Antisense Oligodeoxynucleotide Group Research Articles

Effects of HOXA1 Gene Antisense Oligonucleotides on Growth of Esophageal Cancer Cells

To investigate the effect of antisense oligodeoxynucleotides (ASODN) of Homeobox A1 gene ( HOXA1) on proliferation, apoptosis, invasion and migration of esophageal carcinoma cells. The expression of HOXA1 protein in normal esophageal epithelial cells Het-1A and esophageal cancer TE-1, EC9706 and Eca109 cells was detected by Western blot. Screening of highly expressed of HOXA1 protein esophageal squamous cell carcinoma cells for follow-up experiments. HOXA1 antisense oligonucleotide (ASODN) chains, sense oligodeoxynucleotides (SODN) chain, and nonsense oligodeoxy nucleotides (N-ODN) chain were designed. The screened esophageal squamous cell carcinoma cells with high expression were divided into HOXA1 ASODN group (5, 10, 15 μmol/L HOXA1 ASODN transfected Eca109 cells), control group (conventional culture medium, no cell transfection), SODN group (cells transfected with 15 μmol/L of SODN) and N-ODN group (cells transfected with 15 μmol/L N-ODN). Cell viability, apoptosis rate and invasion and migration ability were detected by methylthiazolyldiphenyl-tetrazolium bromide (MTT) method, flow cytometry, transwell chamber respectively; The expression of HOXA1, phosphorylation serine/threonine kinase (p-AKT), proliferating cell nuclear antigen (PCNA), matrix metalloproteinase 2 (MMP-2) and B-cell lymphoma2 (Bcl-2) associated X protein (Bax) protein was detected by Western blot. Compared with normal esophageal epithelial cells Het-1A, the expression of HOXA1 protein in human esophageal squamous cell carcinoma cells TE-1, EC9706 and Eca109 was significantly higher ( P<0.05). The expression of HOXA1 protein was the highest in Eca109 cells, therefore, Eca109 cells were selected for follow-up experiments. The expression of HOXA1 protein in Eca109 cells transfected with HOXA1 ASODN was significantly decreased ( P<0.05). After transfection of Eca109 cells with HOXA1 ASODN, the viability of Eca109 cells decreased with the increase of concentration and time, the difference was significant compared with the control, SODN and N-ODN groups ( P<0.05). 15 μmol/L HOXA1 ASODN significantly inhibited cell viability. After 15 μmol/L HOXA1 ASODN was transfected into Eca109 cells, the invasion and migration abilities of cells were significantly decreased, the apoptosis rate was increased, the expressions of p-AKT, PCNA and MMP-2 were significantly decreased, and the expression of Bax was significantly increased ( P<0.05). Antisense oligodeoxynucleotides of HOXA1 gene can inhibit the proliferation, invasion and migration of esophageal cancer cells, and induce apoptosis. The mechanism is related to the inhibition of PI3K/AKT signaling pathway.

Effect of Cdc42 on myocardial ischemia-reperfusion of rats.

To investigate the effects and their possible mechanisms of cell division cycle 42 (Cdc42) to neonatal rat myocardial cells subjected to the ischemia-repefusion. Neonatal rat cardiomyocytes were cultured and then subjected to the ischemia-reperfusion. Experimental groups 1. Control group; 2. Ischemia-repefusion group (I/R group); 3. Oligofectamine group (Oli group); 4. Oligofectamine and antisense oligodeoxynucleotide (AS-ODN) group (As group); 5. Oligofectamine and missense oligodeoxynucleotide (MS-ODN) group (Ms group); 6. SP600125 and Oligofectamine and AS-ODN group (SP600125/As group); 7. SP600125 and Oligofectamine and MS-ODN group (SP600125/Ms group). The cardiacmyocyte apoptosis rate was detected by AnnexinV/PI with flow cytometry. Cdc42, JNK, p-JNK, Bax and Bcl-2 were detected by western blot. In comparison with control group, Cdc42, the cardiacmyocyte apoptosis rate and phosphorylation of JNK were increased and the ratio of Bcl-2/Bax was reduced in the I/R group; Cdc42, the cardiacmyocyte apoptosis rate and phosphorylation of JNK in As group was lower than the I/R group, Oli group and the Ms group, and the ratio of Bcl-2/Bax was the highest in the four groups; Cdc42, cardiacmyocyte apoptosis rate, phosphorylation of JNK and the ratio of Bcl-2/Bax showed no differences in the I/R group, Oli group and the Ms group. Compared with As group, phosphorylation of JNK was lower in the SP600125/As group, phosphorylation of JNK in SP600125/Ms group was lower than the Ms group, and it showed no differences between the SP600125 &amp; As group and the SP600125 &amp; Ms group. Cdc42 in myocardial I/R can promote cardiacmyocyte apoptosis rate. AS-ODN of Cdc42 can decrease the cardiacmyocyte apoptosis rate in I/R. Cdc42 may played a role in myocardial I/R via JNK , Bcl-2 and Bax signal pathway.

