Therapeutic advances in 5q-linked spinal muscular atrophy / Avanços terapêuticos na atrofia muscular espinhal ligada ao cromossomo 5q

ABSTRACT Spinal muscular atrophy (SMA) is a severe and clinically-heterogeneous motor neuron disease caused, in most cases, by a homozygous mutation in the SMN1 gene. Regarding the age of onset and motor involvement, at least four distinct clinical phenotypes have been recognized. This clinical variability is, in part, related to the SMN2 copy number. By now, only supportive therapies have been available. However, promising specific therapies are currently being developed based on different mechanisms to increase the level of SMN protein; in particular, intrathecal antisense oligonucleotides that prevent the skipping of exon 7 during SMN2 transcription, and intravenous SMN1 insertion using viral vector. These therapeutic perspectives open a new era in the natural history of the disease. In this review, we intend to discuss the most recent and promising therapeutic strategies, with special consideration to the pathogenesis of the disease and the mechanisms of action of such therapies.

RESUMO A atrofia muscular espinhal (AME) é uma grave doença dos neurônios motores, de grande variabilidade clínica e causada na maioria dos casos por mutação em homozigose no gene SMN1. Pelo menos quatro fenótipos clínicos distintos são reconhecidos com base na idade de início e no grau de envolvimento motor. Tal variabilidade clínica é em parte relacionada com o número de cópias do gene SMN2. Até recentemente, apenas terapias de suporte estavam disponíveis. Atualmente, terapias especificas estão sendo desenvolvidas com base em diferentes mecanismos para aumentar o nível de proteína SMN; em particular oligonucleotídeos antissenso por via intratecal e inserção de cópia do gene SMN1, via endovenosa, usando vetor viral. Nesta revisão, objetivamos discutir as mais recentes e promissoras estratégias terapêuticas, com consideração especial aos aspectos patogênicos da doença e aos mecanismos de ação de tais terapias.

INXS, um longo RNA não codificador de proteínas mediador da apoptose / INXS, a long noncoding RNA that mediates apoptosis

O splicing alternativo do pré-mRNA de BCL-X produz duas isoformas de mRNAs com funções antagônicas, a pró-apoptótica BCL-XS e a anti-apoptótica BCL-XL, cujo balanço regula a homeostasia celular. Entretanto, o mecanismo que regula esse processamento ainda é desconhecido. Nesse trabalho, nós identificamos e caracterizamos um longo RNA não codificador de proteínas (lncRNA) nomeado INXS, que é transcrito a partir da fita oposta do locus genômico de BCL-X, sendo menos abundante em linhagens celulares tumorais e tecidos tumorais de pacientes quando comparados com os respectivos pares não tumorais. INXS é um RNA unspliced de 1903 nts, é transcrito pela RNA Polimerase II, possui cap 5', está enriquecido na fração nuclear das células e se liga à proteína Sam68 do complexo modulador de splicing. O tratamento de células tumorais 786-O com cada um de três agentes indutores de apoptose aumentou a expressão endógena do INXS, levando ao aumento expressivo da proporção entre os mRNAs de BCL-XS / BCL-XL, e ativação das caspases 3, 7 e 9. Estes efeitos foram anulados na presença do knockdown do INXS. Da mesma forma, a superexpressão ectópica do INXS causou uma mudança no splicing favorecendo a isoforma BCL-XS e ativação das caspases, aumentando os níveis da proteína BCL-XS e conduzindo as células à apoptose. Utilizando um modelo in vivo, cinco injeções intra-tumorais do INXS durante 15 dias causaram uma regressão acentuada no volume dos xenotumores. Portanto, INXS é um lncRNA que induz a apoptose, sugerindo que essa molécula seja um possível alvo a ser explorado na terapia contra o câncer

