Methods to Study RNA-Chromatin Interactions.

RNA plays a fundamental role in the organization of chromatin as well as the regulation of gene expression. Although the chromatin is pervasively attached by both coding and noncoding RNAs, the impact of these chromatin-associated RNAs (caRNAs) on gene expression and cellular functions and their underlying mechanisms have just begun to be unraveled. One approach to understand the potential mechanism of gene regulation by caRNAs is to identify the caRNA-associated genomic regions. Several groups have developed methods to capture RNA-chromatin interactions in either one RNA vs the whole genome, i.e., "one-to-all" or all RNAs vs the whole genome, i.e., "all-to-all" manner. In this chapter, we discuss several state-of-the-art methods highlighting the principles behind them, the experimental procedures, the advantages and limitations, and their applications. Our goal is to provide an overview and guide to researchers interested in exploring caRNAs using these techniques.

Identification of HnRNPC as a novel Tau exon 10 splicing factor using RNA antisense purification mass spectrometry.

Alternative splicing in Tau exon 10 generates 3 R- and 4 R-Tau proteoforms, which have equal abundance in healthy adult human brain. Aberrant alternative splicing in Tau exon 10 leads to distortion of the balanced 3 R- and 4 R-Tau expression levels, which is a causal factor to trigger toxic Tau aggregation, neuron dysfunction and patient death in a group of neurodegenerative diseases known as tauopathies. Hence, identification of regulators upstream of the Tau exon 10 splicing events are crucial to understanding pathogenic mechanisms driving tauopathies. In this study, we used RNA Antisense Purification with Mass Spectrometry (RAP-MS) analysis to identify RNA-binding proteins (RBPs) that interact with the Tau pre-mRNA near exon 10. Among the newly identified RBP candidates, we show that knockdown of hnRNPC induces Tau exon 10 skipping whereas overexpression of hnRNPC promotes Tau exon 10 inclusion. In addition, we show that hnRNPC interacts with the poly-uridine (U-tract) sequences in introns 9 and 10 of Tau pre-mRNA. Mutation of these U-tract motifs abolished binding of hnRNPC with Tau pre-mRNA fragment and blocked its impact on Tau exon 10 inclusion. These findings indicate that hnRNPC binds and utilizes these U-tract motifs located in introns 9 and 10 of Tau pre-mRNA to promote Tau exon 10 inclusion. Intriguingly, high hnRNPC expression level is associated with progressive supranuclear palsy (PSP), a sporadic tauopathy with pathological accumulation of Tau species that contain exon 10, which suggests a putative therapeutic role of hnRNPC for PSP treatment. [Figure: see text].

Predictive value of using plasma long non-coding RNAs ANRIL and HOXA11-AS for in-stent restenosis.

In-stent restenosis (ISR) can pose serious challenges for cardiologists following coronary stent implantation. Early identification of patients at high risk of ISR is considered to be effective for its prevention. However, factors that can reliably predict the risk of ISR remain elusive at present. The present study aimed to investigate the possible association between plasma long non-coding RNA (lncRNA) levels and ISR. A total of 410 patients with single-vessel lesion who received drug-eluting stents (DES) were included in the present study. After 12-36 months of follow-up, coronary angiography was performed and ISR was defined as >50% diameter stenosis at follow-up. RT-qPCR was used to measure lncRNA expression. Expression of the lncRNA RNA antisense non-coding RNA at the INK4 locus (ANRIL) was found to be upregulated whereas the lncRNA homeobox A11 antisense (HOXA11-AS) was downregulated in the plasma of patients with ISR compared with that from patients without ISR (P<0.001). Logistic regression analysis revealed that ANRIL [odds ratio (OR)=2.95; 95% confidence interval (CI)=1.68-8.08] was an independent risk factor for ISR, whilst HOXA11-AS (OR=0.58; 95% CI=0.48-0.71) was found to be an independent protective factor for ISR. Receiver operating characteristic (ROC) analysis demonstrated that high ANRIL expression [area under the ROC (auROC)=0.755; 95% CI=0.702-0.803] and low HOXA11-AS levels (auROC=0.712; 95% CI=0.657-0.763) predicted a high risk for ISR, and the combined score of ANRIL and HOXA11-AS (auROC=0.844; 95% CI=0.798-0.884) was more efficient at predicting ISR than either ANRIL or HOXA11-AS alone (P<0.001). In conclusion, increased ANRIL and decreased HOXA11-AS expressions were associated with ISR. However, combined ANRIL and HOXA11-AS plasma levels proved to be more effective at predicting ISR compared with either ANRIL or HOXA11-AS alone, suggesting that the multiplex detection of lncRNAs could be used to predict ISR in the future.

