Objective: To explore the probability of vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotides as a developing new therapeutic strategy for glioma. Methods: VEGF protein expression was detected by...Objective: To explore the probability of vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotides as a developing new therapeutic strategy for glioma. Methods: VEGF protein expression was detected by S-P immunohistochemical technique. Tumor cell apoptosis was observed by TUNEL method. Results: Compared with control, VEGF protein expression was inhibited by antisense oligodeoxynucleotides in vitro. And the inhibitory effects increased with the increasing concentration. VEGF positive rate was 82.10% in control group, while in 2.5, 5, 10 mmol/L AODN groups, they were 70.00%, 57.85%, 53.20% respectively. No inhibition effect was found in the cell lines treated with missense and sense oligodeoxynucleotides. In vivo, antisense oligodeoxy-nucleotides therapy also inhibited VEGF protein expression and induced the increase of apoptotic tumor cells. However, it has no effect on tumor cell proliferation. Conclusion: It is hopeful that VEGF antisense oligodeoxynucleotides may be a new gene therapy method to glioma through its antiangiogenesis effect by inhibition of VEGF.展开更多
To study the effect of c myc antisense oligodeoxynucleotides (ODNs) on proliferation of pulmonary vascular pericytes (PC) induced by hypoxia, cell culture, dot hybridization using probe of digoxigenin 11 dUTP labe...To study the effect of c myc antisense oligodeoxynucleotides (ODNs) on proliferation of pulmonary vascular pericytes (PC) induced by hypoxia, cell culture, dot hybridization using probe of digoxigenin 11 dUTP labeled cDNA, 3H thymidine incorporation, immunocytochemical technique and image analysis methods were used to observe the effect of c myc antisense ODNs on expression of c myc gene and proliferating cell nuclear antigen (PCNA), and 3H thymidine incorporation of PC induced by hypoxia. The results showed that hypoxia could significantly enhance the expression of c myc and PCNA ( P <0.01), and elevate 3H thymidine incorporation of PC ( P <0.01), but antisense ODNs could significantly inhibit the expression of c myc and PCNA ( P <0.05), and 3H thymidine incorporation of PC ( P <0.01). It was suggested that hypoxia could promote the proliferation of PC by up regulating the expression of c myc gene, but c myc antisense ODNs could inhibit hypoxia induced proliferation of PC by downregulating the expression of c myc gene.展开更多
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 alternativ...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.展开更多
Objective and Methods: Excessive accumulation of collagen typeⅠ and type Ⅲ causes the formation of keloids and hypertrophic scars. To understand the mechanism by which antisense oligodeoxynucleotide (Oligo) acts on ...Objective and Methods: Excessive accumulation of collagen typeⅠ and type Ⅲ causes the formation of keloids and hypertrophic scars. To understand the mechanism by which antisense oligodeoxynucleotide (Oligo) acts on in vitro transcrption α1 (I) collagen gene, isotopes (α-32pGTP) was incorporated into 2 SP6 in vitro transcription systems. Results and Conclu- sion: Oligo 2 (at the transcription start region) could effectively inhibit in vitro transcription of pGEM3-Col13 and the control (random oligodeoxynucleotides) showed no inhibition. However, oligo 1 (at the transcription start region) obviously inhibited the in vitro transcription of pGEM3-Col14, while Oligo 2, which targeted at the down stream region (about 200 bp) of the promoter showed no significant inhibition effect.展开更多
Objective: Aurora A kinase representing a family of evolutionarily conserved mitotic serine/threonine kinases has been found elevated in human lung adenocarcinoma cell line A549. It is suggested that the overexpressio...Objective: Aurora A kinase representing a family of evolutionarily conserved mitotic serine/threonine kinases has been found elevated in human lung adenocarcinoma cell line A549. It is suggested that the overexpression of Aurora A con- tributes to the carcinogenesis, chromosomal instability (CIN), and de-differentiation of lung cancers. To address its possibility as a therapeutic target for lung cancer, we employed the antisense oligodeoxynucleotide (ASODN) technique to inhibit Aurora A expression and investigate its effects on tumor growth and cell cycle of A549, as well as the chemosensitivity to paclitaxel. Methods: Aurora A ASODN was synthesized and transfected into A549 cells by lipofectAMINE 2000. Aurora A mRNA and protein expression were examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respec- tively. Cell cycle distribution was observed by flow cytometer. MTT assay was used to evaluate cell inhibition ratio before and after transfection. Results: The proliferation of the A549 cells was inhibited by Aurora A ASODN dose and time dependently. It was also observed that the IC50 of A549 cells after 48 hours’ treatment of ASODN was about 300 nmol/L and under such circumstances, the Aurora A mRNA and protein expression significantly decreased (P < 0.05), along with the induction of ac- cumulation of cells in S phase and the G2-M transition. Furthermore, cell inhibition ratio of the combination of Aurora A ASODN and paclitaxel was higher significantly than paclitaxel (P < 0.05) or Aurora A ASODN alone (P < 0.05). Conclusion: Inhibition of Aurora A expression can result in the suppression of cell growth and chemosensitizing activity to paclitaxel in human lung cancer cell line A549.展开更多