BCL-X mRNA alternative splicing generates pro-apoptotic BCL-XS or anti-apoptotic BCL-XL, whose balance regulates cell homeostasis. However, the mechanism that regulates the splice shifting is incompletely understood. Here, we identified and characterized a long noncoding RNA (lncRNA) named INXS, transcribed from the opposite genomic strand of BCL-X, that was less abundant in tumor cell lines and patient tumor tissues compared with non-tumors. INXS is an unspliced 1903 nt-long RNA, is transcribed by RNA Polymerase II, 5'-capped, nuclear enriched and binds Sam68 splicing-modulator. The treatment of tumor cell line 786-O with each of three apoptosis-inducing agents increased endogenous INXS lncRNA, increased BCL-XS / BCL-XL mRNA ratio, and activated caspases 3, 7 and 9. These effects were abrogated in the presence of INXS knockdown. Similarly, ectopic INXS overexpression caused a shift in splicing towards BCL-XS and activation of caspases, increasing the levels of BCL-XS protein and then leading the cells to apoptosis. In a mouse xenograft model, five intra-tumor injections of INXS along 15 days caused a marked regression in tumor volume. INXS is an lncRNA that induces apoptosis, suggesting that INXS is a possible target to be explored in cancer therapies

Growth inhibition effection of perlecan anti-sense cDNA on human laryngeal carcinoma xnograft in nude mice / 临床耳鼻咽喉头颈外科杂志

OBJECTIVE@#To observe growth inhibition effect of perlecan anti-sense cDNA (pAP) on human laryngeal carcinoma xnografted in nude mice. To vertify its antitumor effect and mechanism in vivo, and it may be useful as a biomarker in carcinoma of larynx cancer.@*METHOD@#Created the model of human laryngeal carcinoma xnograft in nude mice. To observe growth of those xnografts in nude mice and draw growth curve of xnografted. The expression of perlecan mRNA and portein in xnografts were examined by RT-PCR and immunohistochemistry.@*RESULT@#Volume of xnografts in the group transfected by the plasmids of pAP were significant small as compared with other two groups made by the wild type cells and phpApr-neol cells (P < 0.05). It was showed that the expression of perlecan mRNA and protein were significantly reduced in the tumor of pAP transfected Hep-2 cells as compared with the tumors transfected by the wild type cells and phβApr-neol cells (P < 0.01).@*CONCLUSION@#These data raise the possibility that pAP many play key roles in the growth of those xnografts in nude mice.

Gene regulation of pteridine reductase 1 in leishmania promasti-gotes and amastigotes using a full-length antisense construct

Pteridine metabolic pathway is unusual features of Leishmania, which is necessary for the growth of parasite. Leishmania has evolved a complex and versatile pteridine salvage network which has the ability of scavenging a wide area of the conjugated and unconjugated pteridines especially folate and biopterin. In this study, we focus on the inhibition of ptr1 gene expression. L. major ptr1 gene was cloned into pcDNA3 and digested using KpnI and BamHI. The gene was subcloned so that antisense will transcribe and called pcDNA-rPTR. Leishmania major was cultured and late logarithmic-phase promastigotes were harvested. The promastigotes were divided into two groups. One group was transfected with 50 micro g of pcDNA-rPTR, whereas the other group was transfected with pcDNA3. Transfected cells were cultured and plated onto semi-solid media. Mouse pritonean macrophages were transfected using pcDNA-rPTR-tansfected promastigotes. Western blotting was performed on mouse transfected pritonean macrophages and extracts from transfected promastigotes of L. major using a L. major ptr1 antibody raised in rabbits. The PTR1 protein was not expressed in pcDNA-rPTR- tansfected promastigotes and mouse macrophage transfected with pcDNA-rPTR- tansfected promastigotes. This approach might be used to study the pteridine salvage pathway in Leishmania or to assess the possibility of using gene expression inhibition in the treatment of leishmaniasis.

BC047440 antisense eukaryotic expression vectors inhibited HepG2 cell proliferation and suppressed xenograft tumorigenicity