Current Status, Prevention and Treatment of BK Virus Nephropathy.

All renal transplant recipients should undergo a regular screening for BK viral (BKV) viremia. Gradual reduction of immunosuppression is recommended in patients with persistent plasma BKV viremia for 3 weeks after the first detection, reflecting the presence of probable or suspected BKV-associated nephropathy. Reduction of immunosuppression is also a primary intervention in biopsy proven nephropathy associated with BKV (BKVN). Thus, allograft biopsy is not required to treat patients with BKV viremia with stabilized graft function. There is a lack of proper randomised clinical trials recommending treatment in the form of switching from tacrolimus to cyclosporin-A, from mycophenolate to mTOR inhibitors or leflunomide, or the additive use of intravenous immunoglobulins, leflunomide or cidofovir. Fluoroquinolones are not recommended for prophylaxis or therapy. There are on-going studies to evaluate the possibility of using a multi-epitope anti-BKV vaccine, administration of BKV-specific T cell immunotherapy, BKV-specific human monoclonal antibody and RNA antisense oligonucleotides. Retransplantation after allograft loss due to BKVN can be successful if BKV viremia is definitively removed, regardless of allograft nephrectomy.

Critical role of SIRT1 upregulation on the protective effect of lncRNA ANRIL against hypoxia/reoxygenation injury in H9c2 cardiomyocytes.

Dysregulation of long non­coding RNA (IncRNA) antisense non­coding RNA in the INK4 locus (ANRIL) is associated with the risk of myocardial infarction (MI). Therefore, the present study aimed to determine the mechanisms underlying this association, which is currently poorly understood, to the best of our knowledge. The current study used an in vitro myocardial ischemia and reperfusion (MI/R) model, in which H9c2 cardiomyocytes were exposed to hypoxia/reoxygenation (H/R), which demonstrated that ANRIL expression was downregulated and that ANRIL positively regulated sirtuin 1 (SIRT1) expression following H/R injury. Subsequently, it was demonstrated that ANRIL upregulated SIRT1 expression by sponging microRNA­181a (miR­181a). In addition, ANRIL overexpression reduced lactate dehydrogenase release and apoptosis of H9c2 cardiomyocytes exposed to H/R, indicating that ANRIL prevented H/R­induced cardiomyocyte injury. Moreover, both miR­181a overexpression and SIRT1 knockdown significantly decreased the protective effects of ANRIL on H/R­induced cardiomyocyte injury, thus demonstrating that SIRT1 upregulation via sponging miR­181a is a critical mechanism that mediates the function of ANRIL. These results provided a novel mechanistic insight into the role of ANRIL in H/R­injured cardiomyocytes and suggested that the ANRIL/miR­181a/SIRT1 axis may be a therapeutic target for reducing MI/R injury.

Myostatin coordinating the proliferation and differentiation of adipose and skeletal muscle cells and energy metabolism balance based on gene chip technology / A miostatina na coordenação da proliferação e diferenciação de células musculares adiposas e esqueléticas e no equilíbrio do metabolismo energético com base na tecnologia de chips genéticos / Msrn, basado en la tecnología de chips genéticos, coordina la fertilización y el metabolismo de las células musculares grasas y óseas

