Identification and characterization of a novel group of natural antisense transcripts from RNA1.2 gene locus of human cytomegalovirus / 中华医学杂志(英文版)

Background@#Natural anti-sense transcripts (NATs), which are transcribed from the complementary DNA strand of annotated genes, exert regulatory function of gene expression. Increasing studies recognized anti-sense transcription widespread throughout human cytomegalovirus (HCMV) genome, whereas the anti-sense transcription of RNA1.2 gene locus has never been investigated. In this study, the transcription of the RNA1.2 anti-sense strand was investigated in clinically isolated HCMV strain.@*Methods@#Strand-specific high-through RNA-sequencing (RNA-seq) was performed to find possible anti-sense transcripts (ASTs). For analyzing and visualization of RNA-seq data sets, Integrative Genomics Viewer software was applied. To confirm these possibilities, Northern blotting and rapid amplification of cDNA ends (RACE) were used.@*Results@#Transcription of the opposite strand of RNA1.2 gene locus was detected by RNA-sequencing using RNAs extracted from human embryonic lung fibroblasts infected with HCMV clinical isolate HAN. At least three HCMV NATs, named RNA1.2 AST 1, RNA1.2 AST2, and RNA1.2 AST3, were characterized by Northern blotting and RACE analyses. These RNA1.2 ASTs orientated from the complementary strand of RNA1.2 locus during the late phase of HCMV infection. The 5′- and 3′-termini of these transcripts were located within the opposite sequence of the predicted RNA1.2 gene.@*Conclusion@#A cluster of novel NATs was transcribed from the opposite sequence of the HCMV RNA1.2 gene region.

Promotion effects of survivin-ASODN on apoptosis of SMMC-7221 cell and its mechanism / 吉林大学学报(医学版)

Objective To study the influence of survivin targetedly inhibited with antisense oligonucleotide (ASODN)technique on the apoptosis of hepatoma carcinoma cells SMMC-7221,and to clarify the mechanism of promotion effect of survivin-ASODN on the apoptosis of SMMC-7721 cells.Methods The ASODN sequence of survivin marked by FAM fluorescein was designed and synthized. The SMMC-7721 cells were transfected by different concentrations (100,200,300,400,and 600 nmol· L-1 )of survivin-ASODN (ASODN transfection groups),at the same time blank control group and blank liposome control group and sense oligonucleotide (SODN) control group were set up.The apoptotic rates and the changes of cell cycle of the SMMC-7721 cells 24,48,and 72 h after transfected with different concentrations of survivin-ASODN were detected by FCM. The expression levels of survivin were measured by Western blotting method.Results Compared with each control group,24 h after transfection,the apoptotic rates of survivin-ASODN transfected SMMC-7221 cells were increased,the growth of cells was inhibited (P<0.05),and the effects had time-dose dependent tendency.48 h after transfection,the hypodiploid apoptotic peak appeared in ASODN transfection groups before G1 phase, the number of the cells at G0/G1 phase was decreased (P<0.05)and the number of the cells at G2/M phase wsa increased (P<0.05). Compared with each control group,the survivin expression levels in the SMMC-7721 cells in ASODN transfection groups were decreased (P<0.05 ), and the effects of survivin-ASODN was time-dose dependent (P<0.05 ). Conclusion Survivin-ASODN can block the expression of survivin in SMMC-7721 cells and inhibit the proliferation of SMMC-7721 cells by changing the cell cycle and increasing apoptosis in a time-dose dependent manner.