The biological functions of the BC047440 gene highly expressed by hepatocellular carcinoma (HCC) are unknown. The objective of this study was to reconstruct antisense eukaryotic expression vectors of the gene for inhibiting HepG2 cell proliferation and suppressing their xenograft tumorigenicity. The full-length BC047440 cDNA was cloned from human primary HCC by RT-PCR. BC047440 gene fragments were ligated with pMD18-T simple vectors and subsequent pcDNA3.1(+) plasmids to construct the recombinant antisense eukaryotic vector pcDNA3.1(+)BC047440AS. The endogenous BC047440 mRNA abundance in target gene-transfected, vector-transfected and naive HepG2 cells was semiquantitatively analyzed by RT-PCR and cell proliferation was measured by the MTT assay. Cell cycle distribution and apoptosis were profiled by flow cytometry. The in vivo xenograft experiment was performed on nude mice to examine the effects of antisense vector on tumorigenicity. BC047440 cDNA fragments were reversely inserted into pcDNA3.1(+) plasmids. The antisense vector significantly reduced the endogenous BC047440 mRNA abundance by 41 percent in HepG2 cells and inhibited their proliferation in vitro (P < 0.01). More cells were arrested by the antisense vector at the G1 phase in an apoptosis-independent manner (P = 0.014). Additionally, transfection with pcDNA3.1(+)BC047440AS significantly reduced the xenograft tumorigenicity in nude mice. As a novel cell cycle regulator associated with HCC, the BC047440 gene was involved in cell proliferation in vitro and xenograft tumorigenicity in vivo through apoptosis-independent mechanisms.

Inhibition of leishmania major PTR1 gene expression by antisense in escherichia coli

Protozoa related to Trypanosome family including Leishmania, synthesize enzymes to escape from drug therapy. One of them is PTR1 that its enzymatic activity is similar to dihydrofolate reductase [DHFR]. Dihydrofolate reductase - thymidylate synthase has a major role in DNA synthesis, if it is inhibited, the result would be the death of parasite. Since PTR1 activity is similar to DHFR, causes the decrease of inhibition effect of drug. The aim of this study was inhibition of Iranian L. major PTR1 expression with mRNA antisense in prokaryotic system as an approach to appear of the drugs therapeutic effects more. PTR1 gene was ligated to pACYCDuet-1 and pcDNA3 plasmids as sense and antisense plasmids, respectively. Simultaneously transfer of sense and antisense plasmids was done in E. coli strain M15. SDS-PAGE and western blot analysis were carried out to analyze the expression. Sense and antisense plasmids were prepared and confirmed by restriction analysis and PCR then simultaneously transfer of them was done. SDS-PAGE and western blot analysis showed PTR1 gene was inhibited by mRNA antisense in bacterial cells. Expression of PTR1 gene in sense plasmid was inhibited successfully by antisense plasmid

Anti-sense nucleic acid of CyclinD1 induces apoptosis of lung adenocarcinoma cancer cell A549 / 生理学报

To explore the potential of the anti-sense nucleic acid of CyclinD1 in lung cancer therapy, the expression vector containing the anti-sense nucleic acid of CyclinD1 was constructed and named pcDNA3.1-CyclinD1. The A549 cells were transfected with pcDNA3.1-CyclinD1 vectors. After being screened by G418, the stable expression positive clones were obtained. MTT method and flow cytometry technique were used to detect cell proliferation and apoptosis, respectively. The results showed the transfected cells exhibited significantly increased apoptosis and inhibited cell growth, compared with negative control and empty vector groups. To investigate the mechanism for anti-sense nucleic acid of CyclinD1 inducing A549 cells apoptosis, the expression levels of retinoblastoma protein (pRb), adenovirus E2 factor-1 (E2F-1), vascular endothelial growth factor (VEGF), matrix metalloproteinase (MMP)-2 and MMP-9 were detected by Western blot, and the results showed the expressions of these proteins were all decreased significantly in anti-sense nucleic acid of CyclinD transfected group, compared with those in negative control and empty vector groups. In a word, anti-sense nucleic acid of CyclinD1 induces the apoptosis of lung adenocarcinoma cancer cells, and the depressions of pRb, E2F-1, VEGF, MMP-2 and MMP-9 expressions may be the possible mechanism.

Connection of magnetic antisense probe with SK-Br-3 oncocyte mRNA nucleotide detected by high resolution atomic force microscope / 生物医学工程学杂志

The present paper is aimed to detect superparamagnetic iron oxide labeled c-erbB2 oncogene antisense oligonucleotide probe (magnetic antisense probe) connected with SK-Br-3 oncocyte mRNA nucleotide by high resolution atomic force microscope (AFM). We transfected SK-Br-3 oncocyte with magnetic antisense probe, then observed the cells by AFM with high resolution and detected protein expression and magnetic resonance imagine (MRI). The high resolution AFM clearly showed the connection of the oligonucleotide remote end of magnetic antisense probe with the mRNA nucleotide of oncocyte. The expression of e-erbB2 protein in SK-Br3 cells were highly inhibited by using magnetic antisense probe. We then obtained the lowest signal to noise ratio (SNR) of SK-Br-3 oncocyte transfected with magnetic antisense probe by MRI (P<0.05). These experiments demonstrated that the high resolution AFM could be used to show the binding of magnetic antisense probe and SK-Br-3 mRNA of tumor cell nuclear.

