Selection of Effective Bcl-2 Antisense Oligodeoxynucleotides with Computer Software and Experimental Assay

Objective： To explore and investigate the selection of effective antisense oligodeoxynuleotides with the help of computer and RNAstructure folding software. Methods： Bcl-2 gene was used as the target gene and five antisense oligodeoxynuleotides were designed to be bound to Bcl-2 mRNA optimal secondary structure regions that were predicted free from intramolecular fold or instability of free energy. The five antisense oligodeoxynucleotides were studied with experimental assay of leukemia cells, including cell grow assay with tropan blue exclusion, expression of Bcl-2 protein detected with immunochemistry and flowcytometry, Bcl-2 mRNA content detected with RT-PCR technique, as well as apoptosis observed and determined with morphonological method, electrophoresis and flowcytometry. Results： The results showed that two of the five antisense oligodeoxynucleotides were effective antisense oligodeoxynucleotides, which were able to inhibit cell growth in leukemia, to decrease the level of Bcl-2 mRNA and protein, to induce apoptosis of leukemia cells significantly. Conclusion： The computational prediction of antisense efficacy is faster than other methods and more efficient, which can potentially speed the development of sequences for both research and clinical applications.

Cross-species hybridization of woodchuck hepatitis virus-induced hepatocellular carcinoma using human oligonucleotide microarrays

AIM： To demonstrate the feasibility of using woodchuck samples on human microarrays, to provide insight into pathways involving positron emission tomography （PET） imaging tracers and to identify genes that could be potential molecular imaging targets for woodchuck hepatocellular carcinoma. METHODS： Labeled cRNA from woodchuck tissue samples were hybridized to Affymetrix U133 plus 2.0 GeneChips. Ten genes were selected for validation using quantitative RT-PCR and literature review was made. RESULTS： Testis enhanced gene transcript （BAX Inhibitor 1）, alpha-fetoprotein, isocitrate dehydrogenase 3 （NAD＋） beta, acetyI-CoA synthetase 2, carnitine palmitoyltransferase 2, and N-myc2 were up-regulated and spermidine/spermine N1-acetyltransferase was down-regulated in the woodchuck HCC. We also found previously published results supporting 8 of the 10 most up-regulated genes and all 10 of the 10 most downregulated genes. CONCLUSION： Many of our microarray results were validated using RT-PCR or literature search. Hence, we believe that woodchuck HCC and non-cancerous liver samples can be used on human microarrays to yield meaningful results.

Oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus

To compare the oligonucleotide chip, real-time PCR and sequencing for genotyping of hepatitis B virus in Chinese patients with chronic hepatitis B. METHODS： Mixture of samples with different genotypes and clinical serum samples from 126 chronic hepatitis B patients was tested for hepatitis B virus genotypes by oligonucleotide chip, real-time PCR and sequencing of PCR products, respectively. Clinical performances, time required and costs of the three assays were evaluated. RESULTS： Oligonucleotide chips and real-time PCR detected 1% and 0.1% genotypes, respectively, in mixed samples. Of the 126 clinical samples from patients with chronic hepatitis B, genotype B was detected in 41 （33%）, 41 （33%） and 45 （36%） samples, and genotype C in 76 （60%）, 76 （60%） and 81 （64%） samples, by oligonucleotide chip, real-time PCR and sequencing, respectively. Oligonucleotide chip and real-time PCR detected mixed genotypes B and C in 9 samples. Real- time PCR was the rapidest and cheapest among the three assays. CONCLUSION： Oligonucleotide chip and real-time PCR are able to detect mixed genotypes, while sequencing only detects the dominant genotype in clinical samples.

Expression of heparanase mRNA in anti-sense oligonucleotide-transfected human esophageal cancer EC9706 cells

AIM： To investigate the effed3 of anti-sense oligonucleotides （ASODNs） on mRNA expression of heparanase in human esophageal cancer EC9706 cells. METHODS： One non-sense oligonucleotide （N-ODN） and five ASODNs against different heparanase mRNA sites were transfected into EC9706 cells, then the expression of heparanase mRNA in EC9706 cells was studied by in situ hybridization. RESULTS： The expression of heparanase mRNA could be inhibited by ASODNs.There was no significant difference among five ASODNs （P〉0.05）, but there was a significant difference between ASODNs and N-ODN or non-transfected group （ASODNI： 2.25±0.25, ASODN2： 2.21±0.23, ASODN3： 2.23±0.23, ASODN4：2.25±0.24 vs N-ODN： 3.47±2.80 or non- transfected group： 3.51±2.93 respectively, P〈0.05）. CONCLUSION： The expression of heparanase mRNA in EC9706 cells can be inhibited by ASODNs in vivo, and heparanase ASODNs can inhibit metastasis of esophageal squamous cell carcinoma or other tumors by inhibiting the expression of heparanase.