Effects of intrathecally postsynaptic density protein-95 antisense oligodeoxynucleotide on pain behaviors in mice model of neuropathic pain

Objective To investigate the effects of intrathecally postsynaptic density protein-95 (PSD-95) antisense oligodeoxynucleotide on neuropathic pain behaviors of sciatic nerve ligation mouse. Methods Forty-eight male C57BL/6 mice in which intrathecal catheter was successfully implanted were randomly divided into 4 groups (n=12): Sham group, normal saline group (group NS), PSD-95 antisense oligodeoxynucleotide group (group A) and PSD-95 missense oligodeoxynucleotide group (group M). In groups NS, A and M, normal saline 5 μl, antisense oligodeoxynucleotide 5 μg/5 μl and missense oligodeoxynucleotide 5 μg/5 μl were injected intrathecally once a day for 14 d, starting from the 1st day after chronic constriction injury(CCI), respectively. Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWL) were measured on day 1 before CCI and on day 1, 3, 5, 7, 14, 17, 21 after CCI. Results During day 1-day 14, mice in group A maintained the pain thresholds consistent with Sham group [14 d, PWMT(5.69±1.34) g, P>0.05, PWL (9.65±1.44) s, P>0.05]. The withdrawal thresholds in the ipsilateral hind paws of the group A were significantly lower than sham group on day 17 after CCI[PWMT(4.24±1.83) g, P<0.05, PWL(7.18±0.41) s, P<0.05]. However, compared with group NS [PWMT (1.77±0.38) g, P<0.05, PWL (4.33±1.21) s, P<0.05]and group M[PWMT (1.6±0.37) g, P<0.05, PWL(4.38± 0.95) s, P<0.05], the withdrawal thresholds of group A was significantly elevated on day 17 after CCI. Conclusions Intrathecally treated with PSD-95 antisense oligodeoxynucleotide in the development of neuropathic pain induced by CCI completely improved pain behaviors during the course of injection, and the effects of pain relief lasted at least 7 d after no injection. Key words: Neuropathic pain; postsynaptic density protein-95; Antisense oligonucleotide; N-methyl-D-aspartate

Liposome-mediated induction of apoptosis of human hepatoma cells by c-myc antisense phosphorothioate oligodeoxynucleotide and 5-fluorouracil.

The aim of this study was to investigate the effect of a c-myc antisense oligodeoxynucleotide and 5-fluorouracil on the expression of c-myc, invasion and proliferation of HEPG-2 liver cancer cells. HEPG-2 cells were treated with lipiosome-mediated c-myc ADSON and 5-fluorouracil. The proliferation inhibition rate and invasion were measured by MTT and invasion assay, respectively. Cell apoptosis was detected by flow cytometry and expression of c-myc by RT-PCR and immunohistochemistry. The proliferation inhibition rate was significantly higher in the antisense oligodeoxynucleotide added-5-fluorouracil group than single antisense oligodeoxynucleotide or 5-fluorouracil group (p<0.05). G0/G1 cells in the antisense oligodeoxynucleotide group and S cells in the 5-fluorouracil groups were significantly increased than that in the control group, respectively (P<0.01). The amplification strips of PCR products in 5-FU, ASODN and combination groups were significantly weaker than that in the control group (P<0.01). The percentage of c-myc-protein- positive cells were significantly lower in antisense oligodeoxynucleotide, 5-fluorouracil and combination groups than that in the control group (P<0.01). A liposome-mediated c-myc antisense oligodeoxynucleotide and 5-fluorouracil can inhibit the proliferation and invasion of liver cancer cells by reducing the expression of c-myc. A c-myc antisense oligodeoxynucleotide can increase the sensitivity of liver cancer cells to 5-fluorouracil and decrease the dosage of the agent necessary for efficacy, providing an experimental basis for the clinical therapy of liver cancer.

