Epigenetic regulation in pulmonary heart disease by non-coding RNA / 中国药理学通报

Pulmonary heart disease (PHD) is a common cardiovascular disease in China, with high mortality ratein its late stage.Currently, PHD treatments are mainly to delay the disease progression, which cannot effectively improve and cure the disease.Consequently, it is in urgent need to find a new treatment for PHD.Epigenetic regulation plays an important role in the occurrence and pathological process of diseases, which provides a novel idea of developing new drugs by regulating non-coding RNA(ncRNA).However, ncRNA studies in PHD are at the stage of few and shallow, stray and scattered.Therefore, this review for the first time summarizes ncRNA related to various PHD phenotypes, and discusses the epigenetic regulation in PHD by ncRNA.

Antioxidant activities of 10 active compounds from Clematis filamentosa Dunn. and their protective effects on H / 中国药理学通报

Aim To investigate the protective effects of the 10 compounds from Clematis filamentosa Dunn, on H

Tumor microenvironment responsive biomimetic nanoparticles for photodynamic tumor therapy / 药学学报

Inspired by the coordination effects between imidazole and metal ions in hemoglobin, biomimetic nanoparticles were constructed for photodynamic tumor therapy. The photosensitizer of protoporphyrin IX (PpIX) was modified with histidine, which could be self-assembled with Zn2+ to obtain the biomimetic nanoparticles (NPs). Under the conditions of high glutathione and low pH, the biomimetic nanoparticles could be degraded and released for enhanced photodynamic tumor therapy. The structures of NPs were characterized by dynamic light scattering (DLS), UV-visible spectrophotometer (UV-Vis), fluorescence microscope and transmission electron microscope (TEM). The reactive oxygen species (ROS) production ability of NPs was measured by singlet oxygen sensor green (SOSG) test kit. Mouse breast cancer cell lines (4T1 cells) were employed to investigate the subcellular organelle distribution and cytotoxicity of NPs. These results confirmed that NPs possessed a good dispersibility and stability with a uniform structure and particle size at 165 nm. Moreover, MTT assay and live/dead cell staining assay demonstrated that NPs could inhibit the proliferation of 4T1 cells and exhibit a good biocompatability. This research would promote the construction of intelligent biomedicine for tumor precision therapy.

Determination of genome-wide DNA and total RNA methylation in rats with myocardial infarction by mass spectrometry / 药学学报

To detect the methylation level of genome-wide DNA and total RNA in the process of heart failure, we established the method of ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) to observe the change and synchronization of methylation rate of myocardial infarction (MI) tissue and peripheral blood. Animal welfare and experimental process were in accordance with the regulations of the Animal Ethics Committee of Guangzhou Medical University. The rats with myocardial infarction were divided into three groups: 1st, 4th, and 8th week to simulate different levels of cardiac function. And they were euthanized at the same time to keep the same age. DNA and RNA were extracted from infarct marginal tissues and peripheral blood lymphocytes, and then decomposed into single nucleosides by enzymolysis. The methylation rate of DNA and RNA was measured and calculated quantitatively. The results showed a concordant methylation changes in tissue and blood, and the methylation level of genome-wide DNA and total RNA was increased after myocardial infarction in rats. In this study, we obtained the preliminary data of DNA and RNA methylation during the occurrence and development of heart failure, further indicating that epigenetic changes can be used as biomarkers for early diagnosis of heart failure.