Expression of pax5 gene in wildtype zebrafish embryos / 第二军医大学学报

Objective To study the role and related mechanisms of pax5 gene during the development of the embryos, especially in the early development of B cell and midbrain, and to observe the expression of pax5 gene in wild type zebra fish embryos. Methods Total RNA of Tue Un gen wild type zebra fish embryos was extracted to obtain cDNA of pax5 gene by RT-PCR with specific primers. The c DNA of pax5 gene and pCS2+ vector were double digested with EcoR I and Xba I, and then ligated by T4 DNA ligase. The recombinant vector was verified by double digestion, colony PCR screening and sequencing. The verified recombinant vector was then used to synthesize digoxin labeled anti-sense mRNA probe of pax5 gene using T3 RNA polymerase in vitro transcription system. The generated probes were used to detect pax5 gene expression in zebra fish embryos by whole-mount in situ hybridization. Results The pCS2+-pax5 recombinant plasmid and the probe of digoxin-labeled anti-sense mRNA of pax5 gene was successfully constructed. Whole-mount in situ hybridization showed that pax5 gene was expressed in the cerebellum and midbrain hindbrain boundary from 18-72 h post-fertilization (hpf). In the cochlea pax5 gene was expressed from 24-72 hpf and the expression was increased as time went by. From 18-48 hpf pax5 gene is found in the notochord. Conclusion Pax5 gene is highly expressed in the brain and notochord of zebra fish embryos. Pax5 gene expression is found in the cochlea of the Tue bingen wild type zebra fish' s embryos for the first time. It is suggested that pax5 may play an important role in the development of the nervous system in the early zebra fish embryos.

Blocking effects of anti-sense peptide of C5a on the adhesion between pulmonary vascular endothelial cell and neutrophil / 中国免疫学杂志

Objective:To study the blocking effects of anti-sense peptide of C5a on the adhesion between pulmonary vascular endothelial cell(PVEC) and neutrophil resulting from C5a anaphylatoxin.Methods:It was determined by Flowcytometry that the change of adhesion molecule expression on PVEC and the activity of MPO in PMN was determined after adhesion.Results:In response to C5a after interactions with several concentrations of anti-sense peptide R4, the expression of P-Selectin on PVEC decreased significantly and reached the minimum at the concentration of 5 000 ng/ml, and the activity of MPO in PMN reduced by 40% at the concentration of 5 000 ng/ml of anti-sense peptide R4.Conclusion:The results suggested that anti-sense peptide of C5a has significant blocking effects on C5a anaphylatoxin.

THE INHIBITION OF CELL PROLIFERATION IN CARCINOMA OF LARGE INTESTINE BY TRANSFORMING GROWTH FACTOR-ALPHA / 肿瘤研究与临床

Objective To observe the inhibition of transforming growth factor-alpha(TGFa) anti-senseoligodeoxynucleotides on the malignant proliferation of HR8348 cell line. Methods Using the TGFa anti-sense oligodeoxynucleotides, composed of 23 and indifferent oligodeoxynucleotides, to affect the HR8348cell. By observing the cell growth inhibiton, 3H-TdR incorporation,mRNA hybridization and the cell cycleanalysis to identify the inhibiting effects of TGFa anti-sense oligodeoxynucleotides and its mechanism.Results TGFa anti-sense oligodeoxynucleotides can Inhibit the proliferation of HR8348 cell, DNA0synthesis,mRNA expression,and defer the transition period of G,/G, phase to S phase. Conclusion TGFaanti-sense oligodeoxynucleotides can inhibit the malignant proliferation of HR8348 cell effectively.

Molecular Approaches for Brain Tumor Therapy;Gene Transfer and Anti-sense Oligonucleotides

Despite advances in neurosurgery, radiation, and chemotherapy, the prognosis of patients with malignant brain tumors still remains grim. Considerable efforts have been made to develop new therapeutic strategies for malignant brain tumors. One of the promising new therapies for brain tumors is an intervention at molecular level, and several molecular approaches have been shown to have in vitro and in vivo activities. These include the use of retroviral vectors, herpes simplex viruses, adenoviral vectors in gene transfer, and antisense vectors and oligonucleotides. Preclinical studies of retroviral vector have already been extended to clinical trials, clearly demonstrating the clinical potential of these molecular therapies. Here, I discuss the current status of molecular therapy for brain tumors together with future directions for its development.

