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.

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.

Effects of long non-coding RNA Opa-interacting protein 5 antisense RNA 1 on colon cancer cell resistance to oxaliplatin and its regulation of micro RNA-137

BACKGROUND The incidence of colon cancer(CC)is currently high,and is mainly treated with chemotherapy.Oxaliplatin(L-OHP)is a commonly used drug in chemotherapy;however,long-term use can induce drug resistance and seriously affect the prognosis of patients.Therefore,this study investigated the mechanism of Opainteracting protein 5 antisense RNA 1(OIP5-AS1)on L-OHP resistance by determining the expression of OIP5-AS1 and micro RNA-137(miR-137)in CC cells and the effects on L-OHP resistance,with the goal of identifying new targets for the treatment of CC.AIM To study the effects of long non-coding RNA OIP5-AS1 on L-OHP resistance in CC cell lines and its regulation of miR-137.METHODS A total of 114 CC patients admitted to China-Japan Union Hospital of Jilin University were enrolled,and the expression of miR-137 and OIP5-AS1 in tumor tissues and corresponding normal tumor-adjacent tissues was determined.The influence of OIP5-AS1 and miR-137 on the biological behavior of CC cells was evaluated.Resistance to L-OHP was induced in CC cells,and their activity was determined and evaluated using cell counting kit-8.Flow cytometry was used to analyze the apoptosis rate,Western blot to determine the levels of apoptosisrelated proteins,and dual luciferase reporter assay combined with RNA-binding protein immunoprecipitation to analyze the relationship between OIP5-AS1 and miR-137.RESULTS OIP5-AS1 was up-regulated in CC tissues and cells,while miR-137 was downregulated in CC tissues and cells.OIP5-AS1 was inversely correlated with miR-137(P<0.001).Silencing OIP5-AS1 expression significantly hindered the proliferation,invasion and migration abilities of CC cells and markedly increased the apoptosis rate.Up-regulation of miR-137 expression also suppressed these abilities in CC cells and increased the apoptosis rate.Moreover,silencing OIP5-AS1 and up-regulating miR-137 expression significantly intensified growth inhibition of drug-resistant CC cells and improved the sensitivity of CC cells to LOHP.OIP5-AS1 targetedly inhibited miR-137 expression,and silencing OIP5-AS1 reversed the resistance of CC cells to L-OHP by promoting the expression of miR-137.CONCLUSION Highly expressed in CC,OIP5-AS1 can affect the biological behavior of CC cells,and can also regulate the resistance of CC cells to L-OHP by mediating miR-137 expression.

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).

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.

A Novel Vector for Abundant Expression of Antisense RNA, Triplex forming RNA and Ribozyme in vivo

For abundant expression of antisense RNA, triplex forming RNA and Ribozyme in vivo, a novel vector pBSKneo rU6’ was constructed by PCR cloning. This vector contains the intact human snRNA U6 gene expression unit, yet replacing the 61 nt sequence in the middle of U6 snRNA coding region with three restriction enzyme sites. Hela nuclear extract in vitro transcription experiments demonstrated that this vector can effectively express U6 mutant RNA. Containing neo r at the same time, stably transfected pBSKneo rU6’ can be selected easily.

Characteristics of Antisense Non-coding RNA in the INK4 Locus and Its Roles in Disease

Abstract With the development of genome-wide sequencing technology, 195 types of functional long non-coding RNAs （lncRNAs） have so far been found, and their cellular roles are gradually being revealed. Now lncRNAs have become a hotspot in the life science. These small molecules exist in almost all higher eukaryotes, and have very important regulatory roles in these organisms. This review briefly summarizes recent progress in researches on antisense non-coding RNA in the INK4 locus.

Experimental research for specific down-regulated expression of p53 gene by individual antisense RNA in vitro

Objective： To investigate the specific blockage effect of individual antisense RNA on mutant p53 gene in vitro. Methods： The single strand antisense transcription system containing mt-p53 exon 8 sequence （pGEM3zf（＋/-）p53exon8） was constructed. The ligation of antisense RNAwith mt-p53 gene was confirmed by in situ hybridization; MDA-MB-231 human breast cancer cells were transfected with ASp53exon8＇RNA cotionic liposome-mediated. Expression of mt-p53 protein was examined by immunocytochemical staining and Western blot. Cell proliferation was evaluated by MTT assay; Cell cycle distribution was determined by flow cytometry （FCM）; Apoptosis was observed by TUNEL. Results： In transfected MDA-MB-231 cells, hybridization signals were observed in cytoplasm. ASp53exon8＇RNA transfection induced inhibition of cell proliferation, G2/M phase arrest and increasing apoptotic rates. In addition, expression of p53 protein was down-regulated. Conclusion： pGEM3zf（＋/-）p53exon8 was well constructed and ASp53exon8＇RNA can block mt-p53 gene expression specifically and then inhibit MDA-MB-231 cell proliferation in vitro, which may serve as therapeutic means for human malignancy.

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.