Therapeutic effects of artesunate on cytomegalovirus pneumonia in mice / 药学学报

To investigate the therapeutic effect of artesunate on mouse cytomegalovirus pneumonia, the BALB/c-nu mice were infected with murine cytomegalovirus-green fluorescent protein (MCMV-GFP) by nose dropping method. The experimental protocol was approved by the Medical Laboratory Animal Ethics Committee of Guangzhou Medical University. The BALB/c-nu mice were randomly divided into five groups: control group, MCMV pneumonia group, and artesunate (60, 120, and 240 mg·kg-1) groups. The survival rate, weights, and virus loads in lungs among the groups were observed. The degree of histopathologic changes in lungs was assessed directly by hematoxylin-eosin (HE) assay. MCMV-GFP expression was assessed by immunofluorescence. In addition, reverse transcription polymerase chain reaction (RT-PCR) analysis was performed to investigate the content of major immediate early 1 (Mie1) mRNA, and enzyme-linked immunosorbent assay (ELISA) was used to detect the changes of inflammatory factors, interleukin 10 (IL-10), IL-6, and tumor necrosis factor-α (TNF-α). Western blot analysis was used to detect the expression of the changes of nuclear factor-kappa B (NF-κB) signaling pathways in total proteins. Compared with MCMV group, artesunate (120 mg·kg-1) significantly increased body weights of MCMV-infected nude mice over 30 days, and decreased the viral titer in lung homogenate, lung inflammation, and histological severity. Moreover, the administration of artesunate (120 mg·kg-1) could downregulate the expression of phospho-NF-κB (p-NF-κB) p65 in the lungs of mice. The present study suggested that artesunate can protect the immunocompromised mice from MCMV-induced interstitial pneumonia via downregulating NF-κB signaling pathway, thus attenuating inflammation in the lungs.

Design and in vitro evaluation of delivery systems for co-delivery of siRNA and proteins by mimetic exosomes / 药学学报

To prepare the mimetic exosomes and co-delivery proteins and nucleic acids, and achieve efficient and safe co-delivery of multi-component drugs, an optimized formulation was designed by modifying a polylactic acid-glycolic acid copolymer (PLGA) matrix with a cationic lipid excipient dioleyl trimethylammonium propane (DOTAP), and a PLGA/DOTAP nanoparticles packaged protein and nucleic acid was prepared by double emulsion method, and the outermost membrane structure prepared by reverse phase evaporation method and consists of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol and membrane proteins. The structure of the mimetic exosomes is formed by ultrasonic dispersion and extrusion, and analyzed its characteristics and nature of the transfer effect. The size of mimetic exosomes was about 156.13 nm, with negative charge (-18.23 ± 0.57 mV), and it could efficiently co-transfer protein and siRNA, and siRNA could effectively inhibit the expression of target gene Trim28. The mimetic exosomes simulate the structure of exosomes and achieve safe and efficient co-delivery of multi-component drugs.

Lineage reprogramming of fibroblasts into induced cardiac progenitor cells by CRISPR/Cas9-based transcriptional activators

Overexpression of exogenous lineage-determining factors succeeds in directly reprogramming fibroblasts to various cell types. Several studies have reported reprogramming of fibroblasts into induced cardiac progenitor cells (iCPCs). CRISPR/Cas9-mediated gene activation is a potential approach for cellular reprogramming due to its high precision and multiplexing capacity. Here we show lineage reprogramming to iCPCs through a dead Cas9 (dCas9)-based transcription activation system. Targeted and robust activation of endogenous cardiac factors, including GATA4, HAND2, MEF2C and TBX5 (G, H, M and T; GHMT), can reprogram human fibroblasts toward iCPCs. The iCPCs show potentials to differentiate into cardiomyocytes, smooth muscle cells and endothelial cells . Addition of MEIS1 to GHMT induces cell cycle arrest in G2/M and facilitates cardiac reprogramming. Lineage reprogramming of human fibroblasts into iCPCs provides a promising cellular resource for disease modeling, drug discovery and individualized cardiac cell therapy.