PAR-1 regulation of intracellular Ca²(+) mobilization in pulmonary giant cell carcinoma cell line PLA801D/PLA801C / 中华病理学杂志

<p><b>OBJECTIVES</b>To investigate molecular mechanisms of PAR-1 regulation on intracellular Ca²(+) mobilization in lung giant cell carcinoma cells in vitro and its involvement in tumor metastasis.</p><p><b>METHODS</b>Free intracellular Ca²(+) ([Ca²(+)]i) was measured in lung giant cell carcinoma PLA801C and PLA801D cells by confocal microscopy. Sense and anti-sense PAR-1 expression vectors were transfected into PLA801C (C+)and PLA801D(D-) cells, respectively. The effects of PAR-1 expression were investigated by thrombin and TRAP-induced mobilization of [Ca²(+)]i in the C+ and D-cells.</p><p><b>RESULTS</b>There were significant differences of the mean values of [Ca²(+)]i between PLA801D (59.55) and PLA801C cells (35.46, P < 0.01). The mean [Ca²(+)]i of C+ cells (45.77) was significantly higher than that of its control CV cells (35.46, P < 0.05), and the mean [Ca²(+)]i of D-cells (48.42) was significantly lower than that of its control DV cells (59.55, P < 0.05). The peaks of [Ca²(+)]i of C+ and CV cells were 48.19 ± 9.84 and 45.64 ± 9.87 (P < 0.05) respectively at 80 s and 100 s after thrombin treatment, but were 111.31 ± 25.00 and 52.93 ± 11.21 (P < 0.05) respectively at 60 s after TRAP treatment. The peaks of [Ca²(+)]i of D- and DV cells were 40.71 ± 5.89 and 61.07 ± 21.36 (P < 0.05) respectively at 60 s after thrombin treatment, but were 84.98 ± 11.23 and 102.58 ± 21.48 (P < 0.05) respectively at 40 s after TRAP treatment.</p><p><b>CONCLUSIONS</b>The high metastatic potential of PLA801D and PLA801C may be related to [Ca²(+)]i of the tumor cells. PAR-1 may play an important role in the metastasis of lung giant cell carcinoma cells by up-regulating the intracellular Ca²(+).</p>

Inhibitory effects of an antisense PCDGF vector on proliferation and invasion of highly malignant ovarian cancer cells and the related mechanism / 中华肿瘤杂志

<p><b>OBJECTIVE</b>To investigate the inhibitory effects of an antisense PC cell derived growth factor (PCDGF) vector on proliferation and invasion of highly malignant ovarian cancer cell lines Sw626 and A2780 cells, and preliminarily explore the related mechanisms.</p><p><b>METHODS</b>MTT assay and Boyden chamber in vitro invasion assay were employed to detect the changes of proliferation and invasion ability in the Sw626 and A2780 cells transfected with anti-sense PCDGF. The expression levels of cyclin D1 and CDK4 proteins before and after transfection were detected by Western blotting. The effects on the expression and activity of MMP-2 were evaluated by quantitative RT-PCR and zymography, respectively.</p><p><b>RESULTS</b>Comparing with the blank group, the proliferation inhibition rate of the Sw626 and A2780 cells transfected with anti-sense PCDGF was 72.9% and 70.9%, respectively, and the invasion ability was inhibited by 62.9% and 59.0%, respectively. The levels of cyclin D1 and CDK4 protein expression in antisense PCDGF transfected cells were 0.38 +/- 0.08 and 0.37 +/- 0.13, respectively, all significantly lower than 0.84 +/- 0.11 and 0.64 +/- 0.11, respectively, in the blank group (P < 0.01). The MMP-2 mRNA expression level in antisense PCDGF transfected cell group was 0.66 +/- 0.11, not significantly decreased in comparison with 0.89 +/- 0.09 in the blank group (P > 0.05), but the activity of MMP-2 was inhibited significantly.</p><p><b>CONCLUSION</b>The antisense PCDGF vector may inhibit markedly the proliferation and invasion of highly malignant ovarian cancer cells, and partially reverses their malignant phenotype. It seems to be related with down-regulating the expression of cyclin D1 and CDK4 and inhibiting the activity of MMP-2. Our findings indicate that PCDGF may become a new target for antisense gene therapy of ovarian cancer.</p>