Influence of leucine-rich repeats and immunoglobulin-like domains 3 antisense oligodeoxynucleotide on proliferation in nude mice transplanted tumor of human cervical cancer

Objective Leucine-rich repeats and Immunoglobulin-like domains (LRIGs) gene family is a group of recently discovered tumor-associated genes,which acts in a negatingve feedback loop of epidermal growth factor receptor (EGFR) and inhibits the deterioration and proliferation of cancer.The present study investigated the effect of LRIG3 silencing on tumor growth and EGFR expression of the human cervical carcinoma xenografts in nude mice.Methods The Hela229 cells were inoculated subcutaneously into nude mice.The mice were divided into three groups after intratumoral injection of saline,LRIG3 antisense oligodeoxynucleotide (ASODN),and LRIG3 MSODN,respectively.Results Compared to MSODN and saline groups,the tumors in ASOND group grew faster and showed larger size,and demonstrated more deeply stained nucleolus by HE staining.LRIG3 protein expression in the tumors of saline,MSODN and ASODN group were 0.2302 ± 0.0108,0.2203 ± 0.0183 and 0.1532 ± 0.0132,respectively,the expression was significantly down-regulated in ASODN group compared to the other two groups (P ＜ 0.05).EGFR protein expression in the tumors in ASODN group was 1.2730 ± 1.1116,and significantly elevated compared to that in saline and MSODN group,which was 0.9212 ±0.0796 and 0.9904 ±0.0830,respectively (P ＜ 0.05).Conclusion Inhibition of LRIG3 gene expression can accelerate human cervical carcinoma xenograft growth and elevate EGFR expression in xenograft tumors. Key words: Leucine-rich repeats and Immunoglobulin-like domains 3 ; Epidermal growth factor receptor; Nude

Effects of autocrine vascular endothelial growth factor (VEGF) in non-small cell lung cancer cell line A549

It is reported that the autocrine loop of the vascular endothelial growth factor (VEGF) is crucial for the survival and proliferation of non-small cell lung cancer (NSCLC) tumors. In this study we aimed to systematically investigate the role of autocrine vascular VEGF in NSCLC cell line A549 through inhibition of endogenous VEGF. A549 cells were transfected with florescence-labeled VEGF oligodeoxynucleotide with lipofectamine. For the experimental group, cells were transfected with VEGF anti-sense oligodeoxynucleotide (ASODN), sense oligodeoxynucleotide (SODN) and mutant oligodeoxynuleotide (MODN) respectively. For the control group cells were mock transfected with lipofectamine or culture medium. At indicated time point after transfection, the expression levels of VEGF mRNA and protein in A549 cells were analyzed by RT-PCR and ELISA respectively. Cell viability was measured by the MTT assay. Cell cycle distribution was detected by flow cytometry. As revealed by RT-PCR assay, the mRNA level of VEGF in cells transfected with ASDON was significantly lower than the other four groups (P < 0.05) at 24 and 48 h after transfection. ELISA assay yielded similar result with significantly decreased level of VEGF protein expression (P < 0.05). The survival fraction of A549 cells transfected with ASDON was significantly lower than the other four groups (P < 0.05) at 24 h after transfection. Also the percentage of G2 phase cells of ASODN group was significantly lower than other four groups. Our data indicate that VEGF expression is efficiently inhibited in A549 cells by ASODN transfection and this inhibition leads to inhibited cell growth and impaired cell cycle distribution.

Role of phosphatidylinositol 3-kinase p110.BETA. in spinal dorsal horn neurons in the development of arthritic pain in rats: relationship with TRPV1 and ASICla

