Post-transcriptional gene silencing: Basic concepts and applications

Post-transcriptional gene silencing (PTGS)-mediated gene silencing exploits the cellular mechanism whereintranscripts having sequence similarity to the double-stranded RNA (dsRNA) molecules present in the cell will besubjected to degradation. PTGS is closely related to natural processes such as RNA-mediated virus resistance andcross-protection in plants. Gene silencing and the cellular machinery for affecting this phenomenon might haveevolved as a natural protective measure against viral infection in plants. In PTGS, small interfering RNA (siRNA)molecules of 21–23 nucleotides length act as homology guides for triggering the systemic degradation of transcriptshomologous to the siRNA molecules. PTGS phenomenon, first discovered in transgenic petunia plants harbouringchalcone synthase gene and termed co-suppression, has been subsequently exploited to target specific gene transcripts for degradation leading to manifestation of desirable traits in crop plants. Targeted gene silencing has beenachieved either through the introduction of DNA constructs encoding dsRNA or antisense RNA or by deploying cosuppression constructs producing siRNAs against the transcript of interest. Understanding the mechanism of genesilencing has led to the development of several alternative strategies for inducing gene silencing in a precise andcontrolled way. This has paved the way for using PTGS as one ofthe chief functional genomicstools in plants and hashelped in unraveling the mechanism of many cellular processes and identifying the focal points in pathways, besides,opening new vistas in genetic engineering of plants for human benefits. PTGS has shown great potential in silencingthe deleterious genes efficiently so that value-added plant products could be obtained. Thus, PTGS has ushered in anew era in the genetic manipulation of plants for both applied and basic studies. In this review, we have outlined thebasics of RNAi-mediated gene silencing and summarized the work carried out at our institute using this approach, ascase studies. In particular, adopting RNAi-mediated gene silencing (a) as a method to restore fertility in transgenicmale sterile lines developed based on orfH522 gene from sunflower PET1-CMS source, (b) as a tool to suppress theproduction of toxic proteins, ricin and RCA, in castor, and (c) as an approach to induce bud necrosis virus resistancein sunflower has been discussed. Examples from other plant systems also have been mentioned to exemplify theconcept and utility of gene silencing in crop plants.

Study on network pharmacology mechanism of Leonurus japonicus in treating dysmenorrhea caused by cold coagulation and blood stasis / 中草药

Objective: To establish a chemical constitution-disease target-metabolic signaling pathway network of Leonurus japonicus, and explore the mechanism of multi-component, multi-target and multi-pathway of L. japonicus for the treatment of dysmenorrhea caused by cold coagulation and blood stasis. Methods: TCMSP database and Swiss Target Prediction server were used to obtain the chemical components and action targets of L. japonicus. Combined with DrugBank, DisGeNET, TTD and other databases, the action targets of L. japonicus for treating dysmenorrhea symptoms wtih cold coagulation and blood stasis were obtained. Through DAVID's website, GO function enrichment analysis and KEGG pathway analysis were conducted on the targets, and cytoscape 3.6.0 was used to construct the network diagram of the active component-dysmenorrhea target-metabolic signaling pathway of L. japonicus, to explore the mechanism of action of L. japonicus in treating dysmenorrhea with cold coagulation and blood stasis. Furthermore, Western blotting experiments were conducted to verify the effect of L. japonicus on the expression of PTGS1 and PTGS2 proteins in the uterine tissues of the model rats with cold coagulation and blood stasis dysmenorrhea. Results: Eight potential active ingredients of L. japonicus were obtained, including 22 dysmenorrhea related disease targets, main targets PTGS1, PTGS2, ALOX5, PLAG2A, AKR1C3, etc. A total of 71 GO items were obtained by GO functional enrichment analysis (P < 0.05), including 46 biological process (BP) items, four cell component (CC) items, and 21 molecular function (MF) items. There were 22 possible targets for the treatment of dysmenorrhea, and seven signaling pathways were obtained through KEGG pathway enrichment and screening (P < 0.05), involving arachidonic acid metabolism, linoleic acid metabolism, steroid hormone biosynthesis, etc. L. japonicus significantly increased the protein expression levels of PTGS1 and PTGS2 in the uterus of the model rats (P < 0.05), which confirmed some of the predicted results of network pharmacology. Conclusion: L. japonicus may treat dysmenorrhea with cold coagulation and blood stasis by acting on arachidonic acid metabolism pathway.

