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 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

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

MicroRNAs (miRNAs) are a class of highly abundant non-coding RNA molecules that are involved in several biological processes. Many miRNAs are often deregulated in several diseases including cancer. There is substantial interest in exploiting miRNAs for therapeutic applications. In this editorial, we briefly review current advances in the use of miRNAs or antisense oligonucleotides (antagomirs) for such therapies. One of the key issues related to therapy using miRNAs is degradation of naked particles in vivo. To overcome this limitation, delivery systems for miRNA-based therapeutic agents have been developed, which hold tremendous potential for improving therapeutic outcome of cancer patients.
Drug Delivery Systems ; methods ; Gene Expression Regulation, Neoplastic ; Genetic Therapy ; Humans ; MicroRNAs ; genetics ; metabolism ; therapeutic use ; Neoplasms ; genetics ; metabolism ; therapy ; Oligonucleotides, Antisense ; therapeutic use

OBJECTIVETo explore the influence of knock-down of miR-21 expression on the radiosensitivity of radioresistant glioma SHG-44 cells and its mechanism.

METHODSRadioresistant cell line SHG-44(R) cells were established from radiosensitive parental SHG-44 glioma cells. Then the expression levels of miR-21 in SHG-44(R) and SHG-44 were determined by quantitative real-time PCR. The effect of miR-21 on the radiosensitivity was assessed in SHG-44(R) with miR-21 inhibitor to decrease the miR-21 expression.

RESULTSmiR-21 was up-regulated 1.49-fold in SHG-44(R) cells relative to the SHG-44 cells. But after transfection with As-miR-21, the miR-21 expression in SHG-44(R) cells was knocked down significantly. As-miR-21 combined with radiation could synergistically enhanced mitotic death and apoptosis of SHG-44(R) cells, with SER of D(0) and D(q) being equal to 1.48 and 1.54, respectively. Expression levels of caspase-3 in the radiation group, AS-miR-21 transfection group and combination group were 2.24 ± 0.14, 2.05 ± 0.19 and 5.72 ± 0.45, respectively, and its expression in the combination group was higher than that in the other two groups.

CONCLUSIONSmiR-21 may be involved in the formation of radioresistance of glioma cells and as a potential target for enhancing the response of radioresistant-glioma cells to radiotheapy.

Apoptosis ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Gene Expression Regulation, Neoplastic ; Gene Knockdown Techniques ; Glioma ; genetics ; metabolism ; pathology ; Humans ; MicroRNAs ; genetics ; metabolism ; Oligonucleotides, Antisense ; genetics ; metabolism ; Radiation Tolerance ; Transfection

OBJECTIVETo elucidate the regulatory mechanism underlying proliferation and anti-apoptosis in NSCLC by overexpression of miR-21.

METHODSReal-time PCR was used to measure miR-21 abundance in non-small cell lung cancer (NSCLC) tumor samples and adjacent normal tissues, as well as NSCLC cell lines. Tumor suppressor genes as potential targets of miR-21 were predicted by sequence analysis. Luciferase assay and Western blot were used to assess the regulatory effect. The effect on A549 cell viability and apoptosis by miR-21-induced gene repression was tested by trypan-blue exclusion and flow cytometry.

RESULTSmiR-21 expression was 2.24-fold higher in the NSCLC tumor samples and 3.06-fold higher in the A549 cells than that in the adjacent normal tissues. Sequence prediction and gene expression regulation assays showed that miR-21 could reversely regulate the expression of PDCD4 (P < 0.01). Suppression of miR-21 expression is associated with an elevation of Pdcd4, resulting in a significant reduction of proliferation and the apoptosis rate (2.6%) was increased to 10.9%. Moreover, the anti-proliferation and pro-apoptotic effect by miR-21 suppression could be reversed by PDCD4 knock down.

CONCLUSIONSuppression of the tumor suppressor PDCD4 expression may be one of the important regulatory pathways of the miR-21-mediated cell proliferation and decrease of apoptosis in non-small cell lung cancer.