Objective To evaluate the role of phosphatidylinositol 3-kinase (PI3K) p110β in spinal dorsal horn neurons in the development of arthritic pain (AP) in rats and the relationship with transient receptor potential vanilloid 1 (TRPV1) and acid-sensing ion channel (ASIC)1 a.Methods Forty adult female Sprague-Dawley rats in which intrathecal catheters were successfully placed,aged 3 months,weighing 250-300 g,were randomly divided into 4 groups (n =10 each):control group (group C),group AP,AP + PI3K p110β missense oligo-deoxynucleotide group (group MS) and AP + PI3K p110β antisense oligo-deoxynucleotide group (group AS).AP was induced by injecting complete Freund's adjuvant into the ankle joint cavity of right hindpaw.Normal saline 20 μl,missense oligo-deoxynucleotide 15 μg (20μl) and antisense oligo-deoxynucleotide 15 μg (20 μl) were administered intrathecally once a day for 6 consecutive days starting from the time immediately after arthritis was induced in groups AP,MS and AS,respectively.Mechanical paw withdrawal threshold (MWT) and thermal paw withdrawal latency (TWL) were measured 1 day before operation (T0) and on days 4,7,10 after operation (T1-3).The rats were then sacrificed after the last measurement of pain threshold at T3.L4-6 segment of the spinal cord was removed for detection of expression of PI3K p110β (by Western blot),and TRPV1 and ASICla (by immunohistochemistry)in spinal dorsal horn neurons.Results Compared with group C,MWT and TWL were significantly decreased atT1-3,and the expression of PI3K p110β,TRPV1 and ASIC1a was up-regulated in the other 3 groups(P＜ 0.01).MWT and TWL were significantly higher at T1-3,and the expression of PI3K p110β,TRPV1 and ASIC1a was lower in group AS than in groups AP and MS (P ＜ 0.01).Conclusion PI3K p110β in spinal dorsal horn neurons is involved in the development of AP in rats,and the mechanism is related to up-regulation of TRPV1 and ASIC1a expression in spinal dorsal horn neurons. Key words: 1-Phosphatidylinositol 3-kinase; Arthritis; Pain; Capsaicin; Receptors, cell surface; Ion channels; Posterior horn cells

JNK signaling is the shared pathway linking neuroinflammation, blood-brain barrier disruption, and oligodendroglial apoptosis in the white matter injury of the immature brain.

BackgroundWhite matter injury is the major form of brain damage in very preterm infants. Selective white matter injury in the immature brain can be induced by lipopolysaccharide (LPS)-sensitized hypoxic-ischemia (HI) in the postpartum (P) day 2 rat pups whose brain maturation status is equivalent to that in preterm infants less than 30 weeks of gestation. Neuroinflammation, blood–brain barrier (BBB) damage and oligodendrocyte progenitor apoptosis may affect the susceptibility of LPS-sensitized HI in white matter injury. c-Jun N-terminal kinases (JNK) are important stress-responsive kinases in various forms of insults. We hypothesized that LPS-sensitized HI causes white matter injury through JNK activation-mediated neuroinflammation, BBB leakage and oligodendroglial apoptosis in the white matter of P2 rat pups.MethodsP2 pups received LPS (0.05 mg/kg) or normal saline injection followed by 90-min HI. Immunohistochemistry and immunoblotting were used to determine microglia activation, TNF-α, BBB damage, cleaved caspase-3, JNK and phospho-JNK (p-JNK), myelin basic protein (MBP), and glial fibrillary acidic protein (GFAP) expression. Immunofluorescence was performed to determine the cellular distribution of p-JNK. Pharmacological and genetic approaches were used to inhibit JNK activity.ResultsP2 pups had selective white matter injury associated with upregulation of activated microglia, TNF-α, IgG extravasation and oligodendroglial progenitor apoptosis after LPS-sensitized HI. Immunohistochemical analyses showed early and sustained JNK activation in the white matter at 6 and 24 h post-insult. Immunofluorescence demonstrated upregulation of p-JNK in activated microglia, vascular endothelial cells and oligodendrocyte progenitors, and also showed perivascular aggregation of p-JNK-positive cells around the vessels 24 h post-insult. JNK inhibition by AS601245 or by antisense oligodeoxynucleotides (ODN) significantly reduced microglial activation, TNF-α immunoreactivity, IgG extravasation, and cleaved caspase-3 in the endothelial cells and oligodendrocyte progenitors, and also attenuated perivascular aggregation of p-JNK-positive cells 24 h post-insult. The AS601245 or JNK antisense ODN group had significantly increased MBP and decreased GFAP expression in the white matter on P11 than the vehicle or scrambled ODN group.ConclusionsLPS-sensitized HI causes white matter injury through JNK activation-mediated upregulation of neuroinflammation, BBB leakage and oligodendrocyte progenitor apoptosis in the immature brain.