Objective To evaluate the effect of human telomerase reverse transcriptase(hTERT) gene antisense oligodeoxynucleotide (ASON) on telomerase activity in K562 cells.Methods Telomerase activity was detemined by polymerase...Objective To evaluate the effect of human telomerase reverse transcriptase(hTERT) gene antisense oligodeoxynucleotide (ASON) on telomerase activity in K562 cells.Methods Telomerase activity was detemined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA) in K562 cells treated with ASODN and hTERTmRNA expression was detected by reverse transcriptase polymerase chain reaction (RT-PCR). Results The hTERTmRNA level was decreased,and telomerase activity was significantly inhibited when the K562 cells were treated with ASODN for 48 h. Conclusion It is suggested that hTETR ASODN might specifically inhibit telomrase activity of K562 cells at translation level,and it is further proved that hTERT gene has significant correlation with telopmerase activity.展开更多
To investigate the potential utility of nuclease--resistant oligodeoxynucleotides (S-ODN) as anew class of antiviral agents. Methods: Two antisense phosphorothioate analogues (20--iner) complementary to thesequences o...To investigate the potential utility of nuclease--resistant oligodeoxynucleotides (S-ODN) as anew class of antiviral agents. Methods: Two antisense phosphorothioate analogues (20--iner) complementary to thesequences of the first AUG codon and 5’ terminus of NSS of JEV SA--14 genome have been synthesized and their effects on CPE, viral antigen expression and virus plaque formation were tested in vitro. Results: The resultsshowed that 1. 0 pmol/L of S-ODN greatly deferred the onset of CPE in cultured BHKZI cells for at least 48 h.Addition of 5. 0 pmol/L or more S--ODN to culture medium fluid, 2 h prior to 100TCID,,virus inoculum, notablysuppressed viral antigen expression in the cells by making it lower than the limit of EIA detection in 48 h. The inhibition lasted more than 96 h. Viral plaque assay results demonstrated that S-ODN were most effect’ive within 18h with plaque inhibition rate over 90% by 5. 0 pmol/L S--ODNI. The inhibitory activity soon faded in 24 h. In addition, high concentrations (up to 80. 0 pmol/L) of S--ODN did not show any obvious cytotoxicity in 6 d by usingtrypan blue dye exclusion method. Conclusion: The specific synthetic S--ODN transitorily inhibited JEV replicationin BHK--ZI cells with characteristics of specificity and S--ODN dose--dependence.展开更多
Purpose: To investiagate the effect of antisense oligonucleotides (ODN) to Nogo-A mRNA expression in oligodendrocytes and to establish the base for further research of repair of optic nerve injury.Method: (1)Oligodend...Purpose: To investiagate the effect of antisense oligonucleotides (ODN) to Nogo-A mRNA expression in oligodendrocytes and to establish the base for further research of repair of optic nerve injury.Method: (1)Oligodendrocytes were obtained by inoculating the optic nerve of newborn (2 days)rats and were identified with galactocerebroside(GC) antibody immunocytochemical stain. (2) In order to observe the effects of antisense ODN on cultured cells,we set up five groups, including the groups of three concentration of antisense Nogo-A ODN (2μM、5μM、l0μM),a group with the random sequence added to the medium and the control group. Reverse transcription-polymerase chain reaction(RT-PCR) was adopted to study the effects of ODN on the expression of Nogo-A in oligodendrocytes.Results: (1)Three days after inoculation, a few of round or fusiform shape cells migrated from optic nerve tissue; About 11 days later, the coverlips were completely covered by the cells;The cells identified with GC antibody immunocytochemical stain were positive cells.(2)The result of RT-PCR study showed that antisense Nogo-A ODN could significantly and specifically inhibit the expression of Nogo-A after 24 hours ( P< 0.01). Random sequence has no effect on Nogo-A expresson.Conclusion:Antisense Nogo-A ODN can effectively and specifically inhibit the expression of Nogo-A.展开更多
This paper reviews the significant study on SRS leukemia virus (SRSV) in recent years in China. A series of results about the proteins, nucleic acids and function, CDNA libraries, molecular cloning of SRSV, and inhibi...This paper reviews the significant study on SRS leukemia virus (SRSV) in recent years in China. A series of results about the proteins, nucleic acids and function, CDNA libraries, molecular cloning of SRSV, and inhibitory effect of antisense oligodeoxynucleotides on SRSV are reported. It has proved that SRSV is useful to study the vival etiology and pathogenesis of human leukemial.展开更多