Aged ; Apoptosis ; Apoptosis Regulatory Proteins ; genetics ; metabolism ; Carcinoma, Non-Small-Cell Lung ; metabolism ; pathology ; Cell Line, Tumor ; Cell Proliferation ; Gene Expression Regulation, Neoplastic ; Humans ; Lung Neoplasms ; metabolism ; pathology ; Male ; MicroRNAs ; genetics ; metabolism ; Middle Aged ; Oligonucleotides, Antisense ; genetics ; RNA Interference ; RNA-Binding Proteins ; genetics ; metabolism ; Transfection

This paper was aimed to investigate the biodistribution and ability of free 131-bcl-2/bcl-xl ASON (FA) and anionic long circulation liposomes encapsulated with 131I-bcl-2/bcl-xlASON (NA), in tumor-bearing rats, to image breast cancer. We investigated the tissue distribution of NA in virgin female Sprague-Dawley (SD) rats with n-methyl nitrosourea (MNU)-induced breast cancers in situ. The percentage of the injected dose per gram (%ID/g) was calculated, with the maximum ratios of tumor to blood and tumor to muscle, after injections of NA and FA for 0.5 h, 1 h, 2 h, 3 h, 4 h, 6 h, 12 h and 24 h, respectively. The ability of NA to image breast cancer in tumor-bearing rats was determined using emission computed tomography (ECT). Seventy percent (90/130) SD rats in the study developed mammary tumors after MNU injection with the average latency (NA) (96 +/- 1.2)days. The %ID/g of NA in breast cancer tissue, tumor bearing rats in liver and spleen tumor tissues after 10 hours were (6.23 +/- 0.23) %ID/g, (12.00 +/- 0.26) %ID/g and (18.25 +/- 1.33)% ID/g, respectively. The ratios of tumor to blood 6.29 +/- 0.76 and tumor to muscle 10.55 +/- 0.68 in tumor bearing rats slowly maximized at 10 h post injection of NA, most probably due to the enhanced permeability and retention effect. Hence in radionuclide antisense scintigraphy, the breast cancer in rat was clearly displayed at 10h after iv administration of NA-D. However, tumors were not visualized in rats with the iv injection of NS and NN even at the delayed time. Due to the inhibition of rapid uptake of NA by the reticulo-endothelial system, NA displays valuable pharmacologic properties characterized by the enhanced accumulation in tumor.
Animals ; Female ; Iodine Radioisotopes ; Liposomes ; pharmacokinetics ; Mammary Neoplasms, Experimental ; metabolism ; Oligonucleotides, Antisense ; pharmacokinetics ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Radionuclide Imaging ; methods ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Tissue Distribution ; bcl-X Protein ; metabolism

BACKGROUNDThe signal transducer and activator of transcription 6 (STAT6) expression in lung epithelial cells plays a pivotal role in asthma pathogenesis. Activation of STAT6 expression results in T helper cell type 2 (Th2) cell differentiation leading to Th2-mediated IgE production, development of allergic airway inflammation and hyperreactivity. Therefore, antagonizing the expression and/or the function of STAT6 could be used as a mode of therapy for allergic airway inflammation.

METHODSIn this study, we synthesized a 20-mer phosphorothioate antisense oligonucleotide (ASODN) overlapping the translation starting site of STAT6 and constructed STAT6 antisense RNA (pANTI-STAT6), then transfected them into murine spleen lymphocytes and analyzed the effects of antagonizing STAT6 function in vitro and in a murine model of asthma.

RESULTSIn vitro, we showed suppression of STAT6 expression and interleukin (IL)-4 production of lymphocytes by STAT6 ASODN. This effect was more prominent when cells were cultured with pANTI-STAT6. In a murine model of asthma associated with allergic pulmonary inflammation in ovalbumin (OVA)-sensitized mice, local intranasal administration of fluorescein isothiocyanate (FITC)-labeled STAT6 ASODN to DNA uptake in lung cells was accompanied by a reduction of intracellular STAT6 expression. Such intrapulmonary blockade of STAT6 expression abrogated signs of lung inflammation, infiltration of eosinophils and Th2 cytokine production.

CONCLUSIONThese data suggest a critical role of STAT6 in the pathogenesis of asthma and the use of local delivery of STAT6 ASODN as a novel approach for the treatment of allergic airway inflammation such as in asthma.

Animals ; Asthma ; drug therapy ; metabolism ; Blotting, Western ; Cell Differentiation ; drug effects ; Cells, Cultured ; Female ; Interleukin-4 ; metabolism ; Lymphocytes ; drug effects ; metabolism ; Mice ; Mice, Inbred C57BL ; Oligonucleotides, Antisense ; chemistry ; pharmacology ; Phosphates ; pharmacology ; RNA, Antisense ; chemistry ; pharmacology ; Reverse Transcriptase Polymerase Chain Reaction ; STAT6 Transcription Factor ; genetics ; metabolism ; Th2 Cells ; drug effects ; metabolism