Modulation of the prostaglandin-endoperoxide synthase 2 gene expression by variant haplotypes: influence of the 3'-untranslated region

The inducible inflammatory enzyme cycloxigenase-2 is up-regulated in cancer, and favors tumor progression. Cycloxigenase-2 is encoded by the prostaglandin-endoperoxide synthase 2 (PTGS2) gene, which presents sequence variations in the promoter region (PR) and in the 3′-untranslated region (3′-UTR). Different PR (rs689465, rs689466, rs20417) and 3′-UTR (rs5275) variants were generated by site-directed mutagenesis, and combined in haplotypes to access expression levels using a reporter system (luciferase) in human cells (MCF-7 and HEK293FT). Luciferase activity did not differ significantly among PTGS2 PR constructs, except for pAAC (containing variant allele rs20417 C), with 40% less activity than pAAG (wild-type sequence) in MCF-7 cells (P<0.01). Despite the lack of individual significant differences, PTGS2 PR constructs enclosing rs689466 G (pAGG and pAGC) showed an approximate two-fold increase in luciferase activity when compared to those containing rs689466 A (pAAG, pGAC, pAAC and pGAG) in both cell lines (P<0.001 for MCF-7 and P=0.03 for HEK293FT). The effect of PTGS2 3′-UTR sequences varied between MCF-7 and HEK293FT: MCF-7 cells showed significant reduction (40-60%) in luciferase activity (at least P<0.01), whereas HEK293FT cells showed more diverse results, with an average 2-fold increase when combined constructs (PR and 3′-UTR) were compared to respective parental PR sequences. The contribution of 3′-UTR variant (rs5275) was not consistent in either cell line. Despite the modulation of the 3′-UTR, with variable effects of rs5275, the enhancing transcriptional effect of rs689466 G was still detectable (P<0.0001 in MCF-7 or P=0.03 in HEK293FT cells).

Transcriptomic analysis of differential gene expression reveals an increase in COX2 levels during in vitro canine herpesvirus infection / Análise transcriptômica da expressão gênica diferencial revela um aumento nos níveis de COX2 durante a infecção in vitro por herpesvirus canino

ABSTRACT: Canine herpesvirus (CaHV-1) affects canids worldwide, causing death in neonates and immunosuppressed hosts. Acute infection by CaHV-1 can cause reproductive, respiratory, and neurological problems in adult animals. Viral pathogenesis and host genes expressions during of CaHV-1infection are not clearly understood. In the present study, the transcriptome of canine kidney cell Mardin-Darby (MDCK) infected in vitro with canine herpesvirus was explored. For this, RNA sequencing (RNA-seq) of the samples in different moments during infection was carried out. Subsequently, the transcriptomic analysis genes related to cell activities and process involved to viral cycle infection were evaluated until 32h post-inoculation (pi). Among evaluated genes, was verified a significant and gradative increase of the prostaglandin-endoperoxide synthase 2 (PTGS2) or cyclooxygenase 2 (COX2) gene expression, throughout of infection, though differential gene expression analysis and validated by quantitative reverse transcription PCR (RT-qPCR). High COX2 expression is usually induced in response to inflammation, pathogens or activation of the immune system but can be a viral mechanism to favor viral replication. Thus, COX2 level increase can be a favorable factor for viral infection with Cahv-1 virus and the use of selective COX2 inhibitors may be beneficial for limiting the infection or clinical signs by causing interruption of the viral replication cycle during active infection. Additionally, the regulation genes expression differential verified in this study can contribute to determining important targets for inhibiting canine herpesvirus infection either by cellular or viral mechanisms.