AIM: To investigate the combined effects of K-ras antisense oligodeoxynucleotide (K-ras ASODN) specif ic to GTT point mutation at codon 12 and type Ⅰ insulin-like growth factor receptor (IGF-IR) antisense oligodeoxyn...AIM: To investigate the combined effects of K-ras antisense oligodeoxynucleotide (K-ras ASODN) specif ic to GTT point mutation at codon 12 and type Ⅰ insulin-like growth factor receptor (IGF-IR) antisense oligodeoxynucleotide (IGF-IR ASODN) on proliferation and apoptosis of human pancreatic cancer Patu8988 cells in vitro and in vivo. METHODS: K-ras gene point mutation and its style at codon 12 of human pancreatic cancer cell line Patu8988 were detected by using polymerase chain reaction with special sequence primers (PCR-SSP) and sequence analysis. According to the mutation style, K-ras mutation ASODN specifi c to K-ras point mutation at codon 12 was designed and composed. After K-ras ASODN and IGF-IR ASODN treated on Patu8988 cells respectively or cooperatively, the proliferation and morphological change of Patu8988 cells were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony forming assay andtransmission electron microscopy; the expression of K-ras and IGF-IR mRNA and protein in the treated cells was measured by reverse-transcript polymerase chain reaction (RT-PCR) and flow cytometry respectively; apoptosis was determined by flow cytometry. The combined antitumor activity of K-ras ASODN and IGF-IR ASODN was evaluated in BALB/c nude mice bearing human pancreatic cancer inoculated with Patu8988 cells. RESULTS: The results of PCR-SSP and sequence analysis showed that the human pancreatic cancer cell line Patu8988 had point mutation at codon 12, and the mutation style was GGT→GTT. 2-32 μg/mL K-ras ASODN and 2-32 μg/mL IGF-IR ASODN could inhibit Patu8988 cells' growth, induce apoptosis and decrease the expression of K-ras and IGF-IR mRNA and protein alone. However, there was much more effective inhibition of growth and induction of apoptosis by their combination than by each one alone. In tumor bearing mice, the combination of K-ras ASODN and IGF-IR ASODN showed a signif icant inhibitory effect on the growth of transplanted pancreatic cancer, resulting in a statistically signif icant difference compared with each alone. CONCLUSION: It has been found that K-ras ASODN combined with IGF-IR ASODN could cooperatively inhibit the growth of Patu8988 cells, and induce their apoptosis via reinforcing specific down regulation of K-ras and IGF-IR mRNA and protein expression.展开更多
Objective To study the inhibitory effects of caspase-3 mRNA antisense oligodeoxynucleotides (ASODN) on expressions of caspase-3 and it's mRNA in γ-radiation induced apoptotic HL-60 cells, and screen the effective...Objective To study the inhibitory effects of caspase-3 mRNA antisense oligodeoxynucleotides (ASODN) on expressions of caspase-3 and it's mRNA in γ-radiation induced apoptotic HL-60 cells, and screen the effective ASODN. Methods ASODN-1 and ASODN-2 targeting 5′-noncoding region and initial translation region of caspase-3 mRNA were respectively designed, synthesized and introduced into HL-60 cells by means of liposome-mediated transfection followed by 10Gy γ-radiation exposures. TUNEL assay was conducted to investigate the morphologic change and apoptotic percentage of HL-60 cells 18 h later. Immunocytochemical staining and one step RT-PCR were respectively performed to detect the expressions of caspase-3 and it's mRNA. Mismatched oligodeoxynucleotide (MODN) transfected and un-transfected HL-60 cells were taken as control. Results TUNEL assay found that the apoptotic percentages in ASODN-1 and ASODN-2 groups were significantly reduced compared with the control groups (P<0.01) when the final concentration of both ASODNs was ≥3μmol/L. Immunocytochemistry showed that caspase-3 positive cell percentages were reduced but the average gray values increased significantly compared with the control groups (P<0.01). RT-PCR showed expressions of caspase-3 mRNA was decreased after ASODN transfection. Furthermore, ASODN-1 proved more effective in inhibiting HL-60 cell apoptosis than ASODN-2 (P<0.01). Conclusion Caspase-3 mRNA ASODNs can prevent HL-60 cells from apoptosis induced by γ-radiation and reduce expression of caspase-3 and its mRNA. These effects are dose dependent in a certain range.展开更多
Objective:To study the effect of VEGF antisense oligodeoxynucleotide(VEGF ASODN)on VEGF expression in acute monocyte leukemic cell line U937 in vitro.Methods:U937 cells were incubated with VEGF ASODN(final concentra-t...Objective:To study the effect of VEGF antisense oligodeoxynucleotide(VEGF ASODN)on VEGF expression in acute monocyte leukemic cell line U937 in vitro.Methods:U937 cells were incubated with VEGF ASODN(final concentra-tion as follows:10,20 and 30 μmol/L respectively)or scrambled sequence,compared with negative control.The expression of VEGF mRNA was measured by semi-quantitative RT-PCR,VEGF protein was measured by Western blot.Results:VEGF ASODN obviously inhibited expression of VEGF mRNA in U937 cell,compared with scrambled sequence and negative control(P<0.05).And the inhibition effect was most remarkable after 24 h,which is related with the dose of VEGF ASODN(P<0.05).Scrambled sequence groups had no significant difference compared with negative control groups(P>0.05).VEGF ASODN obvi-ously inhibited expression of VEGF protein,compared with scrambled sequence and negative control(P<0.05).Conclusion:The expressions of VEGF at mRNA and protein levels in leukemic cell line U937 are down-regulated after being treated with VEGF ASODN.展开更多
