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

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 test the efficiency of transfecting (99)Tc(m)-labeled anti-miR208b oligonucleotide into early hypertrophic cardiac myocytes in vitro.

METHODSThe anti-oligonucleotide targeting miR208b (AMO) was synthesized and modified with LNA followed by conjugation with N-hydroxysuccinimidyl S-acetyl-meraptoacetyl triglycine (NHS-MAG3) and radiolabeling with (99)Tc(m). NHS-MAG3-LNA-AMO and labeled AMO were purified with Sep-Pak C18 column chromatography, and the former was examined for UV absorption at the 260 nm using Gene Quant DNA/RNA calculator. The labeling efficiency, radiochemical purity, stability and molecular hybridization activity were analyzed. An angiotensin II-induced cell model of hypertrophic cardiac myocytes was transfected with (99)Tc(m)-NHS-MAG3-LNA-AMO via liposome, and the relative expression of miRNA208b and retention ratio of the labeled AMO in early hypertrophic cells were determined.

RESULTSThe labeling efficiency and radiochemical purity of the labeled AMO after purification exceeded 84% and 86%, respectively. The radio- chemical purities of the labeled AMO incubated in serum and normal saline for 12 h were both higher than 80%, and the labeled AMO showed a capacity to hybridize with the target gene. In the hypertrophic model of cardiac myocytes, the retention ratio of labeled AMO at 6 h was higher than 20%.

CONCLUSIONThe (99)Tc(m)-labeled antisense probe can be efficiently transfected into hypertrophic cardiac myocytes in vitro, which provides an experimental basis for subsequent radionuclide imaging studies.

Humans ; Isotope Labeling ; Liposomes ; MicroRNAs ; genetics ; Myocytes, Cardiac ; Oligonucleotides ; Oligonucleotides, Antisense ; Oligopeptides ; Radiopharmaceuticals ; Silicon Dioxide ; Succinimides ; Transfection

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

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

DNA, Viral ; genetics ; Hep G2 Cells ; Hepatitis B virus ; drug effects ; Humans ; Oligonucleotides, Antisense ; pharmacology

OBJECTIVETo investigate the effect of functional blocking of endogenous miR-23a with a specific antisense oligonucleotide (ASO) on the proliferation and invasiveness of gastric adenocarcinoma cell line MGC803 in vitro.

METHODSA specific ASO targeting miR-23a, namely ASO-23a, was transfected into MGC803 cells to block endogenous miR-23a. The mRNA level of miR-23a in the transfected cells was detected with quantitative real-time PCR. The changes of cell proliferation following the transfection were detected with MTT assay and colony formation assay, and TUNEL assay and Transwell assay were employed to evaluate the changes in cell apoptosis and invasiveness, respectively.

RESULTSQuantitative real-time PCR demonstrated efficient functional blocking of endogenous miR-23a in MGC803 cells by ASO-23a. Suppression of miR-23a with ASO-23a obviously inhibited cell growth, colony formation and invasiveness of MGC803 cells and significantly enhanced the cell apoptosis.

CONCLUSIONASO-23a can efficiently block the function of endogenous miR-23a in MGC803 cells to inhibit cell proliferation and invasion and promote cell apoptosis.

Adenocarcinoma ; genetics ; pathology ; Apoptosis ; Cell Line, Tumor ; Cell Proliferation ; Humans ; MicroRNAs ; genetics ; Oligonucleotides, Antisense ; Stomach Neoplasms ; genetics ; pathology ; Transfection

The effect of BHC80 (a component of BRAF-HDAC complex) on development was not well studied, because BHC80 gene knock-out mice died in one day after birth. Interestingly, zebrafish embryos can live, even if their important organs like cardiac system has severe dysfunction, as 25%-40% O2 are supplied through their skin. Therefore, a model of BHC80 gene knock-down zebrafish embryos was established to explore the effect of BHC80 on the early embryonic development. BHC80-morpholino antisense oligonucleotides 2 (BHC80-MO2) was designed and injected into zebrafish embryos to interrupt the correct translation of BHC80 mRNA at one or two cells stage, which was proved by RT-PCR analysis. Two control groups, including non-injection group and control-MO (con-MO) injection group, and four different doses of BHC80-MO2 injection groups, including 4 ng, 6 ng, 8 ng and 10 ng per embryo were set up. The embryonic heart phenotype and cardiac function were monitored, analyzed and compared between con-MO and BHC80-MO2 groups by fluorescence microscope in vmhc:gfp transgenic zebrafish which express green fluorescent protein in ventricle. The results showed that BHC80-MO2 microinjection effectively knocked down the BHC80 gene expression, because the BHC80-MO2 group emerged a new 249 bp band which reduced 51 bp compared to 300 bp band of con-MO group in RT-PCR analysis, and the 51 bp was the extron 10. The abnormal embryo rate rose with the increase of BHC80-MO2 dosage. The proper BHC80-MO2 injection dosage was 8 ng per embryo, as minor embryos had abnormal phenotype in 4 ng and 6 ng per embryo groups and most embryos died in 10 ng per embryo group. BHC80-MO2 embryos exhibited abnormal cardiac phenotype, including imbalance of the proportion of heart ventricle to atrium, incomplete D-loop, even tubular heart, slow heart rates and cardiac dysfunction. The results from a model of BHC80 gene knock-down zebrafish embryos show that the abnormal cardiac phenotype and cardiac dysfunction of BHC80-MO2 embryos may be one of the probable reasons for the BHC80 gene knock-out mice death, which would provide a good research model to clarify the mechanism of cardiac development.
Animals ; Embryonic Development ; genetics ; Gene Expression Regulation, Developmental ; Gene Knockdown Techniques ; Heart ; embryology ; Histone Deacetylases ; genetics ; Mice, Knockout ; Oligonucleotides, Antisense ; RNA, Messenger ; Zebrafish ; embryology ; Zebrafish Proteins ; genetics

