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Antitumor effects of mutant endostatin are enhanced by Bcl-2 Antisense oligonucleotides in UM-UC-3 bladder cancer cell line

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Chinese Medical Journal 2013年第15期126卷 2834-2839页

作者： REN Ming-hua YU Jing-song SONG Er-lin ZHANG Cheng MA Li JIAO Zhi-xing ZHAO Wei-ming SHAN Yu-juan NI Shao-bin Harbin Med Univ Affiliated Hosp 1 Dept Urinary Surg Harbin 150001 Heilongjiang Peoples R China Chinese Peoples Liberat Army Hosp 211 Dept Urinary Surg Harbin 150080 Heilongjiang Peoples R China Harbin Inst Technol Food Sci & Engn Sch Harbin 150090 Heilongjiang Peoples R China

Background Endostatin is a potent inhibitor of tumor angiogenesis. In the preliminary studies, we developed a mutant endostatin containing Arg-Gly-Asp-Arg-Gly-Asp （RGDRGD） sequences. In this study, we compared the antitumor effects of mutant endostatin and Bcl-2 Antisense oligonucleotides both in combination and individually. Methods The artificially synthesized Bcl-2 ASODN （Antisense oligonucleotides） included a translation-initiation site and was transfected into the bladder cancer cells by Lipofectamine. Cell growth was investigated by the tumor cell growth chart, MTT assay, caspase-3 activity detection assay, AO/EB fluorescein stain, and the annexin V-FITC apoptosis detection assay. In the in vivo study, UM-UC-3 bladder cancer cells were subcutaneously implanted into nude mice and the growth of tumor was examined. The ultrastructure of the tumor tissues in the treated and control groups were observed. Results The cell growth chart showed that the cell population of the treated combination group decreased by 52.04% compared to the control group. The inhibition rate of the treated combination group was （79.66＋6.79）%, whereas those of the individual ASODN and ES groups were （53.39＋3.22）% and （50.22＋5.46）% respectively. In the caspase-3 activity detection using AO/EB fluorescein stain and annexin V-FITC apoptosis detection assay, the co-inhibitory effect was higher than the individual inhibitory effects （P〈0.05）. There were significant differences in the inhibition of the solid tumor growth in the in vivo study. Conclusions Our findings indicated that Bcl-2 Antisense oligonucleotides enhance the antitumor effects of mutant endostatin both in vitro and in vivo. We noted the synergistic effects of Bcl-2 Antisense oligonucleotides combined with mutant endostatin.

关键词： endostatin Bcl-2 Antisense oligonucleotides Arg-Gly-Asp

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Inhibitory effect of 2＇-o-methoxyethyl-modified Antisense oligonucleotides targeting vascular endothelial growth factor A on SKOV3 human ovarian cancer cells

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Chinese Medical Journal 2011年第10期124卷 1573-1575页

作者： FU Yi-bing WEN Ze-qing ZHAO Xing-bo YAN Lei ZHANG Chun-hua WANG Fei Department of Obstetrics and Gynaecology Provincial Hospital Affiliated to Shandong University Jinan Shandong 250021 China

Background Ovarian cancers are often at an advanced stage at diagnosis because early detection is difficult. The poor prognosis of ovarian cancers highlights the crucial need to develop better therapeutic agents and strategies. The objective of this study was to investigate the inhibitory effects of a new modified Antisense oligonucleotides targeting vascular endothelial growth factor A （VEGF-A） in SKOV3 ovarian cancer cells.Methods Antisense oligonucleotides targeting VEGF-A was designed, synthesized and transfected into SKOV3ovarian cancer cells. Western blotting and real-time RT-PCR were used to analyze the inhibitory effects of Antisense oligonucleotides on VEGF-A protein and mRNA expression. Transwell matrix assay was used to detect cell migration inhibition.Results The Antisense oligonucleotides targeting VEGF-A significantly decreased VEGF-A protein and mRNA expression and inhibited cell migration in SKOV3 ovarian cancer cells.Conclusions This new modified Antisense oligonucleotides targeting VEGF-A can decrease VEGF-A expression and inhibit cell migration in SKOV3 ovarian cancer cells. This new oligonucleotides may be a promising therapeutic agent for ovarian cancers

关键词： vascular endothelial growth factor A ovarian cancer Antisense oligonucleotides transfection

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Curcumin synergistically augments bcr/abl phosphorothioate Antisense oligonucleotides to inhibit growth of chronic myelogenous leukemia cells