Objective To study the inhibitory effect of antisense endothelin receptor A (ET-AR) on the proliferation of the vascular smooth muscle cells. Methods The sense, antisense and mismatched ODNs for ET-AR were designed an...Objective To study the inhibitory effect of antisense endothelin receptor A (ET-AR) on the proliferation of the vascular smooth muscle cells. Methods The sense, antisense and mismatched ODNs for ET-AR were designed and synthetized. The study was carried out using MTT method and binding assays. Results ET-AR-ODNs could move successfully across VSMC membranes. Photo-absorption in the MTT test was reduced significantly (P<0.05) in the antisense group at 5μmol/L; the reduction of CPM also occurred in the 125 I-ET-1 specific binding assay; and the sense and mismatched ODNs groups did not show this reduction. Conclusion Our study suggested that the antisense oligomers inhibited the proliferation of VSMCs by hindering the translation of target mRNA and by reducing the production of related protein.展开更多
In the present study,we investigated the synergistic effects of targeted methotrexate-selenium nanostructure containing Myc decoy oligodeoxynucleotides along with X-irradiation exposure as a combination therapy on LNC...In the present study,we investigated the synergistic effects of targeted methotrexate-selenium nanostructure containing Myc decoy oligodeoxynucleotides along with X-irradiation exposure as a combination therapy on LNCaP prostate cancer cells.Myc decoy ODNs were designed based on the promoter of Bcl-2 gene and analyzed by molecular docking and molecular dynamics assays.ODNs were loaded on the synthesized Se@BSA@Chi-MTX nanostructure.The physicochemical characteristics of nanostructures were determined by FTIR,DLS,UV-vis,TEM,EDX,in vitro release,and hemolysis tests.Subsequently,the cytotoxicity properties of them with and without X-irradiation were investigated by uptake,MTT,cell cycle,apoptosis,and scratch assays on the LNCaP cell line.The results of DLS and TEM showed negative charge(−9 mV)and nanometer size(40 nm)for Se@BSA@Chi-DEC-MTX NPs,respectively.The results of FTIR,UV-vis,and EDX showed the proper interaction of different parts and the correct synthesis of nanoparticles.The results of hemolysis showed the hemocompatibility of this nanoparticle in concentrations less than 6 mg/mL.The ODNs release from the nanostructures showed a pH-dependent manner,and the release rate was 15%higher in acidic pH.The targeted Se@BSA@Chi-labeled ODN-MTX NPs were efficiently taken up by LNCaP cells by targeting the prostate-specific membrane antigen(PSMA).The significant synergistic effects of nanostructure(containing MTX drug)treatment along with X-irradiation showed cell growth inhibition,apoptosis induction(~57%),cell cycle arrest(G2/M phase),and migration inhibition(up to 90%)compared to the control.The results suggested that the Se@BSA@Chi-DEC-MTX NPs can potentially suppress the cell growth of LNCaP cells.This nanostructure system can be a promising approach for targeted drug delivery and chemoradiotherapy in prostate cancer treatment.展开更多
The Myc gene is the essential oncogene in triple-negative breast cancer(TNBC).This study investigates the synergistic effects of combining Myc decoy oligodeoxynucleotides-encapsulated niosomes-selenium hybrid nanocarr...The Myc gene is the essential oncogene in triple-negative breast cancer(TNBC).This study investigates the synergistic effects of combining Myc decoy oligodeoxynucleotides-encapsulated niosomes-selenium hybrid nanocarriers with X-irradiation exposure on the MDA-MB-468 cell line.Decoy and scramble ODNs for Myc transcription factor were designed and synthesized based on promoter sequences of the Bcl2 gene.The nanocarriers were synthesized by loading Myc ODNs and selenium into chitosan(Chi-Se-DEC),which was then encapsulated in niosome-nanocarriers(NISM@Chi-Se-DEC).FT-IR,DLS,FESEM,and hemolysis tests were applied to confirm its characterization and physicochemical properties.Moreover,cellular uptake,cellular toxicity,apoptosis,cell cycle,and scratch repair assays were performed to evaluate its anticancer effects on cancer cells.All anticancer assessments were repeated under X-ray irradiation conditions(fractionated 2Gy).Physicochemical characteristics of niosomes containing SeNPs and ODNs showed that it is synthesized appropriately.It revealed that the anticancer effect of NISM@Chi-Se-DEC can be significantly improved in combination with X-ray irradiation treatment.It can be concluded that NISM@Chi-Se-DEC nanocarriers have the potential as a therapeutic agent for cancer treatment,particularly in combination with radiation therapy and in-vivo experiments are necessary to confirm the efficacy of this nano-drug.展开更多