OBJECTIVE: To construct FA targeted magnetic nanocomplex (FA-MNP-MMP-9-ASODN) loading matrix metalloproteinase 9 (MMP-9) antisense oligonucleotide (ASODN) and evaluate its targeting capacity and efficiency of gene transfection to folate receptor (FR) positive NPC. METHOD: FA-MNP-MMP-9-ASODN was constructed by MMP-9-ASODN coupling with FA-MNP prepared by our research team through the aldehyde-ammonia condensation reaction. To analyze the feasibility of ASODN coupling with nanocarrier agarose gel electrophoresis. Two kinds of HNE-1 and CNE-2 cells and implanted tumors phagocytosis of FA-MNP-MMP-9-ASODN were observed by MRI on tumor-bearing nude mice, iron staining and TEM. To analyze gene transfection of the vector by observing FITC in the cell. RESULT: The electrophoresis results revealed ASODN successfully coupling with FA-MNP. HNE-1 cell can effectively ingest the nanocomposite,with more FITC in the cell, but CNE-2 cell had not uptake for the nanocomposite, with no FITC in the cell. By comparing with CNE-2 tumor, HNE-1 tumor also can efficiently swallow the nanocomposite. CONCLUSION FA-MNP-MMP-9-ASODN nanocomplex is constructed successfully with good FA targeting ability and gene transfection.
Animals ; Folic Acid ; genetics ; Genetic Vectors ; Matrix Metalloproteinase 9 ; genetics ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Nanocomposites ; Neoplasm Transplantation ; Oligonucleotides, Antisense ; genetics ; Transfection

OBJECTIVETo explore the effect of microRNA-21 (miR-21) antisense oligonucleotide on the biological characteristics of human cervical squamous carcinoma cell lines SiHa in vivo and in vitro.

METHODSSpecific phosphorothioate antisense oligodeoxynucleotides targeting miR-21 were synthesized and transfected into cervical cancer cells in vitro. Expression of miR-21 in SiHa after transfection was detected by real-time RT-PCR. The cell proliferation was evaluated by MTT assay and colony formation experiment. The cell apoptosis was analyzed by annexin V-FITC/PI analysis. The inhibitory effect of miR-21 antisense oligonucleotide on tumor growth was evaluated by tumor growth curves and immunohistochemistry (MaxVision method). H-E staining was used to document morphological changes and fluorometric TUNEL assay was to detect the apoptotic activity.

RESULTSAfter the transfection of antisense miR-21, the expression of miR-21 decreased along with an obvious growth inhibition, compared with that of the control groups (P < 0.05). Colony formation of both cell lines was markedly inhibited with antisense miR-21 (55.6% ± 1.4%), as compared with that in the negative group (98.3% ± 2.0%, P < 0.05). Flow cytometry assay showed that antisense miR-21 expression significantly enhanced the cell apoptosis (6.7% ± 1.3% and 29.4% ± 1.7%, P < 0.05). The tumor-forming rates of miR-21 transfected group, and negative control groups were 3/8 and 6/8, respectively (P < 0.05). Ki-67 proliferative marker staining decreased significantly (42% vs 90%) in the transfected group compared with negative control groups. Extensive dead tumor cells were seen in the miR-21 transfected cells along with a marked increase of apoptosis (P < 0.05).

CONCLUSIONTargeted antisense oligonucleotide miR-21 effectively suppresses the growth of cervical carcinoma SiHa cells both in vitro and in vivo through an induction of apoptosis.

Animals ; Apoptosis ; Carcinoma, Squamous Cell ; genetics ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Female ; Humans ; Ki-67 Antigen ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; MicroRNAs ; genetics ; metabolism ; Neoplasm Transplantation ; Oligonucleotides, Antisense ; metabolism ; Transfection ; Uterine Cervical Neoplasms ; genetics ; metabolism ; pathology