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Acta Pharmacologica Sinica 2007年第1期28卷 105-110页

作者： Kun-zhong ZHANG~2 Jian-hua XU~(2,4) Xiu-wang HUANG~2 Li-xian WU~2 Yu SU~2 Yuan-zhong CHEN~32 Institute of Clinical Pharmacology,School of Pharmacy,Fujian Medical University,Fujian 350004,China 3 Fujian Institute of Hematology Fujian 350004,China 2Institute of Clinical Pharmacology School of Pharmacy Fujian Medical University Fujian 350004 China 2Institute of Clinical Pharmacology School of Pharmacy Fujian Medical University Fujian 350004 China 2Institute of Clinical Pharmacology School of Pharmacy Fujian Medical University Fujian 350004 China 2Institute of Clinical Pharmacology School of Pharmacy Fujian Medical University Fujian 350004 China 2Institute of Clinical Pharmacology School of Pharmacy Fujian Medical University Fujian 350004 China 3Fujian Institute ofHematology Fujian 350004 China

Aim:To investigate the growth inhibition effect of the combination of bcr/ablphosphorothioate Antisense oligonucleotides(PS-ASODN)and curcumin(cur),and the possible mechanisms of cur on the chronic myelogenous leukemia cell lineK562.Methods:The K562 cell line was used as a P210bcr/abl-positive cell model invitro and was exposed to different concentrations of PS-ASODN(0-20 μmol/L),cur(0-20 μmol/L),or a combination of both.Growth inhibition and apoptosis ofK562 cells were assessed by MTT assay and AO/EB fluorescent staining,respec-tively.The expression levels of P210bcr/abl,NF-κ3 and heat shock protein 90(Hsp90)were assessed by Western blot.Results:Exposure to cur(5-20 μmol/L)and PS-ASODN(5-20 μmol/L)resulted in a synergistic inhibitory effect on cell growth.Growth inhibition was associated with the inhibition of the proliferation and in-duction of apoptosis.Western blot analysis showed that the drugs synergisti-cally downregulated the level ofP210bcr/abland NF-κB.Cur downregulated Hsp90,whereas no synergism was observed when cur was combined with PS-ASODN.Conclusion:PS-ASODN and cur exhibited a synergistic inhibitory effect on thecell growth of K562.The synergistic growth inhibition was mediated throughdifferent mechanisms that involved the inhibition of P210bcr/abl.

关键词： combination drug therapy curcumin abl genes Antisense oligonucleotides chronic myelogenous leukemia bcr-abl fusion proteins

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Antisense oligonucleotides-induced local blockade of T-bet expression leads to airway inflammation in rats

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Acta Pharmacologica Sinica 2006年第5期27卷 561-567页

作者： Gang WANG~2 Chun-tao LIU~(3,4) Zeng-li WANG~3 Li-li JIANG~5 Cun-liang YAN~6 Feng-min LUO~3 ~2Department of Integrated Traditional Chinese and Western Medicine.West China Hospital.Sichuan University,Chengdu 610041,China.~3Department of Respiratory Medicine,West China Hospital,Sichuan University.Chengdu 610041.China ~5Department of Pathology,West China Hospital.Sichuan University,Chengdu 610041,China:~6Department of Laboratory Medicine.Shenzhen Hospital,Peking University.Shenzhen 518036,China DepartmentoflntegratedTraditionalChineseandWesternMedicine WestChinaHospitalSichuanUniversityChengdu610041China DepartmentofRespiratoryMedicine WestChinaHospitalSichuanUniversityChengdu610041China