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(CC...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.展开更多
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 ...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.展开更多
文摘Objective: To explore the probability of vascular endothelial growth factor (VEGF) antisense oligodeoxynucleotides as a developing new therapeutic strategy for glioma. Methods: VEGF protein expression was detected by S-P immunohistochemical technique. Tumor cell apoptosis was observed by TUNEL method. Results: Compared with control, VEGF protein expression was inhibited by antisense oligodeoxynucleotides in vitro. And the inhibitory effects increased with the increasing concentration. VEGF positive rate was 82.10% in control group, while in 2.5, 5, 10 mmol/L AODN groups, they were 70.00%, 57.85%, 53.20% respectively. No inhibition effect was found in the cell lines treated with missense and sense oligodeoxynucleotides. In vivo, antisense oligodeoxy-nucleotides therapy also inhibited VEGF protein expression and induced the increase of apoptotic tumor cells. However, it has no effect on tumor cell proliferation. Conclusion: It is hopeful that VEGF antisense oligodeoxynucleotides may be a new gene therapy method to glioma through its antiangiogenesis effect by inhibition of VEGF.
基金This project was supported by a grant from the National Natural Sciences Foundation of China(No.395 70 2 89) .
文摘To study the effect of c myc antisense oligodeoxynucleotides (ODNs) on proliferation of pulmonary vascular pericytes (PC) induced by hypoxia, cell culture, dot hybridization using probe of digoxigenin 11 dUTP labeled cDNA, 3H thymidine incorporation, immunocytochemical technique and image analysis methods were used to observe the effect of c myc antisense ODNs on expression of c myc gene and proliferating cell nuclear antigen (PCNA), and 3H thymidine incorporation of PC induced by hypoxia. The results showed that hypoxia could significantly enhance the expression of c myc and PCNA ( P <0.01), and elevate 3H thymidine incorporation of PC ( P <0.01), but antisense ODNs could significantly inhibit the expression of c myc and PCNA ( P <0.05), and 3H thymidine incorporation of PC ( P <0.01). It was suggested that hypoxia could promote the proliferation of PC by up regulating the expression of c myc gene, but c myc antisense ODNs could inhibit hypoxia induced proliferation of PC by downregulating the expression of c myc gene.
文摘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.
文摘Objective and Methods: Excessive accumulation of collagen typeⅠ and type Ⅲ causes the formation of keloids and hypertrophic scars. To understand the mechanism by which antisense oligodeoxynucleotide (Oligo) acts on in vitro transcrption α1 (I) collagen gene, isotopes (α-32pGTP) was incorporated into 2 SP6 in vitro transcription systems. Results and Conclu- sion: Oligo 2 (at the transcription start region) could effectively inhibit in vitro transcription of pGEM3-Col13 and the control (random oligodeoxynucleotides) showed no inhibition. However, oligo 1 (at the transcription start region) obviously inhibited the in vitro transcription of pGEM3-Col14, while Oligo 2, which targeted at the down stream region (about 200 bp) of the promoter showed no significant inhibition effect.
基金Hubei Provincial Science and Technology Key Program Foundation (No. 2004AA304B08)
文摘Objective: Aurora A kinase representing a family of evolutionarily conserved mitotic serine/threonine kinases has been found elevated in human lung adenocarcinoma cell line A549. It is suggested that the overexpression of Aurora A con- tributes to the carcinogenesis, chromosomal instability (CIN), and de-differentiation of lung cancers. To address its possibility as a therapeutic target for lung cancer, we employed the antisense oligodeoxynucleotide (ASODN) technique to inhibit Aurora A expression and investigate its effects on tumor growth and cell cycle of A549, as well as the chemosensitivity to paclitaxel. Methods: Aurora A ASODN was synthesized and transfected into A549 cells by lipofectAMINE 2000. Aurora A mRNA and protein expression were examined by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot respec- tively. Cell cycle distribution was observed by flow cytometer. MTT assay was used to evaluate cell inhibition ratio before and after transfection. Results: The proliferation of the A549 cells was inhibited by Aurora A ASODN dose and time dependently. It was also observed that the IC50 of A549 cells after 48 hours’ treatment of ASODN was about 300 nmol/L and under such circumstances, the Aurora A mRNA and protein expression significantly decreased (P < 0.05), along with the induction of ac- cumulation of cells in S phase and the G2-M transition. Furthermore, cell inhibition ratio of the combination of Aurora A ASODN and paclitaxel was higher significantly than paclitaxel (P < 0.05) or Aurora A ASODN alone (P < 0.05). Conclusion: Inhibition of Aurora A expression can result in the suppression of cell growth and chemosensitizing activity to paclitaxel in human lung cancer cell line A549.