ABSTRACT Myoblasts fuse into multinucleated muscle fibers to form and promote the growth of skeletal muscle. In order to analyze the role of myostatin (MSTN) in body fat, skeletal muscle cell proliferation and differentiation and energy metabolism, this study will use the antisense RNA technology of gene chip technology to study it. The results showed that the MSTN gene regulated the growth and proliferation of myoblasts and affected the development of skeletal muscle by affecting the expression of Cdc42, bnip2, p38 and other genes; knockout or overexpression of the MSTN gene would lead to a trend of fat-related genes from fat synthesis to fat decomposition; after the MSTN gene was knocked down, the expression levels of cpti-b, PPARG and other genes in the cells were corresponding after MSTN overexpression, the relative expression of the PPARG gene decreased. It is suggested that the knockout or overexpression of MSTN may affect lipid accumulation, and cpti-b and PPARG may directly regulate lipid level. It is hoped that this experiment can provide a reference for the study of MSTN effect on fat deposition.

RESUMO Os mioblastos se fundem eM fibras musculares multinucleadas para formar e promover o crescimento do músculo esquelético. A fim de analisar o papel da miostatina (MSTN) na gordura corporal, proliferação de células musculares esqueléticas e diferenciação e metabolismo energético, este estudo utilizará a tecnologia anti-RNA de chips genéticos para estudá-la. Os resultados mostraram que o gene MSTN regulava o crescimento e a proliferação de mioblastos e afetava o desenvolvimento do músculo esquelético, afetando a expressão de Cdc42, bnip2, p38 e outros genes; a eliminação ou sobrexpressão do gene MSTN conduziria a uma tendência de os genes adiposos sintetizarem a gordura até sua decomposição; após a eliminação do gene MSTN, os níveis de expressão de cpti-b, PPARG e outros genes nas células mostraram-se correspondentes após a sobrexpressão do gene MSTN, e a expressão relativa do gene PPARG diminuiu. Sugere-se que a eliminação ou sobrexpressão da MSTN possa afetar a acumulação de lipídeos, e o cpti-b e o PPARG podem regular diretamente o nível lipídico. Espera-se que esta experiência possa fornecer uma referência para o estudo do efeito da MSTN sobre a deposição de gordura.

RESUMEN Los mioblastos se funden en fibras musculares multinucleadas para formar y promover el crecimiento del músculo esquelético. A fin de analizar el papel de la miostatina (MSTN) en la grasa corporal, proliferación de células musculares esqueléticas y diferenciación y metabolismo energético, este estudio utilizará la tecnología anti-RNA de chips genéticos para estudiarla. Los resultados mostraron que el gen MSTN regulaba el crecimiento y la proliferación de mioblastos y afectaba el desarrollo del músculo esquelético, afectando la expresión de Cdc42, bnip2, p38 y otros genes; la eliminación o sobreexpresión del gen MSTN conduciría a una tendencia de que los genes adiposos sinteticen la grasa hasta su descomposición; después de la eliminación del gen MSTN, los niveles de expresión de cpti-b, PPARG y otros genes en las células se mostraron correspondientes después de la sobreexpresión del gen MSTN, y la expresión relativa del gen PPARG disminuyó. Se sugiere que la eliminación o sobreexpresión de la MSTN pueda afectar la acumulación de lipídos, y el cpti-b y el PPARG pueden regular directamente el nivel lipídico. Se espera que esta experiencia pueda proveer una referencia para el estudio del efecto de la MSTN sobre el depósito de grasa.

Knockdown of long non­coding RNA ANRIL inhibits the proliferation and promotes the apoptosis of Burkitt lymphoma cells through the TGF­ß1 signaling pathway.