The role of stem cell-derived exosomes in repairing myocardial injury / 生理学报

At present, it is generally believed that the paracrine effect of stem cells in the repair of myocardial injury is one of the important ways for stem cell therapy. Exosomes are phospholipid bilayer-enclosed nanovesicles that secreted by cells under physiological and pathological conditions. Cargo loaded into exosomes including protein, lipids and nucleic acids can be delivered to recipient cells. Therefore, exosomes are recognized as important mediators for intercellular communication. It has been suggested that exosomes from stem cells (eg. embryonic stem cells, induced pluripotent stem cells, cardiac progenitor cells, mesenchymal stem cells and cardiosphere-derived cells) have protective effects against heart injury. In this review, we summarized recent research progresses on stem cell-derived exosomes in myocardial injury, including the therapeutic effects and mechanism.

Progress in histone H3 methylation and heart development / 中国病理生理杂志

Congenital heart disease is one of the main types of birth defect.The mammalian heart developmen-tal progress requires precise gene patterning in time and space.In addition to the gene sequence,recent research showed that the regulation of core cardiac gene expression has been proved to be closely related to cardiac transcription factors as well as the modification of genomic architecture of the histone.Methylation of histone might be the key nodes in the regula-tion of cardiac gene expression and chromatin structure.This review focuses on the role of histone H 3 methylation in heart development process,which may lay a foundation for the prediction of epigenetic modification of congenital heart disease.

Exosomes from LPS treated mesenchymal stem cells regulate proportion of Ly6C monocyte population / 中国药理学通报

Aim To determine the effect of exosomes from lipopolysaccharide-treated human bone marrow mesenchymal stem cells on proportion of Ly6Chigh and Ly6Clow monocytes/macrophages in inflammatory micro- environment. Methods BMSCs were obtained by gra-dient centrifugation, identified and then treated with li-popolysaccharide for 48 h. The exosomes were purified from conditional medium with or without LPS treatment and identified by CD63 protein using Western blot and transmission electron microscope. The diameters and concentration were detected by Nanoparticle Trafficking Analysis ( NTA ) . The monocytes/macrophages were sorted from bone marrow of the mice by magnetic beads. Cells were co-cultured with exosomes for 24 hours, and then treated with LPS for 48 hours. The proportion of Ly6C monocytes/macrophages was detec-ted by flow cytometry. Inflammatory cytokines in cell supernatant were investigated using ELISA. Results BMSCs surface markers CD44, CD90 were positively detected, but CD34, CD45 were not expressed. BM-SCs presented adipogenic differentiation ability. Exo-somes were positively expressing CD63 protein, and NTA showed that the diameters of exosomes were up to (82.4 ± 3.7 ) nm. BMSCs stimulated by LPS pro- duced more exosomes ( P < 0.01 ) . Exosomes from BMSCs with or without LPS treatment could increase the ratio of Ly6Chigh monocytes (P<0.01) and down-regulate the ratio of Ly6Chigh macrophages (P<0.05), and the effect of LPS treated-exosomes was more signif-icant than untreated-exosomes (P<0.05). Moreover, the concentration of IL-6 was also elevated under exo-somes treatment ( P <0.05 ) . Conclusions Human bone marrow mesenchymal stem cells-derived exosomes contribute to the regulation of Ly6Chigh monocytes/mac-rophages, indicating that they could be involved in the therapeutic treatment of inflammatory diseases.

Exosomes as potential drug delivery tools for treatment of cardiovascular diseases / 中国药理学与毒理学杂志