非编码RNA在肺纤维化过程中的调控作用

背景:截至目前,肺纤维化的发病机制不明,治疗手段或者药物有限。大量研究发现,非编码RNA在肺纤维化的发生发展过程中发挥重要作用。目的:综述近年来非编码RNA在肺纤维化发病机制中的调控作用,深入了解肺纤维化的发病机制。方法:由第一作者应用计算机检索2000-2024年出版的文献,以“非编码RNA,微小RNA,长链非编码RNA,环状RNA,肺纤维化,综述”等为中文检索词检索中国知网、万方和维普数据库;以“ncRNA,miRNA,lncRNA,circRNA,Pulmonary fibrosis,review”等为英文检索词检索PubMed数据库,按照入选标准最终共纳入65篇文献进行综述。结果与结论:肺纤维化作为一种慢性且通常致命的肺部疾病,不仅可能自发产生,也可能是其他疾病的继发结果,主要特点是肺部间质中细胞外基质的异常增生和大量积累。非编码RNA是指由基因组转录出来的RNA分子,这些RNA分子并不涉及蛋白质的编码过程,凭借其调节能力已成为各种生物学现象中的一线分子参与者之一。非编码RNA在肺纤维化的发病机制中扮演着关键角色,微小RNA(miRNA)、长链非编码RNA(lncRNA)和环状RNA(circRNA)可以通过影响基因表达、转录后修饰以及细胞间的信号传导,参与到肺纤维化的发展过程中,为后续探索肺纤维化疾病的具体分子发生机制提供了新方向,为研发肺纤维化疾病的新靶向药物提供了理论依据及新思路。

Inhibitory effect of IGF-Ⅱ antisense RNA on malignant phenotype of hepatocellular carcinoma

INIRODUCTIONAccording to the therapeutic effect and strategy ofantisense RNA for hepatoccllular carcinoma(HCC),we have specifically synthesized partialcDNA of human insulin-like growth factor Ⅱ(IGF-Ⅱ)and constructed IGF-Ⅱ cDNA antisenseeukaryotic expression vector.The constructedvector was introduced into hepatoma cell lineSMMC-7721 to block the intrinsic IGF-Ⅱexpression.The biological behavior changes ofhepatoma cells were observed.All these

Non-coding RNAs in acute ischemic stroke:from brain to periphery

Acute ischemic stroke is a clinical emergency and a condition with high morbidity,mortality,and disability.Accurate predictive,diagnostic,and prognostic biomarkers and effective therapeutic targets for acute ischemic stroke remain undetermined.With innovations in high-throughput gene sequencing analysis,many aberrantly expressed non-coding RNAs(ncRNAs)in the brain and peripheral blood after acute ischemic stroke have been found in clinical samples and experimental models.Differentially expressed ncRNAs in the post-stroke brain were demonstrated to play vital roles in pathological processes,leading to neuroprotection or deterioration,thus ncRNAs can serve as therapeutic targets in acute ischemic stroke.Moreover,distinctly expressed ncRNAs in the peripheral blood can be used as biomarkers for acute ischemic stroke prediction,diagnosis,and prognosis.In particular,ncRNAs in peripheral immune cells were recently shown to be involved in the peripheral and brain immune response after acute ischemic stroke.In this review,we consolidate the latest progress of research into the roles of ncRNAs(microRNAs,long ncRNAs,and circular RNAs)in the pathological processes of acute ischemic stroke–induced brain damage,as well as the potential of these ncRNAs to act as biomarkers for acute ischemic stroke prediction,diagnosis,and prognosis.Findings from this review will provide novel ideas for the clinical application of ncRNAs in acute ischemic stroke.

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.

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.

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.

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 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.

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.

Retrovirus-mediated antisense RNA to bcl-2 alter the biological behavior of stomach carcinoma MGC-803 cell lines

AIM To demonstrate whether bcl 2 gene can affect or alter biological behavior of a stomach carcinoma cell line MGC 803. METHODS To transduct a retrovirus containing bcl 2 antisense RNA to MGC 803 cells and then to analyse the Bcl 2 protein expression in the cells by Western blotting. To observe the morphology alteration, detect the G1 phase arrest by FCM, inhibition of proliferation by MTT method and tumorigenicity in nude mice. RESULTS The MGC anti bcl 2 cells shows contact inhibition, morphological alteration, from round or near round to shuttle like, decelerated growth rate, G1 phase arrest and weakened tumorigenicity in nude mice unlike the control (MGC neo cells). CONCLUSION Antisense RNA to bcl 2, not only can induce apoptosis, but also reverse the biological behavior of MGC 803 cells. This would be a potential application to the gene therapy for stomach cancers.

THE EFFECT OF ANTISENSE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) RNA ON THE PROLIFERATION OF HUMAN GLIOMA CELLS AND INDUCTION OF CELL APOPTOSIS

Objective: To study the effect of antisense EGFR RNA on the growth of human glioma cells in vitro and evaluate the feasibility of targeting EGFR gene for gene therapy of gliomas. Methods: Southern and Northern blot analysis, in situ hybridization and immunohistochemical staining were used to detect the integration and expression of antisense EGFR constructs. MTT assay and the average number of AgNOR for evaluation of cell proliferation, and the TUNEL method and ultrastructural change for observation of cell apoptosis. Results: Exogenous antisense EGFR cDNA was integrated into the genome of glioma cells and highly expressed, which resulted in a dramatic decrease of endogenous EGFR mRNA and GEPR protein levels. Clones with high expression of the antisense construct showed a lower proliferation activity and the induction of apoptosis in vitro. Conclusion: This study suggests that EGFR plays an important role in the genesis of gliomas; it may be used as a target for antisense gene therapy of gliomas.