RESUMO: O herpesvírus canino (CaHV-1) afeta os canídeos em todo o mundo, causando morte em neonatos e hospedeiros imunossuprimidos. A infecção aguda por CaHV-1 pode causar problemas reprodutivos, respiratórios e neurológicos em animais adultos. A patogênese viral e expressão de genes hospedeiros durante a infecção por CaHV-1 ainda não são bem compreendidos. No presente estudo, o transcriptoma de células de rim canino Madin-Darby (MDCK) infectadas in vitro com herpesvirus canino foi explorado. Para isso, foi realizado o sequenciamento de RNA (RNA-seq) de amostras coletadas em diferentes momentos durante a infecção. Subsequentemente, a análise transcriptômica dos genes relacionados à atividade celular e aos processos envolvidos no ciclo de infecção do vírus foram avaliadas até 32 horas após a inoculação (pi). Dentre os genes avaliados, constatamos uma elevação significativa e gradativa da expressão da Prostaglandina-endoperoxide sintase 2 (PTGS2) ou ciclooxigenase 2 (COX2), ao longo da infecção viral, foi verificada por análise de expressão gênica diferencial e validada por resultados de transcrição reversa por PCR quantitativo (RT-qPCR). O aumento da expressão de COX2 geralmente é induzida em resposta a inflamação, patógenos ou ativação do sistema imune, mas também pode ser um mecanismo para favorecer a replicação viral. Assim, o aumento do nível de COX2 pode ser um fator favorável à infecção viral pelo vírus CaHV-1 e o uso de inibidores seletivos da COX2 pode ser benéfico para limitar a infecção ou os sinais clínicos, causando a interrupção do ciclo de replicação viral durante a infecção ativa. Além disso, a regulação diferencial da expressão dos genes verificados neste estudo podem contribuir para determinar alvos importantes para inibir a infecção por herpesvírus canino, seja por mecanismos celulares ou virais.

Explore mechanism of Compound Uncaria Hypotensive Tablet for hypertension based on network pharmacology / 中国中药杂志

This paper aimed to predict the active ingredients and action targets of Compound Uncaria Hypotensive Tablet for hypertension based on network pharmacology, and discuss its possible "multi-components, multi-targets, and multi-pathways" mechanism for treatment of hypertension. The integrative pharmacological platform of traditional Chinese medicine (TCM-IP) was used to construct the component target-disease target network of Compound Uncaria Hypotensive Tablet, and the internet analysis method was used to screen the key nodes, on which the pathway enrichment analysis was carried out to explore its possible biological process in the treatment of hypertension. Target network analysis showed that, 35 predicted active ingredients of Compound Uncaria Hypotensive Tablet had a strong interaction with the prostaglandin endogenous peroxidase synthase (PTGS1, PTGS2), ATP synthetase (ATP1A1, ATP5A1, ATP5C1, ATP5B) and other 29 major proteins. Network enriched analysis showed that Compound Uncaria Hypotensive Tablet participated in the regulation of hypertension in different processes of pathology, through 15 pathways such as regulating blood pressure, G protein coupled receptor activation, adrenergic myocardial cell signal transduction and platelet activation. This study revealed the potential active compounds and possible mechanism of Compound Uncaria Hypotensive Tablet for treatment of hypertension, providing theoretical references for further systematic laboratory experiments on effective compounds and action mechanism of Compound Uncaria Hypotensive Tablet.

Polimorfismos no gene da cicloxigenase-2 e câncer de mama: impactos sobre a resposta terapêutica e a evolução clínica

O curso clínico do câncer de mama é altamente variável e, nesse contexto, a busca por novos biomarcadores que possam auxiliar na predição de resposta é essencial. O presente estudo teve como objetivo avaliar o impacto dos polimorfismos mais frequentes do gene PTGS2 (rs689465, rs689466, rs20417 e rs20417) e seus haplótipos sobre a resposta terapêutica e a sobrevida do câncer de mama. O estudo envolveu uma coorte de mulheres brasileiras com câncer de mama unilateral e não metastático (...), que foram submetidas à ressecção tumoral (...) ou à quimioterapia neoadjuvante (...) como primeira abordagem terapêutica. O DNA genômico foi extraído de amostras de sangue periférico, e utilizado para genotipagem dos polimorfismos pelas técnicas de PCR em tempo real e PCR-RFLP. A distribuição genotípica foi avaliada quanto à aderência ao princípio de Hardy-Weinberg, e os genótipos individuais foram usados para inferência dos haplótipos. Os desfechos primários avaliados foram: resposta patológica à quimioterapia neoadjuvante, sobrevida livre de doença (SLD) e sobrevida global (SG). A associação entre genótipos ou haplótipos e variáveis histopatológicas ou desfechos de reposta patológica foi avaliada pelas razões de chance (OR) e seus respectivos intervalos de confiança de 95 por cento (IC95 por cento), com ajuste para covariáveis em modelos de regressão logística. O impacto dos genótipos ou haplótipos nas curvas de SLD e SG foi avaliado pelo método de Kaplan-Meier, em modelos multivariados de regressão de riscos proporcionais de Cox com cálculo das razões de risco ajustadas (HR) e respectivos IC95 por cento. (...)