Exosomes serve as vesicles to deliver protein, lipids, nucleic acids or other cellular components, to neighboring or distant cells. Recent studies have highlighted the potential therapeutic effects of stem cell-derived exosomes on cancer and cardiovascular diseases. Our previous studie-shave investigated the role of stem cell-derived exosomes in cardiac protection. Mesenchymalstem cells released miR-22-enriched exosomes after ischemic preconditioning and these exosomes showed protective effects oncardiomyocytes.MiR-21-conaining exosomes were secreted by H2O2-treated cardiac progenitor cells and protected cardiomyocytes from H2O2-induced apoptosis. Heat-shock lead to the production ofheat shock factor 1-enriched exosomes from cardiac stem cells, which reducedapoptosis of cardiomyocytes. Given these important effects of exosomes in intercellular communications, exosomes have been proposed as a vector for drug delivery or other therapeutic purposes. However, cells secretea limited number of exosomes, which has hampered the development of exosomes for research and clinical application.Synthetic exosome-mimics by cellextrusion or cell membrane-cloaked nanoparticles, which canbe fabricated on a large-scale, provide novel platforms fordrug delivery. Two Korean groups fabricated exosome-mimetic nanovesicles by extruding monocytes or macrophages through a serial of filters and utilized these exosome-mimetics for the delivery of anti-tumor drug. Recently,cell membrane-cloaked nanoparticles have emergedas a potential tool for drug delivery with the advantages ofimmunocompatibility, stability and targeting capabilityfor the treatment of cancer. In summary, exosomes or exosome-mimics may serve as potential therapeutic tools for the treatment of cardiovascular diseases.

Genetic and epigenetic predispositions underlying cardiovascular outcomes among patients treated with clopidogrel and aspirin / 中国药理学与毒理学杂志

Coronary artery disease (CAD)is a major cause of death and disability worldwide, and consumes a considerable amount of medical resources every year.Clopidogrel is a first-line antiplate-let therapy for CHD, butit is associated with substantial variability in PK and pharmacodynamics re-sponse. To date, gene variants explain only a smallproportion of the variability.The study aimed to identify new genetic loci-modifying antiplatelet response to clopidogrel in Chinese patients with CAD by a systematic analysis combining antiplatelet effects and PK, and further to investigate the PON1 gene promoter DNA methylation and genetic variations possibly influencing clinical outcomes in pa-tients undergoing PCI. We identified novel variants in two transporter genes (SLC14A2rs12456693, ATP-binding cassette [ABC]A1 rs2487032) and in N6AMT1 (rs2254638) associated with P2Y12 reac-tion unit (PRU) and plasma active metabolite (H4) concentration. These new variants dramatically im-proved the predictability of PRU variability to 37.7％. The associations between these loci and PK pa-rameters of clopidogrel and H4 were observed in additional patients, and its function on the activation of clopidogrel was validated in liver S9 fractions (P<0.05). Rs2254638 was further identified to exert a marginal risk effect formajor adverse cardiac events in an independent cohort.Multivariate logistic regression analysis indicated that PON1methylation level at CpG site-161 (OR=0.95; 95％ CI=0.92–0.98;P<0.01)and the use of angiotensin converting enzyme inhibitors(OR=0.48;95％ CI=0.26–0.89;P<0.01) were associated with decreased risk of bleeding events. In conclusion, new genetic variants were systematically identified as risk factors for the reduced efficacy of clopidogrel treatment.The ab-normal expression of DNA methylation-regulating key genes in the pharmacokinetic and pharmacody-namics pathways of clopidogrel and aspirin may modify clinical outcomes in dual antiplatelet-treated pa-tients undergoing PCI.

Progress in genetic and epigenetic research on in-stent restenosis after percutaneous coronary interventions / 中华医学遗传学杂志

Coronary heart disease is one of the most important causes of death in human, and consumes vast medical resources. Percutaneous coronary intervention (PCI) has been a significant breakthrough for its treatment. However, clinical application has been hampered by in-stent restenosis (ISR). Although drug eluting stent (DES) has reduced the occurrence of restenosis, incidence of ISR is still about 5% to 10%. The main reasons for restenosis after PCI are hyperplasia of vascular endothelial cells and smooth muscle cell migration. The exact mechanism of personalized differences in restenosis is not clear yet, but there may be a variety of risk factors. In addition to aging, smoking and diabetes, an increasing number of studies have found that genetic and epigenetic factors play an important role in ISR. In this article, authors have reviewed genetic and epigenetic factors on the progression of ISR, which may help to determine the genetic risk factors in patients with ISR after PCI.