Effects of repeated intrathecal CREB antisense oligodeoxynucleotide on the expression of NR2A in spinal cord in a mouse model of neuropathic pain

Objective To investigate the effects of repeated intrathecal cyclic AMP response elementbinding protein (CREB) antisense oligodeoxynucleotide (ODN) on the expression of NR2A in spinal cord in mice with neuropathic pain produced by chronic constrictive injury of the sciatic nerve (CCI).Methods Forty C57BL/6 male mice in which intrathecal catheter was successfully implanted were randomly divided into 4 groups ( n =10 each):normal saline group (group NS),CREB sense ODN group (group S),CREB missense ODN group (group M),and CREB antisense ODN group (group A).In groups NS,S,M and A,normal saline 5μl,sense ODN 5 μg/5 μl,missense ODN 5 μg/5 μl and antisense ODN 5 μg/5 μl were injected intrathecally once a day for 6 days,starting from the 1st day after CCI,respectively.Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured on day 1 before CCI and on day 1,3,5 and 7 after CCI.Five mice from each group were sacrificed on day 7 and 14 after CCI and the lumbar segment of the spinal cord (L3-5 )was removed for determination of NR2A expression using Western blot and RT-PCR.Results Compared with the baseline value,no significant change was found in PWMT and PWTL on day 1-7 after CCI in group A ( P ＞0.05),while PWMT and PWTL were significantly decreased on day 1-7 after CCI in groups NS,S and M (P ＜0.05).Compared with groups NS,S and M,the expression of NR2A mRNA and protein was significantly downregulated on day 7 and 14 after CCI in group A ( P ＜ 0.05).The expression of NR2A mRNA and protein was significantly up-regulated on day 14 after CCI compared with that on day 7 after CCI in all the groups.Conclusion Intrathecal CREB antisense ODN during the development of neuropathic pain can attenuate neuropathic pain and inhibition of the expression of NR2A in mouse spinal cord may be involved in the mechanism. Key words: Oligonucleotides,antisense; Neuralgia; Receptors,N-methyl-D-aspartate

Effect of antisense oligodeoxynucleotide targeted against NF-κB/P65 on cell proliferation and tumorigenesis of gastric cancer

To study the inhibitory effect of nuclear transcription factor-kappa B(NF-κB) antisense oligodeoxynucleotide(ASODN) on the growth and tumorgenesis of human gastric cancer. We synthesized and transfected the ASODN of NF-κB/P65 to gastric cancer cell line. The effect of ASODN of NF-κB/P65 on the proliferation of gastric cancer cells was measured by MTT method. The subcutaneous xenograft model of human gastric cancer was established in nude mice, and the tumor growth curve was observed. The cell proliferation was significantly inhibited in P65 ASODN-transfected group in vitro (P<0.05). In vivo, tumor formation test showed that the tumor volume in nude mice in ASODN group was obviously smaller than in other groups (P<0.05); the apoptosis index (AI) was significantly higher (P<0.001). Simultaneously, MVD in ASODN group was markedly lower than in other groups (P<0.01). NF-κB could be used as a new biological therapeutic target of gastric cancer.

Inhibitory effect of STAT3 antisense oligodeoxynucleotide against human adenocarcinoma of lung derived from A549 cell in nude mice

Objective To investigate the inhibitory effects of antisense oligodeoxynucleotide( ASODN) of signal transducer andactivator of transcription 3 ( STAT3) on development and growth of transplanted lung A549 cell adenocarcinoma in nude mice. MethodsThe inhibitory effects of STAT3 ASODN on A549 cell growth was evaluated by CCK-8 assay. STAT3 ASODN-treated A549 cells wereimplanted subcutaneously into nude mice; the tumor growth was observed and the tumor-forming rate was calculated. After subcutaneousimplantation of the untreated A549 cells,the tumor-bearing mice were randomized into 3 groups: control group,STAT3 ASODN group,and nonsense oligodeoxynucleotide ( NSODN) group,with normal saline,STAT3 ASODN,and STAT3 NSODN injected intatumorally ( 15mg/kg daily for 3 weeks) . The tumor volumes were measured every other day and the tumor weights were determined. The proteinexpressions of STAT3 and p-STAT3 in xenografts were examined by Western blotting analysis. Results STAT3 ASODN significantlyinhibited the growth of A549 cells compared with STAT3 NSODN group ( P 0. 05) . The tumor formation ability of A549 cells treated withSTAT3 ASODN was significantly decreased ( with the tumor inhibitory rate being 75. 8% ) . Moreover,the growth of transplanted tumorwas also significantly inhibited in STAT3 ASODN group as compared with those in NSODN group and control group ( P 0. 05) . Thetumor weights were significantly reduced in STAT3 ASODN group than in STAT3 NSODN group and control group ( with the tumorinhibitory rate being 51. 1% ) . The expression of STAT3 protein and p-STAT3 was also down-regulated in STAT3 ASODN group.Conclusion STAT3 ASODN can suppress the tumorigenic ability of A549 cells and inhibit the growth of transplanted tumor in nudemice.