Breast cancer is a very heterogeneous disease, with great variability in its clinical evolution. Despite the advances in molecular classification of tumors at diagnosis and the availability of treatment options that helped reducing its global mortality, new biomarkers are still needed to improve response prediction, and individualize therapy. The present study aimed to evaluate the four most common PTGS2 polymorphisms (rs689465, rs689466, rs20417 and rs20417), as well as their haplotypes, on the therapeutic response and survival of breast cancer patients. The study population was a cohort of Brazilian women diagnosed with unilateral and non-metastatic breast cancer (...), submitted to tumor resection (...) or neoadjuvant chemotherapy (...) as their first therapeutic approach. Genomic DNA was extracted from peripheric blood, and used for genotyping. The genotypic distribution was evaluated for adherence to the Hardy-Weinberg principle, and genotypes were used for haplotype inference. The primary outcomes were: tumoral and pathological complete response to neoadjuvant chemotherapy (tCR and pCR, respectively), disease free-survival (DFS) and overall survival (OS). The association of genotypes or haplotypes with histopathological features at diagnosis and with tCR or pCR was assessed by odds ratios (OR) and respective 95 per cent confidence intervals (95 per cent CI). When other covariates were detected, the association was validated in logistic regression models, with calculation of adjusted OR (ORadjusted). The impact of genotypes or haplotypes DFS and OS was evaluated using Kaplan-Meier curves and multivariate Cox proportional hazards regression models for calculation of adjusted hazard ratios (HRadjusted) and respective 95 per cent CI. (...)

Caracterização de polimorfismos do gene PTGS2 na população brasileira e avaliação quanto ao risco de desenvolvimento de câncer de mama

A enzima inflamatória ciclooxigenase-2 (COX-2) encontra-se com alta expressão em diversos tipos de câncer e está associada à progressão, potencial de invasão e metástase. A COX-2 é codificada pelo gene PTGS2, que é polimórfico e apresenta variações na região promotora (RP) e na região 3’-não traduzida (3´-UTR). Tais variantes podem contribuir para aumentar a transcrição ou a estabilidade do RNAm, favorecendo um efeito pró-inflamatório e podendo aumentar o risco de desenvolvimento de câncer. A freqüência de polimorfismos em PTGS2 varia entre grupos étnicos e, até o momento, não há informações quanto à distribuição genotípica em brasileiros saudáveis ou com câncer. Este trabalho teve como objetivo geral a caracterização de variações no gene PTGS2 em brasileiros, a fim de avaliar se tais variações podem afetar o risco de desenvolvimento de câncer de mama. O protocolo utilizado foi aprovado pelo Comitê de Ética em Pesquisa do INCA e todos os participantes assinaram o Termo de Consentimento Livre e Esclarecido. A etapa de rastreamento de polimorfismos foi realizada em sete fragmentos da região promotora e em três da região 3’-UTR, com os métodos de dHPLC ou PCR-RFLP. Nós recrutamos 282 voluntários sadios com o objetivo de caracterizar as freqüências alélicas e as distribuições genotípicas e avaliar as possíveis diferenças entre sexo e cor de pele. Foram identificados nove polimorfismos já descritos em outras populações, com as seguintes freqüências alélicas: -1290AG (0,22), -1195AG (0,15), - 1131GA (0,007), -765GC (0,32), -604TC (0,015), -163CG (0,02) e -62CG(0,007), 8473TC (0,22) e 9850AG (0,01). Todas as distribuições genotípicas estavam em equilíbrio de Hardy-Weinberg (Eq-HW) e não houve diferenças em relação ao sexo. O polimorfismo 8473TC foi o único que apresentou diferença na distribuição genotípica em função da cor de pele (P = 0,034), sendo menos freqüente em brancos do que em pardos ou pretos. Os polimorfismos com freqüência maior que 10% foram selecionados para o estudo caso-controle em câncer de mama que envolveu 131 voluntárias sadias e 216 pacientes. As freqüências alélicas encontradas para as variações -1290AG, -1195AG, -765GC e 8473TC foram, respectivamente, de 0,16 (IC95% 0,11-0,21), 0,13 (IC95% 0,09- 0,18), 0,31 (IC95% 0,25-0,37), 0,24 (IC95% 0,18-0,30) em voluntárias e 0,17 (IC95% 0,13-0,20), 0,1 (IC95% 0,07-0,13), 0,31 (IC95% 0,26-0,35) e 0,31 (IC95% 0,26-0,35) em pacientes. Nós encontramos uma relação significativa ente o polimorfismo 8473TC e o risco para câncer de mama, com uma odds ratio (OR) de 1,73 (IC95% 1,08-2,77; P = 0,024) para a população total do estudo caso-controle e OR de 2,54 (IC95% 1,21-5,32; P = 0,017) apenas entre mulheres brancas. Além dos polimorfismos de PTGS2 outros fatores de risco para câncer de mama, como idade, tabagismo e histórico familiar de câncer foram avaliados...