Impact of CYP2C9 and VKORC1 polymorphism on warfarin response during initiation of therapy / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate potential contributions of genetic variants of cytochrome P-450 2C9 (CYP2C9) and vitamin K expoxide reductase (VKORC1) to the anticoagulation response during the initiation of warfarin therapy in the Han Chinese population.</p><p><b>METHODS</b>A total of 798 Han Chinese patients received long-term warfarin anticoagulant therapy orally after valve replacement in our hospital between 2000 and 2008 were included in this study. Nine single nucleotide polymorphism (SNP) loci [rs12572351 G > A, rs9332146 G > A, rs4917639 G > T, rs1057910 A > C (CYP2C9(*)3), rs1934967 G > T, rs1934968 G > A, rs9923231 C > T (VKORC1-1639 G > A), rs2359612 G > A and rs10871454 C > T] in 2 genes including CYP2C9 and VKORC1, which were possibly correlated with warfarin pharmacokinetics and pharmacodynamics through literature retrieval, were selected and analyzed. Warfarin steady-state dose requirement, time to the INR (the international normalized ratio) within the therapeutic range and percent of the INR of more than 3.5 were compared among genotype subgroups. SNaPshot technique was used to detect gene SNPs; Hardy-Weinberg genetic equilibrium test was used to test population representativeness.</p><p><b>RESULTS</b>CYP2C9(*)3 genotype did not affect the required warfarin dose while it was associated with increased risk of bleeding when treated with routine dosage regimen during the initiation of treatment. The allelic mutation frequency at VKORC1 gene rs10871454G > A and VKORC1-1639G > A SNP loci was 92.04% and 88.03%, respectively and rs10871454 was in perfect linkage disequilibrium with-1639. Patients with VKORC1 rs10871454 genetic mutation required lower warfarin dose in the first 28 days of therapy. VKORC1-1639 genetic polymorphism was also associated with shorter time to the INR within the therapeutic range and increased risk of over-anticoagulation.</p><p><b>CONCLUSION</b>Detecting genetic polymorphism of CYP2C9 and VKORC1 could guide clinical use of warfarin to reduce the risk of adverse reactions including bleeding in patients receiving chronic anticoagulation therapy.</p>

Screening and identification of auto-antigen RHDAG1 of rheumatic heart disease / 南方医科大学学报

<p><b>OBJECTIVE</b>To identify the candidate auto-antigen of rheumatic heart disease as a molecular marker for this disease.</p><p><b>METHODS</b>The total RNA of the heart tissue of patients with rheumatic heart disease was extracted and reverse-transcribed into long cDNA to construct the phage expression library. The library was screened using the serum from patients with active rheumatic fever, and the positive clone was identified and analyzed by bioinformatics and expressed in vitro. The expressed products were evaluated with Western blotting and its cross-reactivity was assessed.</p><p><b>RESULTS</b>The phage expression library of the heart tissue of patients with rheumatic heart disease was constructed, with the titer of the primary library of 3.3×10(6) pfu/ml, recombinant rate of 99%, and 81% of the inserted segments were larger than 1 kb. An auto-antigen RHDAG1 was identified by screening, which was homologous to keratin 18. RHDAG1 was detected in the serum of patients with active rheumatic fever and of those with rheumatic heart disease, but not in the serum of healthy subjects.</p><p><b>CONCLUSION</b>Phage display library can be an effective strategy to screen the auto-antigens of rheumatic heart disease. The auto-antigen RHDAG1 can be a candidate molecular biomarker of rheumatic heart disease and/or rheumatic fever.</p>