Aim:To explore whether local blockade of T-box expressed in T cells (T-bet)expression in the lungs could lead to airway inflammation.Methods:Twenty-fourrats were randomly divided into 4 groups:saline group,ovalbumin (OVA)-sensi-tized group,nonsense group,and the antisense group.The OVA-sensitized ratswere sensitized and challenged with OVA,and the rats in the nonsense andantisense groups were subjected to an aerosol delivery of the nonsense andAntisense oligonucleotides (AS-ODN) of T-bet (0.1%,w/v).The levels of inter-feron-γ(IFN-γ),interleukin-4(IL-4),and IL-5 in the bronchoalveolar lavage fluid(BALF) were detected by ELISA,and the mRNA and the protein expression ofT-bet and GATA-3 genes were examined by in situ hybridization and Western blotanalysis,respectively.Results:The expression of T-bet mRNA and protein in thelungs of the rats in the antisense group were inhibited effectively.The lungs ofthe rats in the antisense and OVA-sensitized groups showed eosinophil and lym-phocyte inflammatory infiltration,and eosinophilia located predominantly aroundthe airways.The number of GATA-3 mRNA-positive cells and the level of GATA-3protein in the lungs of the rats in the antisense and the OVA-sensitized groupssignificantly increased.The level of IL-4 and IL-5 in the BALF in the antisenseand OVA-sensitized groups were elevated,but the level of IFN-γ decreasedmarkedly.Conclusion:antisense ODN-induced local blockade of T-bet expres-sion leads to airway inflammation with a selective alteration in patterns of cytokineexpression and recruitment of eosinophil cells similar to that in the OVA-sensitizedanimals.

关键词： airway inflammation Antisense oligonucleotides GATA3 transcription factor interferon type Ⅱ interleukin-4 interleukin-5 Tbox expressed in T cells

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Inhibition of PCNA Antisense oligonucleotides Mediated by Liposome on mRNA Expression and Proliferation of h-RPE Cells

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2006年第4期26卷 392-395页

作者：陈建斌项楠徐莉莉曾水清 Department of Ophthalmology Tongji HospitalTongji Medial CollegeHuazhong University of Science and TechnologyWuhan 430030China

Summary： The proliferating cell nuclear antigen （PCNA） gene expression was blocked and retinal pigment epithelium （RPE） proliferation was inhibited by using Antisense oligonucleotides （AS-ODN） mediated by liposome, to find a new genetic therapy of proliferative vitreoretinopathy （PVR）. RPE cells cultured in vitro were transfected with synthetic fluorescence labled AS-ODN mediated by liposome-Lipofectamine, and the intracellular distribution and persistence time of AS-ODN were dynamically observed. AS-ODN （0.07, 0.28 and 1.12 μ mol/L and sense oligonucleotides （S-ODN with the same concentrations as AS-ODN） mediated by liposome were delivered to the RPE cells cultured in vitro, and CPM values were measured by ^3H-TdR incorporation assay and analyzed statistically by variance by comparison with blank control group. Expression ofPCNA mRNA in RPE cells was detected by in situ hybridization after the treatment of different concentrations of PCNA AS-ODN and S-ODN, and the average optic density （AOD） was measured by image analysis system and was subjected to q-test and correlation analysis with CPM. Our results showed that AS-ODN mediated by liposome could quickly aggregate in cellular plasma and nuclei in 30 min and 6 h, and stayed for as long as 6 days. AS-ODN （0.28 and 1.12 μmol/L） markedly suppressed proliferation of RPE cells in a dose-dependent manner with the difference being statistically significant （P〈0.05 and P〈0.01, repectively） as compared with blank control group. AOD was well correlated with CPM （r=0.975）. It is concluded that liposome could increase transfection efficiency of AS-ODN in RPE cells, and AS-ODN could sequence-specifically suppress PCNA mRNA expression and proliferation of human RPE cells.

关键词： human retinal pigment epithelium cell Antisense oligonucleotides proliferating cell nuclear antigen

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Effect of Dexamethasone and Aquaporin-1 Antisense oligonucleotides on the Aquaporin-1 Expression in Cultured Human Trabecular Meshwork Cells

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Journal of Huazhong University of Science and Technology(Medical Sciences) 2006年第1期26卷 137-140页

作者：彭洁张虹李涛李中国吴云霞 Department of Ophthalmology Tongji Hospital Tongji Medical College Huazhong University of Science and Technology Wuhan 430030 China Institute of Integrated Traditional Chinese Medicine and Western Medicine Tongji Medical College Huazhong University of Science and Technology Wuhan 430030 China