Burkitt lymphoma (BL) has a high mortality rate and its treatment is currently limited to chemotherapy combined with immunotherapy. The long non­coding RNA antisense non­coding RNA in the INK4 locus (ANRIL) has been identified as an oncogene that can regulate cell proliferation and apoptosis in multiple types of cancer. However, the function of ANRIL in BL remains unknown. The present study aimed to determine the effect of ANRIL on cell proliferation and apoptosis in BL. Reverse transcription­quantitative PCR was used to analyze the expression levels of ANRIL in BL cells. The effect of ANRIL knockdown on BL cells was determined using Cell Counting Kit­8, flow cytometric, western blotting, immunofluorescence staining and Hoechst staining assays. The results revealed that ANRIL silencing inhibited the proliferation and promoted the apoptosis of BL cells. In addition, the expression levels of cyclin D1, E2F transcription factor 1 and Bcl­2 were downregulated, while the expression levels of cyclin­dependent kinase inhibitor 1A, Bcl­2­associated X protein, cleaved­caspase­9/pro­caspase­9 and cleaved­caspase­3/pro­caspase­3 were upregulated. Furthermore, the knockdown of ANRIL activated the TGF­ß1 signaling pathway, as evidenced by the upregulated expression levels of TGF­ß1, phosphorylated (p)­SMAD2/3/SMAD2/3, p­SMAD1/SMAD1 and sphingosine­1­phosphate receptor 2. Moreover, the protective effect of ANRIL silencing in BL could be inhibited by the TGF­ß receptor type I/II dual inhibitor, LY2109761. In conclusion, the findings of the present study suggested that the knockdown of ANRIL may inhibit cell proliferation and promote cell apoptosis in BL by regulating the TGF­ß1 signaling pathway, which may provide a novel target for the treatment of BL.

Long non-coding RNA SATB2-AS1 inhibits microRNA-155-3p to suppress breast cancer cell growth by promoting breast cancer metastasis suppressor 1-like.

BACKGROUND: Long noncoding RNAs (lncRNAs) and microRNAs (miRNAs) play vital roles in human cancers. Nevertheless, the effects of lncRNAs and miRNAs on breast cancer (BC) remain to be further investigated. This study was designed to testify the roles of lncRNA antisense transcript of SATB2 protein (SATB2-AS1) and microRNA-155-3p (miR-155-3p) in BC progression. METHODS: Levels of SATB2-AS1, miR-155-3p and breast cancer metastasis suppressor 1-like (BRMS1L) in BC were determined. The prognostic role of SATB2-AS1 in BC patients was assessed. The screened cells were respectively introduced with altered SATB2-AS1 or miR-155-3p to figure out their roles in malignant phenotypes of BC cells. The effect of varied SATB2-AS1 and miR-155-3p on BC cells in vivo was observed. Dual luciferase reporter gene assay and RNA-pull down assay were implemented to detect the targeting relationship of SATB2-AS1, miR-155-3p, and BRMS1L. RESULTS: SATB2-AS1 and BRMS1L were decreased while miR-155-3p was increased in BC cells and tissues. Patients with lower SATB2-AS1 expression had poor prognosis. Elevated SATB2-AS1 and inhibited miR-155-3p were able to restrain malignant behaviors of BC cells in vitro, as well as decelerate tumor growth in vivo. Oppositely, inhibited SATB2-AS1 and amplified miR-155-3p had converse effects on BC cell growth. MiR-155-3p mimic abrogated the impact of overexpressed SATB2-AS1. SATB2-AS1 could sponge miR-155-3p, and BRMS1L was the target gene of miR-155-3p. CONCLUSION: Elevated SATB2-AS1 and inhibited miR-155-3p could suppress the malignant phenotypes of BC cells, thereby restricting the development of BC.

RNA Therapy: Current Status and Future Potential.

Recent studies identified diverse RNAs including noncoding RNAs and their various action mechanisms in the cells. These RNAs regulate a variety of cellular pathways and are therefore expected to be important targets for the treatment of human diseases. Along with their extensive functional studies, RNA-based therapeutic techniques have developed considerably in recent years. After years of research and various trial and error, antisense RNAs and small interfering RNAs-based drugs have been developed and are now being used in the clinic. In addition, active research is ongoing to develop drugs based on RNA aptamer and messenger RNA. Along with the development of these RNA-based drugs, diverse strategies have been developed to transport RNA drugs into the cells efficiently. RNA therapy has many advantages over existing small molecule or monoclonal antibody-based therapies, including its potential to target all genes in the cells. This review will introduce the history of RNA therapy, and explain the basic concepts of RNA therapy and RNA-based drugs on the market or clinical trials. In addition, the future potential of RNA therapy will be discussed.