Detection of hepatitis B virus genotypes using oligonucleotide chip among hepatitis B virus carriers in Eastern China

AIM： TO determine the genotype distribution of hepatitis B virus （HBV） with a newly oligonucleotide chip assay among the HBV carriers in Eastern China. METHODS： An assay using oligonucleotide chip was developed for detection of HBV genotypes in serum samples from HBV DNA-positive patients in Eastern China. This method is based on the principle of reverse hybridization with Cy5-labeled amplicons hybridizing to type-specific oligonucleotide probes that are immobilized on slides. The results of 80 randomly chosen sera were confirmed by direct sequencing. RESULTS： HBV genotype B, C and mixed genotype were detected in 400 serum samples, accounting for 8.3% （n = 33）, 83.2% （n = 333）, and 8.5% （n = 34）, respectively. The evaluation of the oligonucleotide assay showed 100% concordance with the amplicon phylogenetic analysis except 9 mixed genotype infections undetected by sequencing. CONCLUSION： The study indicates that HBV genotype C and B prevail in the Eastern China. It is suggested that the oligonucleotide chip is a reliable and convenient tool for the detection of HBV genotyping.

Identification of osteopontin as the most consistently over-expressed gene in intrahepatic cholangiocarcinoma: Detection by oligonucleotide microarray and real-time PCR analysis

AIM: To investigate the molecular pathways involved in human cholangiocarcinogenesis by gene expression profiling. METHODS: Oligonucleotide arrays (Affymetrix U133A) were used to establish a specific gene expression profile of intrahepatic CCC in comparison to corresponding non- malignant liver tissue. To validate the expression values of the most overexpressed genes, RT-PCR experiments were performed. RESULTS: Five hundred and fifty-two statistically differentially expressed genes/ESTs (221 probes significantly up-regulated, 331 probes down-regulated; P < 0.05; fold change > 2; ≥ 70%) were identified. Using these data and two-dimensional cluster analysis,a specific gene expression profile was obtained allowing fast and reproducible differentiation of CCC, which was confirmed by supervised neuronal network modelling. The most consistently overexpressed gene (median fold change 33.5, significantly overexpressed in 100%) encoded osteopontin. Furthermore, an association of various genes with the histopathological grading could be demonstrated. CONCLUSION: A highly specific gene expression profile for intrahepatic CCC was identified, allowing for its fast and reproducible discrimination against non- malignant liver tissue and other liver masses. The most overexpressed gene in intrahepatic CCC was the gene encoding osteopontin. These data may lead to a better understanding of human cholangiocarcinogenesis.

Gene Expression Profile Differences in Gastric Cancer and Normal Gastric Mucosa by Oligonucleotide Microarrays

OBJECTIVE To study the difference of gene expression in gastric cancer (T) and normal tissue of gastric mucosa (C), and to screen for associated novel genes in gastric cancers by oligonucleotide microarrays. METHODS U133A (Affymetrix, Santa Clara, CA) gene chip was used to detect the gene expression profile difference in T and C. Bioinformatics was used to analyze the detected results. RESULTS When gastric cancers were compared with normal gastric mucosa, a total of 270 genes were found with a difference of more than 9 times in expression levels. Of the 270 genes, 157 were up-regulated (Signal Log Ratio [SLR] ≥3), and 113 were down-regulated (SLR ≤-3). Using a classification of function, the highest number of gene expression differences related to enzymes and their regulatory genes (67, 24.8%), followed by signal-transduction genes (43,15.9%). The third were nucleic acid binding genes (17, 6.3%), fourth were transporter genes (15, 5.5%) and fifth were protein binding genes (12, 4.4%). In addition there were 50 genes of unknown function, accounting for 18.5%. The five above mentioned groups made up 56.9% of the total gene number. CONCLUSION The 5 gene groups (enzymes and their regulatory proteins, signal transduction proteins, nucleic acid binding proteins, transporter and protein binding) were abnormally expressed and are important genes for further study in gastric cancers.

