Dendritic Cells ; cytology ; metabolism ; Enzyme-Linked Immunosorbent Assay ; HEK293 Cells ; Humans ; Interferon Regulatory Factor-7 ; metabolism ; Interferon Type I ; metabolism ; Interferon-gamma ; analysis ; Interleukin-6 ; analysis ; Oligonucleotides ; metabolism ; RNA Interference ; RNA, Small Interfering ; metabolism ; Real-Time Polymerase Chain Reaction ; Receptors, Tumor Necrosis Factor ; antagonists & inhibitors ; genetics ; metabolism

OBJECTIVE: To study the safety and efficiency of the transfection of antisense oligonucletide into kidney mediated by lipid microbubbles, and to evaluate its potential clinical application. METHODS: The potential and conditions regarding the transfection self-made lipid microbubbles (CY5)-labeled-oligonucleotide (ODN) or CY5-labeled-ODN connective tissue growth factor (CTGF) into the rat kidney were evaluated. Th e safety was evaluated by HE staining, liver and renal function tests. The transfection efficiency was evaluated by fluorescence microscopy. Th e expression of CTGF was detected by RT-PCR and Western blot. RESULTS: Self-made lipid microbubble and/or ultrasound significantly enhanced the efficiency of gene transfer and expression in the kidney. Especially, 85%-90% of total glomerular could be transfected. CY5-labeled-ODN expression could be observed in glomerular, tubular and interstitial area. Th ere was no significant change in blood tests aft er gene transfer. Levels of LDH in 7 days were decreased compared with that at the fi rst day aft er the transfection (P<0.05). CTGF expression was successfully suppressed by transfection of CTGF-antisense-ODN into kidney. CONCLUSION The ultrasound-mediated gene transfer by self-made lipid microbubble could enhance the efficiency of ODN and expression in the rat kidney. Th is self-made lipid microbubbles supplement may be use for transfection of target genes.
Animals ; Connective Tissue Growth Factor ; genetics ; metabolism ; Kidney ; metabolism ; Lipids ; chemistry ; Microbubbles ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; Rats ; Transfection ; Ultrasonics

OBJECTIVETo observe the effect of anti-survivin oligonucleotides (ASODN) on the invasion and growth of peritoneally implanted ovarian cancer cell xenografts in nude mice.

METHODSNude mouse models bearing peritoneally implanted ovarian cancer cell (SKOV3) xenografts were established and subjected to intraperitoneal injection of survivin ASODN or saline (control). The number and weight of the intraperitoneal xenografts were compared between the two groups.The expressions of interleukin (IL-6), signal transducer and activator of transcription3 (STAT3), phosphorylated STAT3 (p-STAT3), and survivin protein in the tumor tissues were detected with Western blotting in both groups.

RESULTSCompared with those in the control group, the number and weight of the intraperitoneal xenografts were significantly reduced in ASODN group (P<0.05). ASODN treatment also resulted in significantly lowered protein levels of IL-6, STAT3, p-STAT3, and survivin in the tumor tissues (P<0.05).

CONCLUSIONSurvivin ASODN can suppress the invasion and migration capacity of ovarian cancer cells and inhibit peritoneal metastasis of the tumor in nude mice possibly though down-regulation of IL-6/STAT3 signaling pathway.

Animals ; Cell Line, Tumor ; Down-Regulation ; Female ; Humans ; Inhibitor of Apoptosis Proteins ; antagonists & inhibitors ; metabolism ; Interleukin-6 ; metabolism ; Mice ; Mice, Nude ; Neoplasm Transplantation ; Oligonucleotides ; pharmacology ; Ovarian Neoplasms ; pathology ; therapy ; STAT3 Transcription Factor ; metabolism