Long non-coding (lnc)RNA PAPAS overexpression inhibits tumor growth in papillary thyroid carcinoma by downregulating lncRNA HOTTIP.

The long non-coding (lnc)RNA pre-ribosomal RNA antisense transcripts (PAPAS) can repress ribosomal RNA synthesis, although its role in human disease is currently unknown. The present study aimed to investigate the function of PAPAS in papillary thyroid carcinoma (PTC). PAPAS was downregulated, while the lncRNA HOXA transcript at the distal tip (HOTTIP) was upregulated in patients with PTC. PAPAS and HOTTIP were inversely associated in PTC. PAPAS overexpression led to the downregulation of HOTTIP in PTC cells, while PAPAS expression was not significantly affected by HOTTIP overexpression, suggesting that PAPAS was upstream of HOTTIP. PAPAS expression level decreased, while HOTTIP expression level increased with the increase of clinical staging. PAPAS overexpression led to the inhibition of cell proliferation, while HOTTIP overexpression increased proliferation of PTC cells, and HOTTIP overexpression decreased the effects of PAPAS overexpression. lncRNA PAPAS overexpression may inhibit tumor growth in papillary thyroid carcinoma by downregulating lncRNA HOTTIP.

Effects of transfection with antisense microRNA-155 oligonucleotides on the growth of a human cutaneous squamous cell carcinoma cell line A431 / 中华皮肤科杂志

Objective To evaluate effects of antisense oligonucleotides against microRNA-155 (miRNA-155) on the proliferation,apoptosis,migration and invasion of a human cutaneous squamous cell carcinoma cell line A431.Methods A431 cells were divided into 3 groups:nonsense oligonucleotide group transfected with nonsense control oligonucleotides using liposomes,antisense oligonucleotide group transfected with antisense oligonucleotides against microRNA-155 using liposomes,and blank control group treated with Dulbecco's minimum essential medium (DMEM) containing Lipofectamine 2000.Real-time quantitative polymerase chain reaction (qRT-PCR)was performed to determine the expression of miRNA-155 in A431 cells:Methyl thiazolyl tetrazolium (MTT) assay was conducted to estimate cellular proliferative activity at 24,36,72,96 and 120 hours after transfection,flow cytometry to detect apoptosis and cell cycle changes,and Transwell assay to evaluate the migration and invasion of A431 cells.Statistical analysis was carried out by one-way analysis of variance (ANOVA) for intergroup comparisons and by least significant difference (LSD)-t test for multiple comparisons.Results After transfection,there were significant differences in the expression of miRNA-155 among the nonsense oligonucleotide group,antisense oligonucleotide group and blank control group (0.98 ± 0.02,0.28 ± 0.18,1.00 ± 0.01 respectively,F =634.57,P ＜ 0.001),and the expression of miRNA-155 was significantly lower in the antisense oligonucleotide group than in the blank control group and nonsense oligonucleotide group (both P ＜ 0.05).At 72,96 and 120 hours,there were significant differences in the survival rate of A431 cells among the 3 groups (all P ＜ 0.05),and the antisense oligonucleotide group showed a significantly lower survival rate of A431 cells compared with the blank control group and nonsense oligonucleotide group (all P ＜ 0.05).Additionally,the proportions of cells at G0/G1 phase and at S phase,and the cellular proliferative index all significantly differed among the 3 groups (F =23.46,36.81,19.35,respectively,P ＜ 0.01).The antisense oligonucleotide group showed significantly higher proportion of cells at G0/G1 phase (74.63％ ± 2.13％),but lower proportion of cells at S phase (9.88％ ± 1.83％) and cellular proliferative index (25.36 ± 2.13) compared with the blank control group(62.92％ ± 2.56％,18.86％ ± 2.78％,37.08 ± 2.56,respectively,all P ＜ 0.05) and nonsense oligonucleotide group (63.75％ ± 3.06％,18.33％ ± 3.72％,36.25 ± 3.06,respectively,all P ＜ 0.05).Additionally,the antisense oligonucleotide group showed significantly lower numbers of migratory cells and invasive cells compared with the blank control group and nonsense oligonucleotide group (all P ＜ 0.05).Conclusion Transfection of A431 squamous cell carcinoma cells with antisense miRNA-155 oligonucleotides can decrease the expression of miRNA-155,effectively inhibit the proliferation,migration and invasion of A431 cells,and promote cell apoptosis.