The Role of β3-adrenergic Receptor Gene in Neuropeptide Y Y5Receptor Antisense Gene Therapy of Diet-induced Obese Rats

Objective: To study the role of β3-adrenergic receptor gene in neuropeptide Y(NPY) Y5 receptor antisense gene therapy of diet-induced obese rats.Methods: The diet-induced obese rats were prepared by feeding a high-nutrition diet. Lateral ventricular was cannulated in obese rats which then received an intraventricular injection of either 5 μg/μl NPY Y5 receptor antisense or 10 μl missense oligodeoxynucleotide or saline of 10 μl respectively in every rat. When the rats were killed, the wet weight of abdominal adipose tissue, the level of serum lipid and lipoprotein were measured. Total RNA from the retroperitoneal adipose tissue was extracted and the level of β3-adrenergic receptor gene mRNA expression was evaluated by RT-PCR.Results: ①The wet weight of abdominal adipose tissue, the levels of serum lipids were greatly higher in diet-induced obese rats than those in normal rats. However, there were much lower β3-adrenergic receptor gene mRNA expression levels in retroperitoneal adipose tissue in diet-induced obese rats as compared with those in normal rats. ②After the diet-induced obese rats were intraventricularly administered with NPY Y5 receptor antisense oligodeoxynucleotide, the levels of β3-adrenergic receptor gene mRNA expression in retroperitoneal adipose tissue of diet-induced obese rats were strikingly up-regulated, whereas the wet weight of abdominal adipose tissue, the levels of serum lipids were markedly reduced.Conclusion: Intraventricular administration of antisense oligodeoxynucleotide to NPY Y5 receptor could significantly reduce the abdominal adipose tissue and the levels of serum lipids in diet-induced obese rats by up-regulating the level of β3-adrenergic receptor gene mRNA expression in retroperitoneal adipose tissue.

Detectingβ-thalassaemia mutations from a single cell by PEP and RDB

Objective :To evaluate the possibility of the technology involving PEP and RDB for detectingβ-thalassaemia multipoint mutations from a single cell simultaneously. Methods: A set of allele specific oligonucleotide (ASO) probes used for detecting 8 familiarβ-thalassaemia mutations (CD41-42. IVS-Ⅱ-654, CD17, TATA box nt-28, CD71-72, TATA box nt-29, CD26, IVS-Ⅰ-5) were immobilized on a strip of nylon membrane. The genome of a individual cell was amplified by primer extension preamplification (PEP) with the mixture of 15-base random oligonucleotides. The aliquots from PEP were used to amplify the objective gene fractions ofβ-thalassaemia gene by nested or semi-nested PCR. The membrane was hybridized with the final amplified products and then treated with Streptavidin-HRP and color development. Results:Totally 30 lymphocytes were picked up from blood samples of 1 healthy female and 4 patients with knownβ-thalassaemia mutations respectively. Each single lymphocyte was lysed in the proteinase K buffer. The amplification efficacy was 94. 0% and alle drop-out (ADO) rate was 8. 0%. Revert dot blot (RDB) was applied to the final amplified products from the 5 participants. The results of diagnosis were the same to the expected, and their genotypes were N/N, CD17(A→T)/N, IVS-Ⅱ-654(C→T)/CD17(A→T), CD41-42(-CTTT)/N and TATA box nt-28(A→G)/N, respectively. Conclusion: The technology involving PEP and RDB could detect multipleβ-thalassaemia mutations from a single cell simultaneously, and the research provides experimental evidences for the feasibility of applying PEP and DNA array technology to screening multiple genetic mutations from a single cell, and will be applied to preimplantation genetic diagnosis and non-invasive prenatal diagnosis forβ-thalassaemia.

Expression profiling of gastric cancer samples by oligonucleotide microarray analysis reveals low degree of intra-tumor variability

AIM： Gene expression profiling provides an unique opportunity to gain insight into the development of different types of gastric cancer. Tumor sample heterogeneity is thought to decrease the sensitivity and tumor specificity of microarray analysis. Thus, microdissection and preamplification of RNA is frequently performed. However, this technique may also induce considerable changes to the expression profile. To assess the effect of gastric tumor heterogeneity on expression profiling results, we measured the variation in gene expression within the same gasbic cancer sample by performing a gene chip analysis with two RNA preparations extracted from the same tumor specimen. METHODS： Tumor samples from six intestinal T2 gastric tumors were dissected under liquid nitrogen and RNA was prepared from two separate tumor fragments. Each extraction was individually processed and hybridized to an Affymetrix U133A gene chip covering approximately 18 000 human gene transcripts. Expression profiles were analyzed using Microarray Suite 5.0 （Affymetrix） and GeneSpring 6.0 （Silicon Genetics）. RESULTS： All gastric cancers showed little variance in expression profiles between different regions of the same tumor sample. In this case, gene chips displayed mean pair wise correlation coefficients of 0.94±0.02 （mean±SD）, compared to values of 0.61±0.1 for different tumor samples. Expression of the variance between the two expression profiles as a percentage of “total change” （Affymetrix） revealed a remarkably low average value of 1.18±0.78 for comparing fragments of the same tumor sample. In contrast, comparison of fragments from different tumors revealed a percentage of 24.4±4.5. CONCLUSION： Our study indicates a low degree of expression profile variability within gastric tumor samples isolated from one patient. These data suggest that tumor tissue heterogeneity is not a dominant source of error for microarray analysis of larger tumor samples, making total RNA extraction an appropriate strategy for performing gene chip expression profiling of gastric cancer.