Effect of impaired glucose tolerance on cardiac dysfunction in a rat model of prediabetes / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>The effect of impaired glucose tolerance (IGT) on cardiac function during the chronic prediabetes state is complicated and plays an important role in clinical outcome. However, the molecular mechanisms are not fully understood. This study was designed to observe cardiac dysfunction in prediabetic rats with IGT and to determine whether glucose metabolic abnormalities, inflammation and apoptosis are linked to it.</p><p><b>METHODS</b>The IGT rat models were induced by streptozocin, and the heart functions were assessed by echocardiography. Myocardial glucose metabolism was analyzed by glycogen periodic acid-Schiff staining, and the pro-apoptotic effect of IGT was evaluated by TUNEL staining. Additionally, caspase-3 activation, macrophage migration inhibitory factor (MIF) and G-protein coupled receptor kinase 2 (GRK2) were detected by Western blotting in cardiac tissue lysates.</p><p><b>RESULTS</b>Area-under-the-curve of blood glucose in rats injected with streptozotocin was higher than that in controls, increased by 16.28%, 38.60% and 38.61% at 2, 4 and 6 weeks respectively (F = 15.370, P = 0.003). Abnormal cardiac functions and apoptotic cardiomyocytes were observed in the IGT rats, the ejection fraction (EF) being (68.59 ± 6.62)% in IGT rats vs. (81.07 ± 4.59)% in controls (t = 4.020, P = 0.002). There was more glucose which was converted to glycogen in the myocardial tissues of IGT rats, especially in cardiac perivascular tissues. Compared to controls, the cleaved caspase-3, MIF and GRK2 were expressed at higher levels in the myocardial tissues of IGT rats.</p><p><b>CONCLUSIONS</b>IGT in the prediabetes period resulted in cardiac dysfunction linked to abnormal glycogen storage and apoptosis. Additionally, MIF and GRK2 may be involved in the pathogenesis of cardiac dysfunction in prediabetes and their regulation may contribute to the design of novel diagnostic and therapeutic strategies for those who have potential risks for diabetic cardiovascular complications.</p>

Clinical application of anticoagulation treatment with warfarin after prosthetic heart valve replacement: a single center-based survey / 南方医科大学学报

<p><b>OBJECTIVE</b>To investigate the clinical application of anticoagulation treatment with warfarin after prosthetic heart valve replacement and compare the effect and safety of different anticoagulant intensities.</p><p><b>METHODS</b>A total of 845 Chinese patients receiving oral warfarin for anticoagulant treatment after prosthetic heart valve replacement in Guangdong General Hospital between 2000 and 2008 were enrolled in this survey. The general data, clinical data, medications, international normalized ratio (INR) and results of echocardiogram of these patients were followed up to observe the incidence of complication of thrombo-embolism and such adverse effect as hemorrhage.</p><p><b>RESULTS</b>All the patients were of Han nationality, and Cantonese accounted for 88.04%. The daily mean maintenance dose of warfarin was 2.92∓0.88 mg in these patients with a median INR of 2.09∓0.39. Of these patients, 44.62% received low-intensity anticoagulant treatment with warfarin with the INR maintained between 1.5 and 2.0, and 56.45% had standard anticoagulant intensity with the INR maintained between 2.0 and 3.0. The total incidence of thrombo-embolism was 4.14%. Severe hemorrhage occurred in 14 cases (1.66%), most frequently in the alimentary tract. The events of hemorrhage were correlated to the type of prosthetic heart valve replacement, occurring more frequently in patients with mechanical prosthetic heart valve replacement than in those with biological ones. No significant difference was found in the incidence of thrombo-embolism and server hemorrhage between the two groups receiving low and standard intensity therapy anticoagulant.</p><p><b>CONCLUSION</b>The effect and safety of low-intensity anticoagulant treatment are comparable to that of standard intensity treatment in Chinese Han patients, and anticoagulation treatment with warfarin is effective and safe to maintain the INR between 1.8-3.0.</p>

Effect of ketamine on transient outward potassium current of isolated human atrial myocytes / 药学学报