Effect of dexmedetomidine on expression of antisense hypoxia-inducible factor-1α during brain injury after asphyxial cardiac arrest and resuscitation in rats / 中华麻醉学杂志

Objective To evaluate the effect of dexmedetomidine on the expression of antisense hypoxia-inducible factor-1α (aHIF-1α) during brain injury after asphyxial cardiac arrest and resuscitation in rats.Methods Twenty-four pathogen-free healthy male Sprague-Dawley rats,weighing 250-280 g,were divided into 3 groups (n =8 each) using a random number table:sham operation group (group S),cardiac arrest and resuscitation group (group R) and dexmedetomidine group (group D).Asphyxial cardiac arrest and resuscitation were performed in R and D groups.The rats were tracheally intubated without clipping the trachea in group S.Dexmedetomidine 4 μg/kg was injected via the tail vein at 5 min before clipping the trachea in group D,while the equal volume of normal saline was given instead in S and R groups.Neurological deficit was assessed and scored (NDS) at 12,24,48 and 72 h after recovery of spontaneous circulation (T1 4).The rats were sacrificed after assessing neurological deficit at T4,brains were removed and hippocampi were isolated for determination of cell apoptosis (by TUNEL),HIF-1α expression (by Western blot) and expression of HIF-1α and aHIF-1α mRNA in hippocampal tissues (using polymerase chain reaction).Apoptosis rate was calculated.Results Compared with group S,the NDS at each time point and apoptosis rate of hippocampal neurons at T4 were significantly increased,and the expression of HIF-1α protein and mRNA and 5'aHIF-1α mRNA was up-regulated in R and D groups (P＜O.05).Compared with group R,the NDS at T2.4 and apoptosis rate of hippocampal neurons at T4 were significantly decreased,the expression of 5'aHIF-1α mRNA was down-regulated,and the expression of HIF-1α protein and mRNA was up-regulated in group D (P ＜ 0.05).Conclusion The mechanism by which dexmedetomidine reduces brain injury after asphyxial cardiac arrest and resuscitation is related to down-regulation of 5'aHIF-1α expression in rats.

The study of plasma biomarker for early diagnosis of Alzheimer disease / 中华老年医学杂志

Objective To extract,detect and validate the BACE1 expression-regulating lncRNA BACE1-AS containing in the plasma of patients with AD in Chinese Han people,so as to provide a research basis for plasma BACE1-AS in AD to be a plasma molecular markers and a new target for treatment.Methods The study included 27 AD patients and 28 normal individuals whose age,sex,education,etc.were matched between AD and controi group.Total RNA extraction of plasma was performed using guanidine isothiocyanate-phenol chloroform method.Target gene amplification was executed by RT-PCR Kit.Gel electrophoresis and its imaging analysis were performed on the RT-PCR amplified products.Target gene amplified products were sequenced,its sequence consistency with gene bank-reported sequence were compared,and differences in target gene transcription between the two groups were statistically analyzed.Results The positive expression rates of BACE1-AS were 18.5％(5/27 cases)in ADgroup and 0.0％ in control group,respectively(P=0.023).In comparison between two groups,there was a significant difference (P =0.023).Gene sequencing confirmed the consistent between BACE1-AS gene sequence of 3 patients with AD and Gene Bank's BACE1-AS sequence.But the two other AD cases showed individual base replacement.Conclusions Compared with the healthy control group,patients with AD show specific BACE1 expression-regulating lncRNA BACE1-AS in plasma of AD patients,which provides theoretical basis for BACE1 AS as a biomarker of AD diagnosis and a new target in therapy of AD.

Zebrafish Whole-Mount In Situ Hybridization Followed by Sectioning.