Understanding the transcriptional control of gene expression using ChIP-chip technology

Gene expression profiling using cDNA or high-density oligonucleotide microarray contributes signifi cantly to our understanding on the transcriptome of a given biological condition. Using this technology, huge number of differentially-expressed genes of interest have been identified in a broad range of circumstances. Making sense biologically on these genes using the recently-improved functional annotation and data integration has leveraged our understanding in diseases and their biological mechanisms. However, understanding the codes encrypt- ed in the cis-aeting regulatory regions and gaining insights into the circuitry of functional regulatory networks on the genomic scale will require additional empirical data sets that are capable of revealing the cohorts or regulons of the transcription and the dynamic progression of molecular events responsible for certain biological function.

The Cytochrome b Polymorphism of Meat Lines Rabbits

The cytochrome b mtDNA was analysed from peripheral whole blood samples of meat lines rabbits. The PCR product--692 bp long fragment of Oryctolagus cuniculus_cytochrome b （0CU07566 GenBank, NCB1, USA） was amplified. Synthetically produced oligonucleotides for the detection ofcyt b were designed by own algorithm： ORYCTO-cyt b-FOR 5＇- CTA TCA GCA ATC CCA TAT ATC -3＇ and ORYCTO-cyt b-REV 5＇- CTT CAT TTG AGG ATT TTG TT -3＇. Based on AluI-RFLP were described two cytochrome b haplotypes--cyt b 430 and cyt b 306. Haplotype cyt b 430 is 571A （190Threonine-T） ＋ 877G （292Alanine-A）. The new haplotype cyt b 306 is presented A571G nucleotide substitution, i.e, 571G （190Alanine-A） ＋ 877G （292Alanine-A）.

Intrahepatic expression of genes related to metabotropic receptors in chronic hepatitis

AIM： To screen for genes related to metabotropic re- ceptors that might be involved in the development of chronic hepatitis. METHODS： Assessment of 20 genes associated with metabotropic receptors was performed in liver speci- mens obtained by punch biopsy from 12 patients with autoimmune and chronic hepatitis type B and C. For this purpose, a microarray with low integrity grade and with oligonucleotide DNA probes complementary to target transcripts was used. Evaluation of gene expression was performed in relation to transcript level, correlation between samples and grouping of clinical parameters used in chronic hepatitis assessment. Clini- cal markers of chronic hepatitis included alanine and aspartate aminotransferase, ~,-glutamyltranspeptidase, alkaline phosphatase and cholinesterase activity, levels of iron ions, total cholesterol, triglycerides, albumin, glucose, hemoglobin, platelets, histological analysis of inflammatory and necrotic status, fibrosis according to METAVIR score, steatosis, as well as anthropometric body mass index, waist/hip index, percentage of adi- pose tissue and liver size in ultrasound examination. Gender, age, concomitant diseases and drugs were also taken into account. Validation of oligonucleotide microarray gene expression results was done with the use of quantitative real-time polymerase chain reaction （qRT-PCR）. RESULTS： The highest （0.002 〈 P 〈 0.046） expres- sion among genes encoding main components of metabotropic receptor pathways, such as the a subunit of G-coupled protein, phosphoinositol-dependent pro- tein kinase or arrestin was comparable to that of an- giotensinogen synthesized in the liver. Carcinogenesis suppressor genes, such as chemokine ligand 4, tran- scription factor early growth response protein 1 and lysophosphatidic acid receptor, were characterized by the lowest expression （0.002 〈 P 〈 0.046）, while the factor potentially triggering hepatic cancer, transcrip- tion factor JUN-B, had a 20-fold higher expression. The correlation between expression of genes of protein kinases PDPK1, phosphoinositide 3-kinase and protein kinase A （Spearman＇s coefficient range： 0.762-0.769） confirmed a functional link between these enzymes. Gender （P = 0.0046） and inflammation severity, mea- sured by alanine aminotransferase activity （P = 0.035）, were characterized by diverse metabotropic receptor gene expression patterns. The Pearson＇s coefficient ranging from -0.35 to 0.99 from the results of qRT-PCR and microarray indicated that qRT-PCR had certainlimitations as a validation tool for oligonucleotide mi- croarray studies. CONCLUSION： A microarray-based analysis of hepa- tocyte metabotropic G-protein-related gene expression can reveal the molecular basis of chronic hepatitis.