BACKGROUND: Several molecular assays have been developed to detect the BRAF V600E mutation in fine needle aspirates (FNAs) for the diagnosis of papillary thyroid cancer. Using a multiplex PCR technique, we evaluated the Anyplex BRAF V600E Real-time Detection (Anyplex) assay and compared its efficacy with that of the Seeplex BRAF V600E ACE Detection (Seeplex) method. METHODS: We tested 258 consecutive FNA specimens using the Seeplex and Anyplex assays. Any conflicting results between the two assays were confirmed by using mutant enrichment with 3'-modified oligonucleotide (MEMO) sequencing. The limits of detection (LODs) and reproducibility for each assay were evaluated with serially diluted DNA from a BRAF V600E-positive cell line. RESULTS: The BRAF V600E mutation was detected in 36.4% (94/258) FNA specimens by either the Seeplex or Anyplex assay. Results for the two assays showed 93.4% (241/258) agreement, with a kappa value of 0.861 (95% confidence interval, 0.798-0.923). Of the eight specimens that were BRAF V600E-positive by the Anyplex assay but not by the Seeplex assay, five were found to be BRAF V600E-positive by MEMO sequencing. The mutation detection rate of the Seeplex and Anyplex assays was 79.0% and 84.0%, respectively, in the FNA specimens diagnosed as malignant (n=81). The LOD as determined by probit analysis was 0.046% (95% confidence interval, 0.019-0.532%). CONCLUSIONS: The Anyplex assay performed better than the Seeplex assay with respect to the detection of the BRAF V600E mutation.
Adult ; Aged ; Asian Continental Ancestry Group/genetics ; Biopsy, Fine-Needle ; DNA/chemistry/metabolism ; DNA Mutational Analysis/*methods ; DNA Primers/*metabolism ; Female ; Humans ; Male ; Middle Aged ; Multiplex Polymerase Chain Reaction ; Oligonucleotides/metabolism ; Polymorphism, Single Nucleotide ; Proto-Oncogene Proteins B-raf/*genetics ; Republic of Korea ; Thyroid Nodule/*metabolism/pathology

OBJECTIVEThe present study was undertaken to design antisense oligodeoxynucleotides (AS-ODNs) of glial glutamate transporter-la (GLT-1a) and to evaluate the effectiveness of the designed AS-ODNs on the expression of GLT-1a.

METHODSFive sequences of GLT-1a AS-ODNs were designed according to the C terminus specific sequences of GLT-1a mRNA using antisense design software of IDT Com- pany. Western blot analysis was used to evaluate the inhibition effects of the five GLT-1a AS-ODNs on the expression of GLT-la.

RESULTSThe sequence of GLT-1a AS-ODNs with sequence of 5'-GGTTCTTCCTCAACACTGCA-3' could specifically inhibit the expression of GLT-1a in the hippocampal CA1 subfield of rats, while it had no effect on the expression of GLT-1b. This sequence showed similar inhibition on the expression of GLT-la in sham and ceftriaxone (Cef)-treated rats. It could also significantly inhibit the cerebral ischemic preconditioning (CIP)-induced up-regulation in the expression of GLT-1a. The magnitude of the inhibition in sham, Cef- or CIP-treated rats was similar by more than 60%.

CONCLUSIONFrom the designed five sequences of GLT-1a AS-ODNs, we obtained an effective sequence which can specifically inhibit the expression of GLT-1a.

Animals ; CA1 Region, Hippocampal ; metabolism ; Excitatory Amino Acid Transporter 2 ; antagonists & inhibitors ; metabolism ; Ischemic Preconditioning ; Oligonucleotides, Antisense ; genetics ; RNA, Messenger ; Rats ; Up-Regulation

OBJECTIVETo observe the effects of miR-224 antisense oligonucleotide (ASO) on the proliferation and apoptosis of gastric cancer cells in vitro and vivo.

METHODSThe expression of miR-224 in the cancer tissues and their adjacent tissues in 120 gastric cancer patients were detected by real-time quantitative PCR. The biological effects of miR-224 ASO on human gastric cancer SGC7901 cells was assessed by MTT assay, clone formation assay, flow cytometry and in vivo experiment in nude mice.