The effects of ketamine on transient outward potassium current (I(to)) of isolated human atrial myocytes were investigated to understand the mechanism of part of its effects by whole-cell patch-clamp. Atrial myocytes were enzymatically isolated from specimens of human atrial appendage obtained from patients under going cardiac valve displacing. Ito is recorded in voltage-clamp modes using the patch-clamp technique at room temperature. Currents signals were recorded by an Axopatch 200B amplifier with the Digidata 1322A-pClamp 9.0 data acquisition system. Ketamine decreased I(to) of human atrial myocytes in a dose-dependent manner. The current-voltage curve was significantly lowered, 30, 100, 300, and 1000 micromol x L(-1) ketamine decreased respectively I(to) current density about (13.62 +/- 0.04)%, (38.92 +/- 0.05)%, (72.24 +/- 0.10)% and (83.84 +/- 0.05)% at the potential of 50 mV, with an IC50 of 121 micromol x L(-1). The I(to) activation curve, inactivation curve and the recovery curve were not altered by ketamine. So, ketamine concentration-dependently decreased I(to) of human atrial myocytes.

High glucose up-regulates GRK2 gene expression in H9C2 cardiomyoblasts in vitro / 南方医科大学学报

<p><b>OBJECTIVE</b>To study the effect of high glucose on GRK2 gene expression in H9C2 cardiomyoblasts in vitro.</p><p><b>METHODS</b>H9C2 cardiomyoblasts were cultured for 72 h in the presence of 0, 5.5, 12.5, 25 or 33 mmol/L glucose (with the osmotic pressure adjusted with monnitol). Semi-quantitative detection of GRK2 gene expression in H9C2 cardiomyoblasts was carried out using RT-PCR and phosph-Akt (Ser473) protein level was measured by Western blotting.</p><p><b>RESULTS</b>Glucose in the culture medium (5.5 to 33 mmol/L) concentration-dependently increased the mRNA expression of GRK2 concentration and decreased phosphorylation Akt (ser473) level in in H9C2 cardiomyoblasts.</p><p><b>CONCLUSION</b>Increased GRK2 gene expression may play an important role in cardiac dysfunction in diabetes.</p>

An efficient method for screening effective siRNAs using dual-luciferase reporter assay system / 南方医科大学学报

<p><b>OBJECTIVE</b>To establish an efficient method for screening effective small interference RNA (siRNA) using dual-luciferase reporter assay system.</p><p><b>METHODS</b>Based on the siRNA expression vector pSilencer-4.1, 3 candidate green fluorescence protein (GFP) gene siRNA expression plasmids, namely pSi-GFPsiRNA1, pSi-GFPsiRNA2, and pSi-GFPsiRNA3, along with the negative control pSi-Negative, were constructed. Using the pGL3-promoter vector, the GFP-luciferase (GFP-LUC) expression plasmid pGL3-GFPf was constructed with the same Kozak consensus translation initiation site and start codon ATG for GFP-LUC coding sequence. The GFP fragment containing the target sequences of 3 GFP siRNAs was introduced into the 3' untranslate region of LUC in the modified pGL3-promoter vector to construct the plasmid pGL3-GFPp. The GFP siRNAs expression plasmids and Renilla luciferase reporter vector pRL-TK were co-transfected with pGL3-GFPf or pGL3-GFPp into the HEK293 cells, respectively. The luciferase activities were determined by dual-luciferase reporter assay, and the GFP mRNA expressions were detected by real-time quantitative PCR.</p><p><b>RESULTS</b>In the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPf, the luciferase activities were reduced obviously, and the reduction was more significant in cells transfected with GFPsiRNA1 compared with the control cells (P<0.01).GFP mRNA levels were also markedly lowered in cells transfected with GFPsiRNA1 as shown by real-time PCR (P<0.01). In addition, the results of dual-luciferase reporter assay and real-time PCR showed that among the groups cotransfected with GFP siRNAs expression plasmids and pGL3-GFPp, the GFP expression was inhibited most obviously by GFPsiRNA1 (P<0.01).</p><p><b>CONCLUSION</b>The dual-luciferase reporter assay system provides a useful method for screening effective siRNAs targeting specific genes.</p>