Long-term stable expression of antisense cDNA of cyclin B1 profoundly inhibits the proliferation of tumor cells and suppresses tumorigenicity in implanted mice / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Cyclin B1 (CLB1) is necessary for mitotic initiation in mammalian cells and plays important roles in cancer development. Therefore, a potential strategy in cancer therapy is to suppress the activity of CLB1 by delivering antisense constructs of CLB1 into tumor cells. In previous CLB1 studies, antisense constructs with a short half life were often used and these constructs might not persistently inhibit CLB1.</p><p><b>METHODS</b>We successfully created a recombinant plasmid encoding the full-length antisense cDNA of mouse cyclin B1 (AS-mCLB1) and transfected this construct to the murine Lewis lung carcinoma (LL/2) and CT-26 colon carcinoma (CT-26) cells. We isolated clones of LL/2 and CT-26 transfectants with stable expression of AS-mCLB1. Reverse transcriptional polymerase chain reaction (RT-PCR) and Western blot were applied to detect the expression of the mRNA and protein levels of CLB1. To further test the efficacy of this strategy in vivo, AS-mCLB1-expressing LL/2 and CT-26 transfectants were implanted into mice.</p><p><b>RESULTS</b>We found the expression of the mRNA and protein levels of CLB1 decrease in these transfectants. The inhibition of CLB1 caused prominent G1 arrest, abnormal morphology, retarded cell growth and an increase in apoptosis. In AS-mCLB1-expressing LL/2 and CT-26 transfectants implanted mice, tumorigenicity was effectively suppressed compared with the controls. In addition, the expression of AS-mCLB1 also significantly increases the survival duration of implanted animals.</p><p><b>CONCLUSION</b>AS-mCLB1 is likely to be useful in future cancer therapy, which may be associated with its ability to down-regulate the expression of CLB1 and then induce G1arrest and apoptosis in tumor cells.</p>

Preparation and quality control of 99mTc labeled MDR1 oligonucleotide DNAs / 生物医学工程学杂志

The aim of this study is to explore the optimal labeling condition of technetium-99m labeled antisense oligonucleotides (ASON) DNA and sense oligonueleotides (SON) DNA against multi-drug resistance gene-1 (MIDR1) mRNA, to prepare its two-step icefrozen kits, and to perform the quality control of technetium-99m labeled ASON and SON DNAs and its two-step icefrozen kits. A 20 mer single-stranded ASON sequence and its SON sequence against MDR1 mRNA were synthesized respectively, both of the ASON and SON DNAs were uniform phosphorothioated for this investigation with a primary amine on the 5'-end via a six-carbon alkyl linker, and then were labeled with technetium-99m by conjugating with the bifunctional chelator S-Acetyl NHS-MAG3 to form ASON- and SON-MAC3 DNAs. The optimal labeling condition was explored by varying the amount of ASON- and SON-MAG3 DNAs, SnCl2.2H2O and buffer, the pH value in the reaction medium was also adjusted. The technetium-99m labeled ASON and SON DNAs' two-step icefrozen kits were developed. The radiochemical purities, labeling stability of ASON- and SON-MAG3 DNAs in vivo and vitro were measured, and stability of the two-step icefrozen kits were also studied. The recycled rates of ASON- and SON-MAG3 DNAs were over 70% (n >6), the two-step icefrozen kits of ASON- and SON-MAG3 DNAs were colourless ice crystal. The radiochemical purities of technetium-99m labeled ASON- and SON-MAG3 DNAs were over 92 %. The radiochemical purities were over 90% after stored at room temperature for 24 hours. The kits were stable within 6 months when stored at 0 degrees C, the radiochemical purities of technetium-99m labeled ASON- and SON-MAG3 DNAs were still over 90%. The two-step icefrozen kits of ASON- and SON-MAG3 DNAs were successfully developed. The radiochemical purities were all over 90%. The labeling method was simple, feasible and efficient with good stability.

