BcMF12 gene regulated by BcA9 promoter in the transgenic pakchoi and confirmed the effect of antisense BcMF12 gene on the pollen development. A conserved BcMF12 gene fragment was amplified from the cDNA of flower buds in pakchoi （Brassica campestris L. ssp. chinensis, syn. B. rapa L. ssp. chinensis） and was fused to the anther specific BcA9 promoter. The plant antisense expression vector was constructed and then introduced into pakchoi via Agrobacterium-mediated transformation. The transgenic plants were screened by antibiotics and molecular analysis. PCR and Southern blot revealed that the antisense BcMF12-GUS fusion gene regulated by BcA9 promoter was integrated into transgenic plants. Northern blot suggested that the expression of BcMF12 gene was down-regulated significantly. The pollen germination rate of transgenic plants with antisense BcMF12 gene decreased as compared with that of the control plants. The expression of the gene BcMF12 related to the pollen development was inhibited by the antisense BcMF12 driven by BcA9 promoter, which consequently affected the pollen development in pakchoi.

Construction of Antisense RNA Expression Plasmid for u-PAR and Its Transfection to Highly Invasive PC-3M Cell Subclones

To evaluate the specific inhibition of antisense u PAR on the u PAR expressions in highly invasive cell subclones and to determine its blocking function in the invasion by those cells, a cDNA fragment of u PAR obtained by RT PCR was inserted into a plasmid vector named pcDNA3 in antisense orientation. Then the antisense u PAR recombinant was transfected into highly invasive cell subclones. The u PAR expression in neo resistant cells was examined by RT PCR and immunohistochemical assay. Compared to the control cells, the content of mRNA and protein of u PAR in transfected cells decreased sharply, and the rate of inhibition was 53 % and 73 %, respectively, indicating that an antisense u PAR might have played a specific inhibitory role in its expression in the cells, which may provide a good cell model for making further investigation of the inhibitory effects of the antisense u PAR on invasion in highly invasive cell subclones of human prostate carcinoma.