The changes in the expression of aquaporin-1 （AQP1） mRNA and protein in cultured human trabecular meshwork （HTM） cells treated with dexamethasone and transfected with Antisense oligonucleotides （AS-ODN） were studied, and the implication of AQP1 regulation in corticosteroid-glaucoma and the possibility of AS-ODN inhibiting the AQP1 expression were evaluated. The cultured HTM cells in vitro were treated with different concentrations of dexamethasone and transfected with oligonucleotides for 5 days respectively. Then, total RNA and protein of HTM cells were extracted. The changes of AQP1 mRNA and protein were demonstrated qualitatively and quantitatively by RT-PCR and Western blot. Band intensities were detected by imaging analysis. There was a parallel relationship between the results of RT-PCR and those of Western blot. The expression levels of AQP1 mRNA and protein in dexamethasone-treated groups were increased initially and decreased later as dexamethasone concentration was stepped up. In the 0.04 μg/mL and 0.4 μg/mL groups, the levels of AQP1 were higher than in control group （0 μg/mL）. In the 4 μg/ mL and 40 μg/mL groups, the AQP1 expression levels were lower than in control group. AS-ODN could down-regulate the expression of AQP1 mRNA and protein in a dose-dependent manner. At 5 μg/mL, down-regulation efficiency reached the maximum. There was no statistically significant difference in the expression of AQP1 mRNA and protein between all sense oligonucleotides groups and control group. It was suggested that dexamethasone may induce the changes of the AQP1 expression in HTM cells to be involved in the occurrence of corticosteroid-glaucoma. AS-ODN can down-regulate the AQP1 expression in HTM cells to some extent.

关键词： trabecular meshwork cells aquaporin-1 dexamethasone Antisense oligonucleotides

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Depressive Effect of the Antisense oligonucleotides of C-myc and PCNA on the Proliferation of VSMC

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South China Journal of Cardiology 2007年第4期8卷 193-197页

作者： Qingxian Li Yanfu Wang Yuhua Liao Huiling Zhang Yanying Jiang Laboratory of Cardiovascular Immunology Institute of Cardiology Union Hospital Tong Ji Medical College Hua Zhong University of Science ＆ Technology Wuhan 430022 China Department of Cardiology The Affiliated Hospital Jining Medical College Jining 272029 China Department of Ophthalmology The Affiliated Hospital fining Medical College fining 272029 China

Objectives To study the depressive effect of the antisense oligonuceotides （ASODN） of c-myc and proliferating cell nuclear antigen （PCNA） on the proliferation of VSMC. Methods Taking the VSMC obtained from rat aorta tho- racalis cultured 4 - 8 generation as research object. The objects were divided into three groups to carry out control study： control group, PCNA ASODN group and c-myc ASODN group. The ASODNs＇ working concentration all were 1 ： 50. The depressive effect of ASODN on VSMC proliferation was investigated by cell counting, MTT and ^3H-TdR incorporation assay; PCNA and c-myc expression were detected by immunohistochemical method after transferring PCNA and c-myc ASODN into VSMC. Results ① PCNA and c-myc ASODN could inhibit the proliferation of VSMC significantly, compared with control group （ P 〈 0. 05）. ②Transferring PCNA and c-myc ASODN into VSMC obtained successfully ; the corresponding gene was inhibited obviously ; compared with control group （ P 〈 0. 05 ）. Conclusions PCNA and c-myc might play a considerable role in the VSMC proliferation process. The corresponding gene could be depressed successfully after transferring PCNA and c-myc ASODN into VSMC, and then the proliferation of VSMC was slowed down. This study presented a beneficial proposal and theoretical fundament for atherosclerotic treatment.

关键词： vascular smooth muscle cell Antisense oligonucleotides PCNA c-myc

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Knockdown of polypyrimidine tract binding protein facilitates motor function recovery after spinal cord injury

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Neural Regeneration Research 2023年第2期18卷 396-403页

作者： Ri-Yun Yang Rui Chai Jing-Ying Pan Jing-Yin Bao Pan-Hui Xia Yan-Kai Wang Ying Chen Yi Li Jian Wu Gang Chen Department of Histology and Embryology Medical School of Nantong UniversityNantongJiangsu ProvinceChina Key Laboratory of Neuroregeneration of Jiangsu Province and the Ministry of Education Co-innovation Center of NeuroregenerationNantong UniversityNantongJiangsu ProvinceChina Center for Basic Medical Research Medical School of Nantong UniversityNantongJiangsu ProvinceChina Department of Anesthesiology Affiliated Hospital of Nantong UniversityNantongJiangsu ProvinceChina