Construction and identification of antisense c-Jun N-terminal kinase 1 eukaryotic fluorescent expressing plasmids and JNK1-/- human embryo lung fibroblasts cell line / 中华劳动卫生职业病杂志

<p><b>OBJECTIVE</b>To construct antisense c-Jun N-terminal kinase 1 (JNK1) eukaryotic fluorescent expressing vector and JNK1-/- human embryo lung fibroblasts cell line.</p><p><b>METHODS</b>Trizol reagent was used to extract total RNA in HELF. The proper primers of JNK1 were chosen and synthesized. RT-PCR and gene recombinant techniques were used to construct the fragment of JNK1. After purification, the PCR products were cut, and JNK1 were inserted reversely into eukaryotic fluorescent expressing vector pEGFP-C1. Enzyme-cutting and DNA auto-sequencing were used to prove the successful construction of JNK1 eukaryotic expressing vector. Then plasmids were extracted and transfected into HELF cells and screen by G418 24 h later. Monoclone was chosen and cultured. Fluorescent imaging and Western blot were used to identify the JNK1-/- HELF cell line.</p><p><b>RESULTS</b>Sequence analysis of pEGFP-C1-as JNK1 plasmids was same as expected. The expression level of JNK1 was inhibited markedly.</p><p><b>CONCLUSION</b>Construction of antisense JNK1 eukaryotic fluorescent expressing vectors and JNK1-/- HELF cell line is successful.</p>

Application of superparamagnetic iron oxide labeled antisense oligodeoxynucleotide probe in cellular magnetic resonance imaging / 中国医学科学院学报

<p><b>OBJECTIVE</b>To prepare the superparamagnetic iron oxide (SPIO)-labeled antisense oligodeoxynucleotide (ASODN) probe and evaluate the application of this probe in cellular magnetic resonance imaging (MRI).</p><p><b>METHODS</b>We prepared the SPIO-labeled ASODN probe using chemical cross linking method to conjugate SPIO to ASODN, detected its configuration by atomic force microscopy, determined the conjugating rate and biology activation by high performance liquid chromatography, and detected the stability by polyacrylamide gel electrophoresis. After that, we transfected the SK-Br3 oncocytes which had over-expression of the c-erbB2 oncogene by this probes, observed the intracellular iron distribution by optical microscope, measured iron content by atomic absorption spectroscopy, and observed the signal change by MRI.</p><p><b>RESULTS</b>Atomic force microscope showed that the SPIO-labeled ASODN probe was mostly spherical and well-distributed, with a diameter of 25-40 nm and a conjugating rate of 100%. This probe had inhered biological activity and stability. In addition, light microscopy revealed an intracellular uptake of iron oxides in the transfected SK-Br3 oncocyte, and the iron content of the group of transfected SK-Br3 oncocytes was significantly higher than those of other contrast groups (all P < 0.01). MRI showed that transfected SK-Br3 oncocyte had the lowest signal among all other cells (all P < 0.05).</p><p><b>CONCLUSIONS</b>We prepared the SPIO-labeled ASODN probe successfully. It can effectively transfect SK-Br3 oncocyte and enter SK-Br3 oncocyte, and thus reduce the signal intension in MRI.</p>

Recombination and identification of sense and antisence CyclinD1 eukaryotic expression vectors and the effects of the vectors on the proliferation of airway smooth muscle cell in asthmatic rats / 药学学报