基金Science and Technology of Guangzhou City (2001-Z-037-01)Guangdong Province (99M1204G)
文摘Objective To evaluate the effect of human telomerase reverse transcriptase(hTERT) gene antisense oligodeoxynucleotide (ASON) on telomerase activity in K562 cells.Methods Telomerase activity was detemined by polymerase chain reaction enzyme-linked immunoassay (PCR-ELISA) in K562 cells treated with ASODN and hTERTmRNA expression was detected by reverse transcriptase polymerase chain reaction (RT-PCR). Results The hTERTmRNA level was decreased,and telomerase activity was significantly inhibited when the K562 cells were treated with ASODN for 48 h. Conclusion It is suggested that hTETR ASODN might specifically inhibit telomrase activity of K562 cells at translation level,and it is further proved that hTERT gene has significant correlation with telopmerase activity.
文摘To investigate the potential utility of nuclease--resistant oligodeoxynucleotides (S-ODN) as anew class of antiviral agents. Methods: Two antisense phosphorothioate analogues (20--iner) complementary to thesequences of the first AUG codon and 5’ terminus of NSS of JEV SA--14 genome have been synthesized and their effects on CPE, viral antigen expression and virus plaque formation were tested in vitro. Results: The resultsshowed that 1. 0 pmol/L of S-ODN greatly deferred the onset of CPE in cultured BHKZI cells for at least 48 h.Addition of 5. 0 pmol/L or more S--ODN to culture medium fluid, 2 h prior to 100TCID,,virus inoculum, notablysuppressed viral antigen expression in the cells by making it lower than the limit of EIA detection in 48 h. The inhibition lasted more than 96 h. Viral plaque assay results demonstrated that S-ODN were most effect’ive within 18h with plaque inhibition rate over 90% by 5. 0 pmol/L S--ODNI. The inhibitory activity soon faded in 24 h. In addition, high concentrations (up to 80. 0 pmol/L) of S--ODN did not show any obvious cytotoxicity in 6 d by usingtrypan blue dye exclusion method. Conclusion: The specific synthetic S--ODN transitorily inhibited JEV replicationin BHK--ZI cells with characteristics of specificity and S--ODN dose--dependence.
基金This work was supported by the Natural Science National Foundation of China (grant number 30270458)the Application Basic Research Foundation of Chongqing Scientific Committee (grant number 41A1152C)the Fifteenth Medicine and Health Scientific Researc
文摘Purpose: To investiagate the effect of antisense oligonucleotides (ODN) to Nogo-A mRNA expression in oligodendrocytes and to establish the base for further research of repair of optic nerve injury.Method: (1)Oligodendrocytes were obtained by inoculating the optic nerve of newborn (2 days)rats and were identified with galactocerebroside(GC) antibody immunocytochemical stain. (2) In order to observe the effects of antisense ODN on cultured cells,we set up five groups, including the groups of three concentration of antisense Nogo-A ODN (2μM、5μM、l0μM),a group with the random sequence added to the medium and the control group. Reverse transcription-polymerase chain reaction(RT-PCR) was adopted to study the effects of ODN on the expression of Nogo-A in oligodendrocytes.Results: (1)Three days after inoculation, a few of round or fusiform shape cells migrated from optic nerve tissue; About 11 days later, the coverlips were completely covered by the cells;The cells identified with GC antibody immunocytochemical stain were positive cells.(2)The result of RT-PCR study showed that antisense Nogo-A ODN could significantly and specifically inhibit the expression of Nogo-A after 24 hours ( P< 0.01). Random sequence has no effect on Nogo-A expresson.Conclusion:Antisense Nogo-A ODN can effectively and specifically inhibit the expression of Nogo-A.
文摘This paper reviews the significant study on SRS leukemia virus (SRSV) in recent years in China. A series of results about the proteins, nucleic acids and function, CDNA libraries, molecular cloning of SRSV, and inhibitory effect of antisense oligodeoxynucleotides on SRSV are reported. It has proved that SRSV is useful to study the vival etiology and pathogenesis of human leukemial.