In situ hybridization is a powerful technique used for locating specific nucleic acid targets within morphologically preserved tissues and cell preparations. A labeled RNA or DNA probe hybridizes to its complementary mRNA or DNA sequence within a sample. Here, we describe RNA in situ hybridization protocol for whole-mount zebrafish embryos.

Construction of adenovirus-mediated anti-sense RNA targeting K-ras exon 1 in SW1990 cell line / 第二军医大学学报

Objective: To construct an adenovirus-mediated anti-sense RNA targeting K-ras exon 1 of SW1990 cell line and observe its effect on cell proliferation and apoptosis after transferred into SW1990 cell line. Methods: K-ras exon 1 cDNA was cloned into shuttle vector pShuttle-CMV and the resultant plasmid was confirmed by enzyme digestion and PCR. Clones with inverted insertion were selected and co-transferred into E. coli BJ5183 with an adenoviral backbone plasmid pAdEasy-1 to produce recombinant plasmid by homologous recombination. Recombinants were then selected and transfected into 293 cell line to produce recombinant adenovirus. Recombinant adenovirus production was confirmed by PCR analysis and was amplified and purified; the virus titer was determined. Ad-LacZ was used to infect SW1990 cells and the infection efficiency was observed by X-gal staining. SW1990 cells was infected with the recombinant adenovirus and their proliferation and apoptosis were determined by MTT and annexin V/PI FCM assay. Results: A 282 bp target gene fragment was acquired by PCR; the titer of recombinant adenovirus was 7.6 × 108 pfu/ml before purification by CsCl2 gradient centrifugation and 5.0 × 1910 pfu/ml after CsCl2 gradient centrifugation. When the recombinant adenovirus was at 100 MOI, the infection efficiency of SW1990 cells nearly reached 100%. The transfection of recombinant adenovirus significantly inhibited SW1990 cell proliferation (P<0.05), with a maximal inhibitory rate of 40.5% 4-5 days after infection; it also significantly increased SW1990 cell apoptosis, with the apoptotic rate being (22.54 ± 5.38) % 72 hours after infection. Conclusion: We have successfully constructed an anti-sense RNA adenovirus vector targeting K-ras exon 1 of SW1990 cell line, paving a way for the anti-sense K-ras gene therapy of pancreatic carcinoma.

Ultrasound-mediated destruction of gene-loaded microbubbles enhanced the transfection of phospholamban antisense RNA into cardiac myocytes / 中华超声影像学杂志

Objective To explore whether ultrasound-mediated microbubbles destruction could enhance anti-sense RNA transfection and expression. Methods Phospholamban antisense RNA eukaryon vector PcDNA 4. 1-asPLB was successfully constructed and it was transfected into cardiac myocytes by various methods including calcium phosphate precipitation, ultrasound exposure and ultrasound-mediated microbubbles destruction. The expression of PLB and sarcoplasmic retculum Ca2+ ATPase (SERCA2a) in cardiac myocytes was tested by RT-PCR and western blot. Results The transfection and expression of PcDNA 4. 1-asPLB increased significantly in cells treated with ultrasound-mediated microbubbles destruction compared to other transfer groups( P ＜0.05). The expression of PLB was inhibited specifically after cardiac myocytes were transfected with PcDNA 4. 1-asPLB. There was no change of PLB expression after cardiac myocytes transfected with PcDNA 4. 1 ( P ＜0.05). Though the expression of SERCA2a never exhibited any changes after PcDNA 4. 1-asPLB transfection, the PLB/SERCA2a ratio decreased markedly. Conclusions As a highly effective antisense RNA transfer method, ultrasound-mediated microbubbles destruction can enhance the transfection and expression of the PcDNA 4. 1-asPLB significantly. The PcDNA4. 1-asPLB transfection inhibits the expression of PLB and result in decrease of PLB/SERCA2a ratio in cardiac myocytes.