ADAM28 Manipulates Proliferation, Differentiation, and Apoptosis of Human Dental Pulp Stem Cells

Introduction The purpose of this study was to investigate the influence of a disintegrin and metalloproteinase 28 (ADAM28) on the proliferation, differentiation, and apoptosis of human dental pulp stem cells (HDPSCs) and possible mechanism. Methods After ADAM28 eukaryotic plasmid and antisense oligodeoxynucleotides (AS-ODNs) were constructed and respectively transfected into HDPSCs by Lipofectamine 2000, the ADAM28 expression levels among diverse groups were estimated by reverse transcription polymerase chain reaction (RT-PCR) and western blotting. Methabenzthiazuron (MTT) and cell cycle assays were used to test the HDPSCs proliferation activity. Annexin V- fluorescein isothiocyanate (FITC)/propidium iodide and alkaline phosphatase analysis were performed respectively to measure apoptosis and the cytodifferentiation level. Immunocytochemistry and western blotting were performed to determine the effects of ADAM28 eukaryotic plasmid on HDPSCs expressing dentin sialophosphoprotein (DSPP), dentin matrix protein 1, and bone sialoprotein. Results ADAM28 could be correctly transcribed, translated, and expressed in HDPSCs. The ADAM28 AS-ODN group displayed the highest optical density value, whereas the eukaryotic plasmid group showed the lowest, which suggested that ADAM28 had a negative regulatory effect on the proliferation of HDPSCs. ADAM28 eukaryotic plasmid could significantly inhibit the HDPSC proliferation, promote specific differentiation of HDPSCs, induce apoptosis, and enhance the DSPP expression, whereas ADAM28 AS-ODN produced the opposite effects. Conclusions Our results proved that ADAM28 might actively participate in manipulating the proliferation, differentiation, and apoptosis of HDPSCs.

Effect of antisense oligodeoxynudeotide targeted against NF-κB/P65 on cell proliferation and tumorgenesis of gastric cancer

AbstractGastric cancer is still a major health problem worldwide due to its frequency, poor prognosis and limited treatment options. To study the inhibitory effect of nuclear transcription factor kappa B(NF-κB) antisense oligodeoxynucleotide(ASODN) on the growth and tumorgenesis of human gastric cancer, we synthesis and transfect ASODN of NF-κB/P65 to gastric cancer cell line. The effect of ASODN of NF-κB/P65 on the proliferation of gastric cancer cells was measured by MTT method. The subcutaneous xenograft model of human gastric cancer was established in nude mice and the tumor growth curve was observed. The cell proliferation was significantly inhibited in P65 ASODN transfected group in vitro (P&lt;0.05). In vivo, tumor formation test showed that the tumor volume in nude mice in ASODN group was obviously smaller than in other groups (P&lt;0.05), the apoptosis index (AI) was significantly higher (P&lt;0.001). Stimultaneously, MVD in ASODN group was markedly lower than in other groups (P&lt;0.01). Our findings suggest that P65 ASODN can inhibit gastric cancer cells proliferation and tumorgenesis by inducing cell apoptosis and inhibiting tumor angiogenesis. NF-κB can be used as a new biological therapeutic target of gastric cancer.