This study is to investigate the expression of CyclinD1 in asthmatic rats and construct expression plasmids of sense and antisense CyclinD1 gene and transfect them to asthmatic airway smooth muscle cell to study the effects of CyclinD1 on the proliferation of airway smooth muscle cells in asthmatic rats. CyclinD1 cDNA was obtained by RT-PCR of total RNA extracted from the airway smooth muscle in asthmatic rats. The sequence was inserted into eukaryotic expression vector pcDNA3.1 (+) to recombinate the sense and antisense pcDNA3.1-CyclinD1 eukaryotic expression vector. The two recombinations and vector were then separately transfected into airway smooth muscle cell in asthmatic rats by using liposome. The expression level of CyclinD1 was certificated by Western blotting analysis. The proliferations of ASMCs isolated from asthmatic rats were examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. Results showed (1) Compared with control group, the content of CyclinD1 was significantly increased; (2) It was comformed by restriction endonucleasa digestion and DNA sequence analysis that the expression plasmid of sense and antisense CyclinD1 were successfully recombinated. There was significant change of CyclinD1 expression between vector and sense CyclinD1 transfected cells, and the expression level of CyclinD1 in ASMC transfected with antisense CyclinD1 was lower than that in vector transfected cells (P <0.01); (3) In the asthmatic groups, compared with the vecter group, the percentage of S + G2M phase, absorbance A value of MTT and the expression rate of PCNA protein in ASMC transfected with pcDNA3. 1-CyclinD1 vector significantly increased. The values decreased remarkably in the pcDNA3,1-as CyclinD1 group. Statistical analysis revealed that there were significant differences in these indicators of cell proliferation in three groups (P <0.01). In the normal groups, statistical analysis revealed that there were significant differences in the percentage of S + G2M phase, a value of MTT and the expression rate of PCNA protein in three groups (P <0.01). Sense CyclinD1 eukaryotic expression vectors could have a positive effect on the proliferation of ASMC, however the antisence one have a negative effect, which implicated that CyclinD1 might contribute to the process of airway smooth muscle cell proliferation.

Highly expressed genes are associated with inverse antisense transcription in mouse.

There is a growing evidence, that antisense transcription might have a key role in a range of human diseases. Although predefined sense-antisense pairs were extensively studied, the antisense expression of the known sense genes is rarely investigated. We retrieved and correlated the expression of sense and antisense sequences of 1182 mouse transcripts to assess the prevalence and to find the characteristic pattern of antisense transcription. We contrasted three Affymetrix MGU74A version 1 mouse genome chips to six MGU74A version 2 chips. For these 1182 transcripts, the version 1 chips contain the antisense sequences of the transcripts presented on the version 2 chips. The original data was taken from the GEO database (GDS431 and GDS432). As the Affymetrix data are semiquantitative, the relative expression levels of antisense partners were analysed. We detected antisense transcription, although the average antisense expression is shifted towards smaller expression values (MGU74A version 1, 516; version 2, 1688). An inverse direct correlation between sense and antisense expression values could be observed at high expression values. At a very high relative expression--above 40,000--the Pearson correlation coefficient is getting closer to -1. Transcripts with high inverse expression ratio may be correlated to the investigated gene (major histocompatibility complex class II trans activator). The ratio of sense to antisense transcripts varied among different chromosomes; on chromosomes 14 and 1 the level of antisense expression was higher than that of sense. We conclude that antisense transcription is a common phenomenon in the mouse genome. The hypothesis of regulatory role of antisense transcripts is supported by the inverse antisense gene expression of highly expressed genes.

Antisense expression of a gene encoding a calcium-binding protein in transgenic tobacco leads to altered morphology and enhanced chlorophyll.

Entamoeba histolytica contains a novel calcium-binding protein like calmodulin,which was discovered earlier,and we have reported the presence of its homologue(s)and a dependent protein kinase in plants.To understand the functions of these in plants,a cDNA encoding a calcium-binding protein isolated from Entamoeba histolytica (EhCaBP)was cloned into vector pBI121 in antisense orientation and transgenic tobacco plants were raised.These plants showed variation in several phenotypic characters,of which two distinct features,more greenness and leaf thickness,were inherited in subsequent generations.The increase in the level of total chlorophyll in different plants ranged from 60% to 70%.There was no major change in chloroplast structure and in the protein level of D1,D2,LHCP and RuBP carboxylase.These morphological changes were not seen in antisense calmodulin transgenic tobacco plants,nor was the calmodulin level altered in EhCaBP antisense plants.