基金Social development foundation of Suzhou, China, No. SZD0614Young teacher foundation of Soochow UniversityFoundation of health department of Jiangsu Province, China, No. Z200622
文摘AIM: To investigate the combined effects of K-ras antisense oligodeoxynucleotide (K-ras ASODN) specif ic to GTT point mutation at codon 12 and type Ⅰ insulin-like growth factor receptor (IGF-IR) antisense oligodeoxynucleotide (IGF-IR ASODN) on proliferation and apoptosis of human pancreatic cancer Patu8988 cells in vitro and in vivo. METHODS: K-ras gene point mutation and its style at codon 12 of human pancreatic cancer cell line Patu8988 were detected by using polymerase chain reaction with special sequence primers (PCR-SSP) and sequence analysis. According to the mutation style, K-ras mutation ASODN specifi c to K-ras point mutation at codon 12 was designed and composed. After K-ras ASODN and IGF-IR ASODN treated on Patu8988 cells respectively or cooperatively, the proliferation and morphological change of Patu8988 cells were analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, colony forming assay andtransmission electron microscopy; the expression of K-ras and IGF-IR mRNA and protein in the treated cells was measured by reverse-transcript polymerase chain reaction (RT-PCR) and flow cytometry respectively; apoptosis was determined by flow cytometry. The combined antitumor activity of K-ras ASODN and IGF-IR ASODN was evaluated in BALB/c nude mice bearing human pancreatic cancer inoculated with Patu8988 cells. RESULTS: The results of PCR-SSP and sequence analysis showed that the human pancreatic cancer cell line Patu8988 had point mutation at codon 12, and the mutation style was GGT→GTT. 2-32 μg/mL K-ras ASODN and 2-32 μg/mL IGF-IR ASODN could inhibit Patu8988 cells' growth, induce apoptosis and decrease the expression of K-ras and IGF-IR mRNA and protein alone. However, there was much more effective inhibition of growth and induction of apoptosis by their combination than by each one alone. In tumor bearing mice, the combination of K-ras ASODN and IGF-IR ASODN showed a signif icant inhibitory effect on the growth of transplanted pancreatic cancer, resulting in a statistically signif icant difference compared with each alone. CONCLUSION: It has been found that K-ras ASODN combined with IGF-IR ASODN could cooperatively inhibit the growth of Patu8988 cells, and induce their apoptosis via reinforcing specific down regulation of K-ras and IGF-IR mRNA and protein expression.
文摘Objective To study the inhibitory effects of caspase-3 mRNA antisense oligodeoxynucleotides (ASODN) on expressions of caspase-3 and it's mRNA in γ-radiation induced apoptotic HL-60 cells, and screen the effective ASODN. Methods ASODN-1 and ASODN-2 targeting 5′-noncoding region and initial translation region of caspase-3 mRNA were respectively designed, synthesized and introduced into HL-60 cells by means of liposome-mediated transfection followed by 10Gy γ-radiation exposures. TUNEL assay was conducted to investigate the morphologic change and apoptotic percentage of HL-60 cells 18 h later. Immunocytochemical staining and one step RT-PCR were respectively performed to detect the expressions of caspase-3 and it's mRNA. Mismatched oligodeoxynucleotide (MODN) transfected and un-transfected HL-60 cells were taken as control. Results TUNEL assay found that the apoptotic percentages in ASODN-1 and ASODN-2 groups were significantly reduced compared with the control groups (P<0.01) when the final concentration of both ASODNs was ≥3μmol/L. Immunocytochemistry showed that caspase-3 positive cell percentages were reduced but the average gray values increased significantly compared with the control groups (P<0.01). RT-PCR showed expressions of caspase-3 mRNA was decreased after ASODN transfection. Furthermore, ASODN-1 proved more effective in inhibiting HL-60 cell apoptosis than ASODN-2 (P<0.01). Conclusion Caspase-3 mRNA ASODNs can prevent HL-60 cells from apoptosis induced by γ-radiation and reduce expression of caspase-3 and its mRNA. These effects are dose dependent in a certain range.
文摘Objective:To study the effect of VEGF antisense oligodeoxynucleotide(VEGF ASODN)on VEGF expression in acute monocyte leukemic cell line U937 in vitro.Methods:U937 cells were incubated with VEGF ASODN(final concentra-tion as follows:10,20 and 30 μmol/L respectively)or scrambled sequence,compared with negative control.The expression of VEGF mRNA was measured by semi-quantitative RT-PCR,VEGF protein was measured by Western blot.Results:VEGF ASODN obviously inhibited expression of VEGF mRNA in U937 cell,compared with scrambled sequence and negative control(P<0.05).And the inhibition effect was most remarkable after 24 h,which is related with the dose of VEGF ASODN(P<0.05).Scrambled sequence groups had no significant difference compared with negative control groups(P>0.05).VEGF ASODN obvi-ously inhibited expression of VEGF protein,compared with scrambled sequence and negative control(P<0.05).Conclusion:The expressions of VEGF at mRNA and protein levels in leukemic cell line U937 are down-regulated after being treated with VEGF ASODN.