RESULTSCompared with the control group (0.50 ± 0.07), miR-224 ASO significantly reduced the miR-224 mRNA expression in the cancer patients (0.09 ± 0.01, P < 0.05). MTT assay results showed that the survival rate of gastric cells at 24 h, 48 h and 72 h was 53.6%, 59.1% and 70.1% in the miR-224 ASO group, and 12.3%, 17.4% and 24.7%, respectively, in the control group (P < 0.05 for all). Clone formation assay revealed that clone formation rate in the miR-224 ASO group was (5.33 ± 0.74)%, significantly lower than the (33.33 ± 8.38)% in the control group (P < 0.05). Flow cytometry indicated that the apoptotic index was (15.68 ± 1.46)% in the miR-224 ASO group and (3.36 ± 0.88)% in the control group (P < 0.01). In addition, the expressions of Bcl2 mRNA and protein were 1.05 ± 0.04 and 0.21 ± 0.03 in the miR-224 ASO group, significantly lower than that in the control group (4.87 ± 0.96 and 0.88 ± 0.09, P < 0.01). The in vivo study further showed that the tumor volume in the experimental group is significantly smaller than that in the control group (P = 0.01).

CONCLUSIONSMiR-224 is overexpressed in human gastric cancer. Reducing the expression of miR-224 can effectively inhibit the growth and promote apoptosis of gastric cancer cells. miR-224 may become a new target for the regulation of gene expression in gastric cancer.

Adult ; Aged ; Animals ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Female ; Humans ; Male ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; MicroRNAs ; genetics ; metabolism ; Middle Aged ; Neoplasm Transplantation ; Oligonucleotides, Antisense ; metabolism ; Proto-Oncogene Proteins c-bcl-2 ; genetics ; metabolism ; RNA, Messenger ; metabolism ; Stomach Neoplasms ; genetics ; metabolism ; pathology ; Tumor Burden

OBJECTIVE: To select specific DNA aptamer for determining ketamine by FluMag-SELEX. METHODS: Based on magnetic beads with tosyl surface modification as solid carrier and ketamine as target, a random ssDNA library with total length of 78 bp in vitro was compounded. After 13 rounds screening, DNA cloning and sequencing were done. Primary and secondary, structures were analyzed. The affinity, specificity and Kd values of selected aptamer were measured by monitoring the fluorescence intensity. RESULTS: Two ssDNA aptamers (Apt#4 and Apt#8) were successfully selected with high and specific abilities to bind ketamine as target with Kd value of 0.59 and 0.66 μmol/L. The prediction of secondary structure was main stem-loop and G-tetramer. The stem was the basis of stability of aptamer's structure. And loop and G-tetramer was the key of specific binding of ketamine. CONCLUSION FluMag-SELEX can greatly improve the selection efficiency of the aptamer, obtain the ketamine-binding DNA aptamer, and develop a new method for rapid detection of ketamine.
Aptamers, Nucleotide/metabolism* ; DNA ; DNA, Single-Stranded/genetics* ; In Vitro Techniques ; Ketamine/metabolism* ; Oligonucleotides ; SELEX Aptamer Technique/methods*

This paper is aimed to investigate the inhibitory effects of hepatitis B virus (HBV) preC and C genes-specific antisense locked nucleic acid (LNA) on HBV replication and expression in transgenic mice. The antisense LNA, which was complementary to the preC and C gene region of HBV, was designed, synthesized, and injected into transgenic mice via the tail vein. Serum HBV DNA was tested with real-time PCR, and Serum HBsAg was tested with time-resolved fluorescence immune assay (TRFIA). Then the expression of HBcAg in the liver was detected with immuneohistochemistry. Serum ALB, ALT, BUN and CRea were measured with an antomatic biochemicall analyzer. It was found that 5 days after LNA injection, serum HBV DNA levels in the dual-target group were reduced by 53.72%, and serum HBsAg levels were decreased by 71.57%. These values were significantly higher than those in the control groups (P<0.05) and the expression levels of HBcAg in the liver were significantly lower than those in the control groups (P<0.05). The result also showed that there were no significant differences discovered in serum ALB, ALT, BUN and CR between the experiment groups and the control groups. The present study provides that antisense LNA targeting to both preC and C genes has shown strong inhibition on HBV replication and expression in transgenic mice, and stronger than target at single gene site.
Animals ; Antiviral Agents ; pharmacology ; DNA, Viral ; blood ; Female ; Gene Targeting ; Hepatitis B Core Antigens ; metabolism ; Hepatitis B Surface Antigens ; blood ; Hepatitis B virus ; drug effects ; genetics ; physiology ; Liposomes ; Male ; Mice ; Mice, Transgenic ; Oligonucleotides ; pharmacology ; Oligonucleotides, Antisense ; pharmacology ; Virus Replication ; drug effects