Adenovirus-mediated antisense HSP70 cDNA transfection inhibits the growth of laryngeal carcinoma Hep-2 cells / 南方医科大学学报

<p><b>OBJECTIVE</b>To construct a recombinant adenovirus vector carrying antisense heat shock protein 70 (HSP70) cDNA and observe its effect on inhibiting the growth of laryngeal carcinoma Hep-2 cells.</p><p><b>METHODS</b>The HSP70 gene fragment encoding the 5' region was cloned reversely into the shuttle plasmid PAdTrack-CMV, and the resultant plasmid was recombined with the backbone plasmid PadEasy-1 in the E.coli Bj5183 cells to generate the recombinant adenovirus vector. The adenovirus were then packaged and amplified in 293 cells, and the viral titer was determined using GFP.</p><p><b>RESULTS</b>The recombinant adenovirus vector carrying antisense HSP70 cDNA was constructed successfully with a viral titer of 8 x 10(9). HSP70 expression of Hep-2 cells was obviously blocked by antisense HSP70 RNA, and Western blotting and immuohistochemistry demonstrated that cells transfected with antisense HSP70 did not express or express HSP70 at low levels. Flow cytometry presented apoptotic peak in the antisense HSP70-transfected cells, but not in the control cells.</p><p><b>CONCLUSION</b>The recombinant adenovirus vector containing antisense HSP70 cDNA can effectively deliver antisense HSP70 gene into Hep-2 cells, suggesting the great potential of this gene therapy strategy in management of human laryngeal carcinoma.</p>

Functional aspects of protease-activated receptor 1 in promoting metastasis of lung cancer / 中华病理学杂志

<p><b>OBJECTIVE</b>To study the functional aspects of protease-activated receptor 1 (PAR-1) gene involved in tumor metastasis.</p><p><b>METHODS</b>Two human lung giant cell carcinoma cell lines PLA801C (low metastasis potential) and PLA801D (high metastasis potential) were chosen as in-vitro human cancer model systems. Sense and anti-sense expression constructs of PAR-1 gene (pC/PAR1s and pC/PAR1as) were transfected into PLA-801C and PLA-801D cells by lipofection. PAR-1 expression was determined by RT-PCR and western blot analysis. MTT growth, flow cytometry analysis, fibronectin adhesion, and matrigel invasion assays were used to study the effect of PAR-1 expression on the proliferation, adhesion, and invasion of the transfected cells.</p><p><b>RESULTS</b>Appropriate up-regulation or down-regulation of protein expression of PAR-1 was observed in both transfected cell lines (PLA801C and PLA801D) to express PAR-1s or PAR-1as, respectively. Expression of the sense PAR-1 markedly increased cellular proliferation, adhesion and invasion of PLA-801C cells. In contrast, anti-sense PAR-1 significantly inhibited cell growth, adhesion and invasion capabilities, along with cell arrest at G0/G1 phase of the PLA-801D cells.</p><p><b>CONCLUSIONS</b>Successful up- and down- regulation of expression of PAR-1 can be achieved by in-vitro transfection of sense and antisense PAR-1 constructs. PAR-1 may enhance metastasis of lung cancer through its regulation of cellular proliferation, adhesion and invasion. Down-regulation of expression of PAR-1 may provide a new therapeutic strategy against lung carcinoma.</p>

Study on combined gene therapy for malignant gliomas transfected with antisense hTERT/PTEN in vitro and in vivo / 中华医学遗传学杂志

<p><b>OBJECTIVE</b>To study inhibitory efficacy of combined gene therapy for malignant gliomas transfected with antisense human telomerase reverse transcriptase (hTERT)/PTEN in vitro and in vivo.</p><p><b>METHODS</b>To construct two adenovirus recons which contained antisense hTERT and wild-type PTEN respectively with high performance homologous recombination system in bacteria. The two adenovirus recons were transfected into U251 human malignant glioma cells combinedly or respectively in vitro and in vivo. U251 cell proliferation in vitro was determined by MTT assay and flow cytometry, tumor growth in vivo was measured by the volume of glioma in nude mice. Telomerase activity was detected by telomeric repeat amplification protocol (TRAP) assay. Expression of hTERT and PTEN protein was detected by Western blotting methods.</p><p><b>RESULTS</b>After transfection in vitro, the growth of U251 cells was inhibited significantly. The inhibitory effect was time-dependent. The strongest inhibition was observed in combined transfection group, at the 6th day, the survival rate was 37.6%, telomerase activity (only 28.8TPG) was inhibited significantly, hTERT protein expression was inhibited significantly too, which was 0.2106, but PTEN protein expression was increased significantly, which was 0.9630. In vivo, the growth of tumors was also effectively inhibited.</p><p><b>CONCLUSION</b>Growth of malignant glioma cells is effectively inhibited after transfection with combined antisense hTERT and PTEN in vitro and in vivo.</p>