Antagonism Function of Anti-Sense Nucleic Acids on IL-6 Signal Transduction in Myeloma Cells / 中国肿瘤生物治疗杂志

Objective: To investigate which step of IL-6 signal transduction pathways in myeloma cells can be taken as the acting target of the antagonists for IL-6. Methods: EMSA and immunoprecipitation were used to detect the activation of transcription factors(TFs)-STAT3, NF-IL-6 and protein kinase ERK in a myeloma cell line-Sko-007 by IL-6. then anti-sense expression plasmids and anti-sense ODN for these signal moleculars were constructed and designed,the effects of these anti-sense nucleic acids on IL-6 signal transduction in Sko-007 cells were analyzed by the same methods. Results: IL-6 signal transduction could be antagonized by these anti-sense nucleic acids at different extent.Conclusion: TFs or protein kinases in IL-6 signal trareduction pathways can be taken as the target for antagonists design.

Hepatocarcinoma specific IL-1? anti-sense RNA inhibits implanted hepatocarcinoma in mice / 中国肿瘤生物治疗杂志

Objective :To construct hepatocarcinoma specific IL-1? anti-sense RNA expression vector and to explore its effect on the growth of implanted hepatocarcinoma H22 cells in mice and the possible mechanism. Methods:Murine IL-1? anti-sense RNA expression vectors pafpIRES2-antiIL-1?1 and pafpIRES2-antiIL-1?2 under the regulation of minimal alpha-feto protein (AFP) promoter and CMV enhancer were constructed,and further verified by PCR,restriction endonuclease analysis and DNA sequencing. H22 cells transfected with pafpIRES2-antiIL-1? 1 or pafpIRES2-antiIL-1? 2 were divided into 3 groups:H22/mock,H22/antiIL-1?1 and H22/antiIL-1?2 group. Expression of IL-1? was detected by RT-PCR. Transfected H22 cells were subcutaneously injected into mice to establish tumor implanted mouse model. Tumor volume was measured; the cytotocixity of spleen NK against H22 cells was detected by MTT. Results:Hepatocarcinoma specific IL-1? anti-sense RNA expression vectors pafpIRES2-antiIL-1?1 and pafpIRES2-antiIL-1?2 were successfully constructed and were verified by PCR,restriction endonuclease analysis and DNA sequencing. IL-1? expression in H22 cells was down-regulated after transfected with IL-1? anti-sense RNA expression vectors,especially with the pafpIRES2-antiIL-1?2 vector. Hepatocarcinoma cells implanted mouse model was successfully established. Tumor volume and growth of tumor in H22/antiIL-1?2 mice was obviously smaller than that in H22/mock mice,and the cytotocixity of spleen NK against H22 cells in H22/antiIL-1?1 and H22/antiIL-1?2 mice was also greatly enhanced. Conclusion:Hepatocarcinoma specific IL-1? anti-sense RNA expression vector pafpIRES2-antiIL-1? was successfully constructed. It effectively inhibits the growth of implanted hepatocarcinoma in mice probably through specifically blocking expression of IL-1? and increasing cytotocixity of spleen NK.

GROWTH INHIBITION OF LUNG CANCER CELL LINE in vitro BY TELOMERASE ANTI-SENSE OLIGONUCLEATIDES / 解剖学报

Objective To study the inhibition of lung cancer cell line by telomerase anti\|sense DNA,and discuss the possibility of using it in clinical treatment. Methods A phosphorothioate oligonucleotide(PS\|ODN) with sequence identical to the repeat sequence of the mammalian telomere 5′\|d(TTAGGG)\|3′ and a control scrambled sequence 5′\|d(TGTGAG)\|3′ were incubated with a lung cancer cell line.The effects of PS\|ODN on cell line growth,colony\|forming and growth shape were detected.The in vivo efficacy of this PS\|ODN was evaluated in a 801\|D nude mouse model.Once tumors were established these animals were administered PS\|ODN or saline for 15 days. Results Telomease anti\|sense DNA inhibit telomerase activation of cell line 801\|D growth and colony\|forming.The activity of the 6\|mer telomere mimic demonstrated a dose dependency.No activity was observed with the scrambled controls.A significant decrease in tumor weight was observed in animals given PS\|ODN, but not followig saline\|treated animale.Conclusion\ These results demonstrated that short hexameric oligonucleotide telomere exerts the growth inhibitory effect on lung cancer cell in vitro and in vivo, and suggest the potential utility of telomerase anti\|sense DNA as cancer cell inhibitors.\;[