The inflammatory enzyme ciclooxigenase-2 (COX-2) is over expressed in several types of cancer and its presence is associated with progression, invasive potential and metastasis. The gene encoding human COX-2 (PTGS2) is polymorphic and many variants have been described in the promoter region and in the 3’-untranslated (3’-UTR) region. These polymorphisms may affect gene transcription or RNAm stability, and thereby favor the synthesis of COX-2 and a pro-inflammatory effect, which might increase the risk of cancer. The frequency of PTGS2 polymorphisms varies between ethnic groups and, until now, there have been no reports about its distribution in Brazilians. The objective of our study was to characterize PTGS2 polymorphisms occurring in the Brazilian population in order to evaluate the role of selected variants as risk factors for breast cancer. The protocol was approved by the local ethics committee and all participants signed a consent form. The screening of PTGS2 polymorphisms was performed in seven fragments of the promoter region and in three fragments in the 3'–UTR region by dHPLC or PCR-RFLP. We recruited 282 healthy volunteers in order to characterize allelic frequencies and genotypic distributions and to evaluate possible differences according to gender or skin color. We identified nine polymorphisms with the following allelic frequencies: -1290AG (0.22), - 1195AG (0.15), -1131GA (0.007), -765GC (0.32), -604TC (0.015), -163CG (0.02), -62CG (0.007), 8473TC (0.22) and 9850AG (0.01). All genotypic distributions followed Hardy-Weinberg equilibrium and no differences were observed in relation to gender. The 8473TC polymorphism was the only one which showed different genotypic distributions in Brazilian color groups (P = 0.034), being less frequent in whites that in intermediate or black volunteers. The polymorphisms with minor allele frequencies higher than 0.10 were selected for the case-control study in breast cancer, which involved 131 healthy women and 216 patients. The frequencies of the variant alleles -1290G, -1195G, -765C and 8473C were respectively: 0.16 (CI95% 0.11-0.21), 0.13 (CI95% 0.09-0.18), 0.31 (CI95% 0.25-0.37) and 0.24 (CI95% 0.18-0.30) among healthy controls and 0.17 (CI95% 0.13-0.20), 0.10 (CI95% 0.07-0.13), 0.31 (CI95% 0.26-0.35) and 0.31 (CI95% 0.26-0.35) among patients. We found a significant association between 8473TC polymorphism and breast cancer risk, with an odds ratio (OR) of 1.73 (CI95% 1.08 - 2.77; P = 0.024) in the general study population and an OR of 2.54 (CI95% 1.21- 5.32; P = 0.017) among white women. Besides the PTGS2 polymophisms, we evaluated the contribution of other possible risk factors for breast cancer, namely: age, smoking and family history of cancer. We found significant association only for age (OR = 1,65; CI95% 1,07-2,54). The results suggest that the polymorphism 8473TC located in the 3‘-UTR region of PTGS2 gene may be a risk factor for development of breast cancer in Brazilian women.