The Regulatory Role of A Disintegrin and Metalloproteinase 28 on the Biologic Property of Human Periodontal Ligament Stem Cells

A disintegrin and metalloproteinase 28 (ADAM28) is considered to be the possible virulence gene for congenital hypoplasia of tooth root. Periodontal ligament stem cells (PDLSCs) are regarded as playing crucial roles in the developing process of the periodontium and are generally used in dental regenerative medicine. This study evaluates the influence of ADAM28 on the biologic property of human PDLSCs (HPDLSCs) and to postulate the possible mechanism of this influence. HPDLSCs were acquired by immunomagnetic bead selection and identified by immunofluorescence detection. After ADAM28 eukaryotic plasmid and antisense oligodeoxynucleotides (AS-ODNs) were constructed and respectively transfected into HPDLSCs by a transfection reagent, the expression differences of ADAM28 among various groups were assessed by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide and cell-cycle assays were used to test the proliferation activity of HPDLSCs. Annexin V-fluorescein isothiocyanate/propidium iodide analysis was performed to detect the apoptotic level. Cell differentiation was tested by measuring the alkaline phosphatase level. Immunocytochemistry and Western blotting were carried out to determine the effects of ADAM28 AS-ODNs on HPDLSCs expressing cementum attachment protein (CAP), osteopontin, and osteocalcin. The ADAM28 eukaryotic plasmid group showed the highest expression level in HPDLSCs, whereas the AS-ODN group displayed the lowest. Furthermore, the overexpression of ADAM28 enhanced the proliferation of HPDLSCs and inhibited the specific differentiation of HPDLSCs, whereas the inhibition of ADAM28 produced the opposite effects and induced apoptosis. ADAM28 AS-ODNs were able to significantly inhibit CAP expression, and ADAM28 had a positive correlation with CAP. Our findings demonstrated that ADAM28 was able to effectively manipulate the proliferation, apoptosis, and differentiation of HPDLSCs.

Study on growth inhibition of implanted tumor in nude mice by antisense oligodeoxynucleotide targeting telomerase RNA.

Many studies have proven that telomerase plays an important role during the occurrence and development of lung cancer, which has been an important target in gene therapy. The aim of this study is to explore the inhibitory effect of antisense oligodeoxynucleotides(ASODN) targeting human telomerase RNA on implanted tumor growth in nude mice. Implanted tumor models were Constructed in nude mice using human lung adenocarcinoma cell line A549. Eighteen Balb/C nude mice with implanted tumors were divided into three groups randomly: antisense oligodeoxynucleotide group(group ASODN), sense oligodeoxynucleotide group(group SODN), normal saline group (group NS), with 6 mice respectively. ASODs or SODNs mixed with Lipofectamine-2000 was injected into tumor directly every 24 h for fourteen days. The inhibitory rates of tumor volume in Group ASODN and Group SODN were 43.94% and 6.91%, respectively. There was significant difference between the two groups(t =6.17, P<0.001). All the animals tolerated the drugs well and there was no nausea, vomiting and subcutaneous hemorrhage to occur during the therapy. The weight of the animals increased slightly at the end of experiment. The results of this study suggest that intratumoral injection of human telomerase ribonucleic acid(hTR) ASOND could effectively inhibit the growth of implanted tumor in nude mice.

Insulin-like Growth Factor I Regulates Apoptosis in Condylar Cartilage

Endogenous insulin-like growth factor-I (IGF-I) is known to affect the growth and development of condylar cartilage. However, the critical effect of IGF-I on cell survival is still unknown. We hypothesized that endogenous IGF-I could regulate the survival of cells of the mandibular condylar cartilage. Mandibular condyles dissected from 12-day-old rats were cultured for 1, 3, and 5 days in medium containing antisense oligodeoxynucleotide (AS-ODN) for IGF-I. Real-time RT-PCR analysis showed that the levels of IGF-I and IGF binding protein (IGFBP)3 mRNAs in the AS-ODN group were significantly decreased. After 3 days' culture, the number of necrotic cells was observed in the undifferentiated mesenchymal cell layer. These cells were TUNEL-positive and confirmed to be apoptotic by electron microscopic observation. Immunoblotting revealed that expression of cleaved caspase3 was increased with AS-ODN. These results may suggest that the cells in the undifferentiated mesenchymal cell layer of the mandibular condyle require IGF-I for survival.