Echinomycin is a small-molecule inhibitor of hypoxia-inducible factor-1 DNA-binding activity, which plays a crucial role in ovarian ovulation in mammalians. The present study was designed to test the hypothesis that hypoxia-inducible factor (HIF)-1alpha-mediated endothelin (ET)-2 expressions contributed to ovarian ovulation in response to human chorionic gonadotropin (hCG) during gonadotropin-induced superuvulation. By real-time RT-PCR analysis, ET-2 mRNA level was found to significantly decrease in the ovaries after echinomycin treatment, while HIF-1alpha mRNA and protein expression was not obviously changed. Further analysis also showed that these changes of ET-2 mRNA were consistent with HIF-1 activity in the ovaires, which is similar with HIF-1alpha and ET-2 expression in the granulosa cells with gonadotropin and echinomycin treatments. The results of HIF-1alpha and ET-2 expression in the granulosa cells transfected with cis-element oligodeoxynucleotide (dsODN) under gonadotropin treatment further indicated HIF-1alpha directly mediated the transcriptional activation of ET-2 during gonadotropin-induced superuvulation. Taken together, these results demonstrated that HIF-1alpha-mediated ET-2 transcriptional activation is one of the important mechanisms regulating gonadotropin-induced mammalian ovulatory precess in vivo.
Animals ; Cells, Cultured ; Chorionic Gonadotropin/*pharmacology ; Echinomycin/*pharmacology ; Endothelin-2/genetics/*metabolism ; Female ; Gonadotropins, Equine/*pharmacology ; Granulosa Cells/drug effects/metabolism ; Humans ; Hypoxia-Inducible Factor 1, alpha Subunit/*antagonists & inhibitors/genetics/metabolism/physiology ; Oligonucleotides/genetics ; Ovary/cytology/drug effects/physiology ; Rats ; Rats, Sprague-Dawley ; Superovulation/*drug effects ; Transcriptional Activation

Papillary thyroid carcinoma (PTC) is the most common malignant thyroid tumor, and 36-69% of PTC cases are caused by mutations in the BRAF gene. The substitution of a valine for a glutamic acid (V600E) comprises up to 95-100% of BRAF mutations; therefore, most diagnostic methods, including allele-specific PCR and real-time PCR, are designed to detect this mutation. Nevertheless, other mutations can also comprise the genetic background of PTC. Recently, a novel and sensitive technique called mutant enrichment with 3'-modified oligonucleotides (MEMO) PCR has been introduced. When we applied allelespecific PCR and MEMO-PCR for the detection of the BRAF V600E mutation, we found an unusual 3' bp deletion mutation (c.1799_1801delTGA) only when using MEMO-PCR. This deletion results in the introduction of a glutamic acid into the B-Raf activation segment (p.V600_K601delinsE), leading to an elevated basal kinase activity of BRAF. This is the first report of a rare 3 bp BRAF deletion in a PTC patient that could not be detected by allele-specific PCR.
Alleles ; Base Sequence ; Female ; Humans ; Lymphatic Metastasis ; Middle Aged ; Oligonucleotides/genetics ; Polymerase Chain Reaction/*methods ; Proto-Oncogene Proteins B-raf/*genetics/metabolism ; Sequence Deletion ; Thyroid Neoplasms/genetics/pathology ; Thyroid Nodule/metabolism