After spinal cord injury(SCI),a fibroblast-and microglia-mediated fibrotic scar is formed in the lesion core,and a glial scar is formed around the fibrotic scar as a res ult of the activation and proliferation of astrocytes.Simultaneously,a large number of neuro ns are lost in the injured area.Regulating the dense glial scar and re plenishing neurons in the injured area are essential for SCI repair.Polypyrimidine tra ct binding protein(PTB),known as an RNA-binding protein,plays a key role in neurogenesis.Here,we utilized short hairpin RNAs(shRNAs)and Antisense oligonucleotides(ASOs)to knock down PTB expression.We found that reactive spinal astrocytes from mice were directly reprogrammed into motoneuron-like cells by PTB downregulation in vitro.In a mouse model of compressioninduced SCI,adeno-associated viral shRNA-mediated PTB knockdown replenished motoneuron-like cells around the injured area.Basso Mouse Scale scores and forced swim,inclined plate,cold allodynia,and hot plate tests showed that PTB knockdown promoted motor function recovery in mice but did not improve sensory perception after SCI.Furthermore,ASO-mediated PTB knockdown improved motor function resto ration by not only replenishing motoneuron-like cells around the injured area but also by modestly reducing the density of the glial scar without disrupting its overall structure.Together,these findings suggest that PTB knockdown may be a promising therapeutic strategy to promote motor function recovery during spinal cord repair.

关键词： Antisense oligonucleotides astrocytes glial scar motoneuron-like cells motor function neurogenesis neuron-like cells polypyrimidine tract binding protein short hairpin RNAs spinal cord repair

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Lipid nanomaterials-based RNA therapy and cancer treatment

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Acta Pharmaceutica Sinica B 2023年第3期13卷 903-915页

作者： Xingcai Zhang Luo Hai Yibo Gao Guocan Yu Yingli Sun School of Engineering and Applied Sciences Harvard UniversityCambridge 02138MAUSA Central Laboratory National Cancer Center/National Clinical Research Center for Cancer/Cancer Hospital&Shenzhen HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeShenzhen 518116China Department of Thoracic Surgery National Cancer Center/National Clinical Research Center for Cancer/Cancer HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100021China State Key Laboratory of Molecular Oncology National Cancer Center/National Clinical Research Center for Cancer/Cancer HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100021China Laboratory of Translational Medicine National Cancer Center/National Clinical Research Center for Cancer/Cancer HospitalChinese Academy of Medical Sciences and Peking Union Medical CollegeBeijing 100021China Key Laboratory of Bioorganic Phosphorus Chemistry&Chemical Biology Department of ChemistryTsinghua UniversityBeijing 100084China University of Chinese Academy of Sciences Beijing 100101China CAS Key Laboratory of Genome Sciences and Information Beijing Institute of GenomicsChinese Academy of Sciences/China National Center for BioinformationBeijing 100101China

We summarize the most important advances in RNA delivery and nanomedicine.We describe lipid nanoparticle-based RNA therapeutics and the impacts on the development of novel drugs.The fundamental properties of the key RNA members are described.We introduced recent advances in the nanoparticles to deliver RNA to defined targets,with a focus on lipid nanoparticles(LNPs).We review recent advances in biomedical therapy based on RNA drug delivery and state-of-the-art RNA application platforms,including the treatment of different types of cancer.This review presents an overview of current LNPs based RNA therapies in cancer treatment and provides deep insight into the development of future nanomedicines sophisticatedly combining the unparalleled functions of RNA therapeutics and nanotechnology.

关键词： Antisense oligonucleotides siRNA miRNA mRNA Cancer treatment Nanomedicine RNA therapy Lipid nanoparticles

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Viral miRNA-mediated activation of hyaluronan production as a drug target against COVID-19

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Acta Pharmaceutica Sinica B 2022年第7期12卷 3195-3197页

作者： Shuai Yang Lu Chen Ying Tong Wenqiang Yu Laboratory of RNA Epigenetics Institutes of Biomedical Sciences&Shanghai Public Health Clinical Center&Department of General SurgeryHuashan HospitalCancer Metastasis InstituteShanghai Medical CollegeFudan UniversityShanghai 200040China Shanghai Key Laboratory of Medical Epigenetics Shanghai 200032China

The global coronavirus disease 2019(COVID-19)pandemic has caused more than 6.1 million deaths until March 24,2022,as reported by the World Health Organization(WHO).Recently,breakthrough infections have appeared in individuals fully vaccinated against SARS-Co V-2^(1),which could be attributed to the rapid mutation of the RNA virus2.Currently,following the Delta variant,the Omicron variant of SARS-Co V-2 is increasingly becoming the dominant epidemic strain in the world.

关键词： COVID-19 Human Identical Sequences Hyaluronan Hymecromone Antisense oligonucleotides

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