Association between Prostaglandin-endoperoxide Synthase 2 (PTGS2) Polymorphisms and Blood Pressure in Korean Population

Blood pressure refers to the force exerted by circulating blood on the walls of blood vessels, and chronical elevation of blood pressure is known as hypertension. Although hypertension is affected by genetic and environmental factors, the genetic background of hypertension is not fully understood. One of the candidate genetic factors, Prostaglandin-endoperoxide synthase 2 (PTGS2), is a membrane-bound enzyme, catalyzing the conversion of arachidonic acid to prostaglandin, and recently SNPs of PTGS2 gene was associated with hypertension in Japanese population. Therefore the association of PTGS2 polymorphisms was investigated with blood pressure in healthy Korean subjects, 470 unrelated individuals randomly selected from Ansung and Ansan cohorts. The 25 SNPs of PTGS2 gene were identified by the sequencing analysis of 24 Korean samples. Among identified polymorphisms, three SNPs (rs689466, -1329A>G; rs5275, +6365T>C; rs4648308, +8806G> A) were selected for further association analysis, and rs689466 located in promoter region was associated with blood pressure as well as triglyceride level in the blood. By in silico analysis, rs689466 locates in v-Myb transcription factor binding site, and the v-Myb site disappears when the SNP is changed from A to G nucleotide. Individuals with A/G and G/G genotype in rs689466 have higher blood pressure than those with A/A genotype, and the regression p-value is 0.008 for systolic and 0.004 for diastolic blood pressure. In summary, the PTGS2 polymorphism (rs689466) is associated with blood pressure in Asian populations based on this and Japanese studies, shedding light on it as a genetic risk marker of hypertension.

A practical approach to the understanding and teaching of RNA silencing in plants

Gene silencing, also called RNA interference (RNAi) is a specific mechanism of RNA degradation involved in gene regulation, development and defense in eukaryotic organisms. It became an important subject in the teaching programs of molecular biology, genetics and biotechnology courses in the last years. The aim of this work is to provide simple and inexpensive assays to understand and teach gene silencing using plants as model systems. The use of transient and permanent transgenic plants for expressing reporter genes, like those derived from jellyfish green fluorescent protein (gfp) encoding gene, provides a nice, colorful and conclusive image of gene silencing. Three experimental approaches to evidence RNA silencing are depicted. In the first approach gene silencing is demonstrated after transient expression of reporter genes in non-transgenic plants. In the second, silencing is triggered against a reporter gene stably integrated into a transgenic plant. The third approach involves the triggering of RNA silencing against endogenous genes using viral vectors. In addition we illustrate systemic gene silencing showing how the silencing signal is spread over a plant and finally it is also demonstrated the suppression of gene silencing. The first group of experiments is recommended to be tough on undergraduate courses, the following two sections are recommended for graduate courses. Hopefully, it will help students to understand this important phenomenon and to unravel the importance of gene silencing as a key gene regulation mechanism and as a molecular and biotechnological tool.

Genetic machinery for RNA silencing and defense against viruses in Citrus

RNA silencing mechanisms are conserved throughout eukaryotic evolution, possibly due to their importance in viral resistance and other aspects of cell biology. Here, we explored the Citrus EST (CitEST) database in search of sequences related to the most important known genes involved in RNA silencing. Transcripts strongly matching Argonaute (AGO), Dicer-like (DCL), Hua enhancer (HEN), and RNA-dependent RNA Polymerase (RdRP) were found in many of the citrus libraries. The reads were clustered and quantified. This shows that post-transcriptional gene silencing apparatus is active in citrus. It seems plausible that a better understanding of the players of RNA silencing in Citrus spp. and related genera may help create new tools to defeat the viral diseases that affect the citrus industry. Functional analyses of these citrus genes would enable the pursuit of this hypothesis.

RNA interference c-erbB-2 and c-raf-1 genes expression combined transfection in the Tca8113 cell lines / 重庆医科大学学报

Objective:To investigate the proliferating inhibition and the mechanisms of dsRNA interference c-erbB-2 and c-raf-1 genes expression combined transfection in the human tongue carcinoma Tca8113 cell lines.Methods:There were 3 groups in our study,control group,normal control group and RNAi experimental group.At different time after liposome-mediated transfection,the cell proliferation,apoptosis,mRNA level,protein expressing level and cell cycle were observed by MTT,RT-PCR and electronic microscope.Results:According to the results of RNAi experimental group,the OD-value were 0.073( P