文摘Objective To study the inhibitory effect of antisense endothelin receptor A (ET-AR) on the proliferation of the vascular smooth muscle cells. Methods The sense, antisense and mismatched ODNs for ET-AR were designed and synthetized. The study was carried out using MTT method and binding assays. Results ET-AR-ODNs could move successfully across VSMC membranes. Photo-absorption in the MTT test was reduced significantly (P<0.05) in the antisense group at 5μmol/L; the reduction of CPM also occurred in the 125 I-ET-1 specific binding assay; and the sense and mismatched ODNs groups did not show this reduction. Conclusion Our study suggested that the antisense oligomers inhibited the proliferation of VSMCs by hindering the translation of target mRNA and by reducing the production of related protein.
基金Zanjan University of Medical Sciences supported the present study(Grant Number:A-12-1244-18).
文摘In the present study,we investigated the synergistic effects of targeted methotrexate-selenium nanostructure containing Myc decoy oligodeoxynucleotides along with X-irradiation exposure as a combination therapy on LNCaP prostate cancer cells.Myc decoy ODNs were designed based on the promoter of Bcl-2 gene and analyzed by molecular docking and molecular dynamics assays.ODNs were loaded on the synthesized Se@BSA@Chi-MTX nanostructure.The physicochemical characteristics of nanostructures were determined by FTIR,DLS,UV-vis,TEM,EDX,in vitro release,and hemolysis tests.Subsequently,the cytotoxicity properties of them with and without X-irradiation were investigated by uptake,MTT,cell cycle,apoptosis,and scratch assays on the LNCaP cell line.The results of DLS and TEM showed negative charge(−9 mV)and nanometer size(40 nm)for Se@BSA@Chi-DEC-MTX NPs,respectively.The results of FTIR,UV-vis,and EDX showed the proper interaction of different parts and the correct synthesis of nanoparticles.The results of hemolysis showed the hemocompatibility of this nanoparticle in concentrations less than 6 mg/mL.The ODNs release from the nanostructures showed a pH-dependent manner,and the release rate was 15%higher in acidic pH.The targeted Se@BSA@Chi-labeled ODN-MTX NPs were efficiently taken up by LNCaP cells by targeting the prostate-specific membrane antigen(PSMA).The significant synergistic effects of nanostructure(containing MTX drug)treatment along with X-irradiation showed cell growth inhibition,apoptosis induction(~57%),cell cycle arrest(G2/M phase),and migration inhibition(up to 90%)compared to the control.The results suggested that the Se@BSA@Chi-DEC-MTX NPs can potentially suppress the cell growth of LNCaP cells.This nanostructure system can be a promising approach for targeted drug delivery and chemoradiotherapy in prostate cancer treatment.
基金supported by Zanjan University of Medical Sciences,Zanjan,Iran(Grant Number:A-12-1244-16&Ethical Code:IR.ZUMS.REC.1399.316).
文摘The Myc gene is the essential oncogene in triple-negative breast cancer(TNBC).This study investigates the synergistic effects of combining Myc decoy oligodeoxynucleotides-encapsulated niosomes-selenium hybrid nanocarriers with X-irradiation exposure on the MDA-MB-468 cell line.Decoy and scramble ODNs for Myc transcription factor were designed and synthesized based on promoter sequences of the Bcl2 gene.The nanocarriers were synthesized by loading Myc ODNs and selenium into chitosan(Chi-Se-DEC),which was then encapsulated in niosome-nanocarriers(NISM@Chi-Se-DEC).FT-IR,DLS,FESEM,and hemolysis tests were applied to confirm its characterization and physicochemical properties.Moreover,cellular uptake,cellular toxicity,apoptosis,cell cycle,and scratch repair assays were performed to evaluate its anticancer effects on cancer cells.All anticancer assessments were repeated under X-ray irradiation conditions(fractionated 2Gy).Physicochemical characteristics of niosomes containing SeNPs and ODNs showed that it is synthesized appropriately.It revealed that the anticancer effect of NISM@Chi-Se-DEC can be significantly improved in combination with X-ray irradiation treatment.It can be concluded that NISM@Chi-Se-DEC nanocarriers have the potential as a therapeutic agent for cancer treatment,particularly in combination with radiation therapy and in-vivo experiments are necessary to confirm the efficacy of this nano-drug.
基金Supported by the National Natural Science Foundation of China(No.81771499)the Natural Science Foundation of Hebei Province,China(No.H2018206099,No.H2021206460)。
文摘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.
基金supported by grants from the National Natural Science Foundation of China(No.81771499)and the Natural Science Foundation of Hebei Province,China(No.H2018206099 and No.H2021206460).
文摘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.