Gene Expression Profiles in Porcine Tissues of Liver and Kidney

Microarray technologies are widely used all over the world for the gene expression analysis in various tissues, but still this technology has some limitations. The problem of eliminated reproducibility of the results, obtained in different laboratories using different platforms, is very relevant nowadays. For revelation of problems ofmicroarrays, the comparative analysis of hepatic and renal gene expression profiles （GEP） was carried out by using swine protein annotated oligonucleotide microarrays （SPAM）. Revealed differences in GEP between kidney and liver of pigs were correlated with functional and histological distinctions of these organs. It was shown that sources of errors in the comparative analysis of organ-specific GEP could be connected to the cross hybridization of one probe to transcripts （cDNA of mRNA） of different genes and to individual variability in gene expression between animals, related with the changeability of influences of exo- and endogenous regulation factors.

先天性低促性腺激素性性腺功能减退症遗传咨询及生殖相关建议专家共识

先天性低促性腺激素性性腺功能减退症(CHH)是一类罕见的影响青春期发育和成年期生育的遗传性疾病,根据是否合并嗅觉异常分为嗅觉异常的卡尔曼综合征和嗅觉正常的CHH。超过50个基因参与CHH发病,遗传模式包括X连锁隐性、常染色体隐性和常染色体显性遗传,至少20%呈寡基因遗传。该类患者及其家庭的遗传咨询成为疾病诊疗中不可或缺的重要部分。

Genotype of ethanol metabolizing enzyme genes by oligonucleotide microarray in alcoholic liver disease in Chinese people

OBJECTIVE: To explore the relationship between genetic polymorphisms of the ethanol metabolizing enzymes and the occurrence of alcoholic liver disease (ALD). METHODS: Sixty-five healthy male controls and 165 alcoholisms (including 122 ALD patients and 43 male alcohol abusers without liver complications defined as alcohol-dependent) were analyzed by polymerase chain reaction and hybridized with oligonucleotide microarray to detect the polymorphisms of the ethanol metabolizing enzymes genes. RESULTS: The frequencies of alcohol dehydrogenase gene 2 * 1 ( ADH2 * 1 ) allele were shown as 37.69%, 46.51% and 59.02% in control, alcohol-dependent and ALD groups respectively; while those of ADH2 * 2 allele were shown as 62.31 %, 53.49% and 40.98% respectively. No ADH2 * 3 was detected in any of the subjects. The frequency of ADH2 * 1 was significantly higher in alcoholisms (ALD group and alcohol-dependent group) than in healthy controls ( P

Liposome-C-erbB_2 antisense oligodoxynucleotides in human ovarian cancer cells

Objective To explore the effects of liposome C erbB 2 antisense phosphorothioate oligodeoxynucleotides (S ODNs) on C erbB 2 proto oncogene expression and cell proliferation in human ovarian cancer cells Methods The effects of liposome C erbB 2 S ODNs on C erbB 2 protein expression, cell cycle and cell proliferation in human ovarian cancer cells were studied by means of flow cytometry and 3H thymidine incorporation Results Liposome C erbB 2 S ODNs can specifically reduce C erbB 2 protein expression in human ovarian cancer cells, accompanied by a 30% inhibition of cell proliferation The effectiveness of liposome C erbB 2 S ODNs on the expression of C erbB 2 was about 40 times higher than that of C erbB 2 S ODNs Conclusions The data suggest that antisense therapy might be a useful method of gene therapy in ovarian cancer The effectiveness of C erbB 2 S ODNs could be greatly increased by adsorption of S ODNs by liposomes

Effect of anti-sense oligodeoxynucleotides homeobox B2 on the proliferation and expression of primary human umbilical vein endothelial cells

Objective: To explore the effect of homeobox B2 (HOXB2) antisense oligodeoxynucleotides (asodn) on the proliferation and expression of primary human umbilical vein endothelial cells (HUVECs). Methods: Various concentrations of HOXB2 asodn modified by thiophosphate transfected the induction of liposome into HUVECs. MTT and RT-PCR methods were employed to determine the effect of different concentrations of asodn on the endothelial proliferation and the expression level of HOXB2 mRNA. Results: After the transfection of HOXB2 asodn, the endothelial proliferation was inhibited in a dose-dependent fashion. Simultaneously, the expression of HOXB2 mRNA decreased significantly. Conclusions: HOXB2 plays an important role in the proliferation of endothelia.