Inhibition of MHCⅡExpression on Human Dermal Fibroblast Through CⅡTA Anti-sense RNA / 中华皮肤科杂志

Objective To investigate the feasibility of using anti-sense RNA against classⅡmajor histocompatibility complex(MHCⅡ)transactivator(CⅡTA),which might regulate MHCⅡexpression,to suppress the relative immune response.Methods Stable transfectants of dermal fibroblasts with pDarⅡ(pDarⅡ-D)were tested for the expression of classic MHCⅡ(HLA-DR,-DP,-DQ)antigens induced with recombinant human interferon-gamma(IFN-?).mRNA abundance of CⅡTA,and classic MHCⅡwas mea-sured by RT-PCR.IL-2mRNA expressed in T cells,stimulated by transfected dermal fibroblasts,was de-termined by mixed lymphocyte reaction.Results When induced with IFN-?,the expression of HLA-DR and-DP antigens on pDarⅡ-D was reduced by95.63%and87.89%,respectively.Meanwhile,the mRNA contents of CⅡTA and classic MHCⅡwere decreased significantly(P

Application of EST Data of Siderophore Regulation Protein-like Gene of Aspergillus oryzae RIB40 / 吉林大学学报(医学版)

Objective: To acquire the new clear color phenotype of Aspergillus oryzae b y th e antisense strategy of siderophore regulation protein (SREP)-like gene. Method s: Construct the cDNA library of Aspergillus oryzae RIB40 and amplify the fr agme nt ac7336f from the every EST clone, which had high homology with SREP gene of o ther species, then construct the eukaryotic expression vector with SREP-like ge ne using antisense strategy. Results: The sequence of this SREP-like gene was a cquired, the vector was successfully constructed. Conclusion: The deduced amino acid sequence of SREP-like gene of Aspergillus oryzae indicated that there is t he high homology with those of SREP genes of Penicillium chrysogenum, Neur ospora crassa and Schizosaccharomyces pombe.

Combination of antisense human telomerase RNA and antisense human telomerase catalytic subunit inhibits cervical cancer Hela cell growth / 中华妇产科杂志

Objective To investigate the inhibitory effect of combination of antisense human telomerase RNA (anti hTR) and antisense human telomerase catalytic subunit (anti hTERT) on proliferation of cervical cancer Hela cell line cultured in vitro Methods Cervical cancer Hela cells were transfected by anti hTR, anti hTERT, anti hTR + anti hTERT, sense hTR, sense hTERT with Oligofectamin TM reagent The proliferation activity of cervical cancer Hela cells was determined using methyl thiazolyl tetrazolium (MTT) The activity of telomerase was tested by polymerase chain reaction telomeric repeat amplification protocol enzyme linked immunosorbent assay (PCR TRAP ELISA) Cell morphologies were observed under fluorescence microscope with acridine orange staining Apoptosis and cell cycle were examined by flow cytometer method (FCM) Results The inhibitory rates on proliferation and apoptotic rates of Hela cells transfected by antisense oligonucleotides were significantly higher compared with those of control, sense oligonucleotides and Oligofectamin TM (all P

Effects of plasminogen activator inhibitor-1 antisense RNA on aorta endothelial cells cultured / 中华老年医学杂志

Objective To investigate the role of antisense RNA of plasminogen activator inhibitor 1 (PAI 1) in regulating the expression of PAI 1 and vascular endothelial growth factor (VEGF) in aorta endothelial cells (EC) cultured in vitro. Methods The second extron of PAI 1 was amplified by polymerase chain reaction(PCR) and the product was inserted into eukaryotic cell expression vector pcDNA3.1(-) to construct PAI 1 antisence RNA recombinant plasmid. The recombinant plasmid was transfected into EC and the PAI 1 expression was detected by immunohistochemistry, Westernblot and ELISA. The effects of PAI 1 variation on VEGF were examined by immunofluorescence method. Results PAI 1 antigen was the lowest (0 017 ng/ml) in cells and the immunofluorescence representing the expression of VEGF in the cytoplasm showed the weakest at the third day after transfection. At the fifth day, PAI 1 antigen increased to 0 093 ng/ml with VEGF expression increased correspondingly. At the seventh day, PAI 1 antigen(0 143 ng/ml) and VEGF increased closed to normal level. Conclusions PAI 1 antisense RNA blocked the translation of PAI 1 proteins effectively and inhibited the expression of VEGF in aorta endothelial cells.