Inhibition of the VEGF expression and cell growth in hepatocellular carcinoma by blocking HIF-1α and Smad3 binding site in VEGF promoter

In order to investigate the inhibitory effects on the vascular endothelial growth factor (VEGF) expression and cell growth in hapatocellular carcinoma (HCC) by blocking HIF-1alpha and Smad3 binding site in the VEGF promoter, antisense oligodeoxynucleotides (ASODN) were designed to block HIF-1alpha and Smad3 binding site in the VEGF promoter. Different concentrations of ASODN and ODN were transfected into HCC cells respectively. The expression of VEGF mRNA and protein was detected by SABC, Western blot and RT-PCR techniques and the inhibitory effects on the expression of VEGF and cell growth of the HCC cells stimulated by the supernatants were determined by using MTT method. Immunohistochestry revealed that after co-inoculation of hepatocellular carcinoma cells with different concentrations of ODN and ASODN for 48 h, there was no significant difference in the expression of VEGF protein between ODN group and control group (P > 0.05), but there was significant difference between ASODN group and control group (P < 0.05). At a concentration of 10 micromol/L ASODN, the difference was very significant (P < 0.01). Western blot and RT-PCR revealed that, after treatment for 48 h at a concentration of 10 micromol/L. the integral gray levels and RNA odds were 59743.2 +/- 10412.5 and 0.783 +/- 0.032 in ODN group, and 38694.5 +/- 10925.1 and 0.468 +/- 0.015 in ASODN group, respectively, with the difference being very significant (P < 0.01). Antisense ODN could inhibit the growth of HCC cells in a concentration-dependent manner. It was concluded that anti-gene technique of aiming at HIF-1alpha action site in the VEGF promoter could suppress the VEGF expression and inhibit HCC cell growth, and it is promising that anti-gene technique works as a new gene therapeutic tool for anti-angiogenesis of HCC.

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

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

Inhibitory effects of transfected Bcl-XL antisense oligodeoxynucleotide on proliferation of esophageal cancer cells and growth of human esophageal carcinoma in nude mice

To investigate the biologic effects of Bcl-XL antisense oligodeoxynucleotide (ASODN) transfected into cultured esophageal carcinoma cells and human esophageal carcinoma xenograft in nude mice. Cationic liposome-mediated ASODN was used to transfect esophageal carcinoma cells. RT-PCR, Western blot, MTT assay, flow cytometry and in-situ apoptosis cells detection (TUNEL detection) were used to systematically study the biological effects of the transfected cells in vitro and in vivo. MTT assay showed that the proliferation of esophageal carcinoma cells in the ASODN group decreased significantly as compared with control (P < 0.05), along with a 57.3% inhibitory rate of Bcl-XL mRNA, a significant decrease of Bcl-XL protein and the apoptosis rates of (31.1 +/- 5.8)% and 35.0% by flow cytometry and TUNEL assay, respectively (P < 0.01, as compared with controls). The growth of human esophageal carcinoma in nude mice was also significantly inhibited in the ASODN group (P < 0.05), along with a significant decrease of Bcl-XL mRNA and protein expression, and also an enhanced apoptosis of the tumor cells in nude mice. Bcl-XL ASODN can effectively inhibit the proliferation of esophageal carcinoma cells in vitro and the growth of the tumor in vivo. The suppression of Bcl-XL expression by ASODN may offer both a therapeutic approach and an important theoretic foundation for gene therapy against esophageal carcinoma.

C-FOS ANTISENSE OLIGODEOXYNUCLEOTIDE OFFSETS BEHAVIORAL NOCICEPTIVE RESPONSES AND BOTH UP-REGULATIONS OF C-FOS PROTEIN AND DYNORPHIN A (1-8) IN DORSAL HORN: A STUDY USING THE FORMALIN TEST IN RATS

The formalin test was used to elicit acute and chronic pain in rats, and antisense oligodeoxynucleotide (AS-ODN) was used as a tool to modulate the expression of nociceptive behavioral and neurochemical responses. AS-ODN complementary to c-Fos mRNA was administered intrathecally (i.t.) 4 h before formalin injection in the experimental group. Normal saline or reverse AS-ODN was pre-administered i.t. at the same time in two control groups (saline and reverse AS-ODN). The results showed that the acute phase of nociceptive behavior showed no change by AS-ODN administration, whereas the tonic phase of nociceptive licking and biting behavior was significantly suppressed by AS-ODN as compared with the saline or the reverse AS-ODN group, respectively (p < .05 and p < .01). At the same time, both Fos-like immunoreactive (FLI) neurons and density of dynorphin-like immunoreactivities (DLI) were decreased significantly (p < .05 and p < .01) in the AS-ODN group as compared with that in two control groups. The results indicate that the long-lasting nociceptive responses elicited by sustained noxious inputs are based on the up-regulation of c-Fos gene expression, which in turn induces the upregulation of Dyn A production. It is proposed that intensified Dyn A production in the dorsal horn may be pivotal for the appearance of chronic pain.