• Entomological Research
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• v.48 no.5
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• pp.384-389
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• 2018

Aphis gossypii Glover is an important insect pest that functions as a viral vector and mediates approximately 45 different viral diseases. As part of a strategy for control of A. gossypii, we investigated the functions of genes using RNAi. To this end, a cDNA library was constructed for various genes and for selecting appropriate targets for RNAi mediated silencing. The cDNA library was constructed using the Gateway cloning system with site-specific recombination of bacteriophage ${\lambda}$. It was used to carry out single step cloning of A. gossypii cDNAs. As a result, a cDNA library with a titer of $8.4{\times}10^6$ was constructed. Since the sequences in this library carry att sites, they can be cloned into various binary vectors. This library will be of value for various studies. For later screening of selected genes, it is planned to clone the library into virus-induced gene silencing (VIGS) vectors, which makes it possible to analyze gene function and allow subsequent transfection of plants. Such transfection experiments will allow testing of RNAi-induced insecticidal activity or repellent activity to A. gossypii, and result in the identification of target genes. It is also expected that the constructed cDNA library will be useful for analysis of gene functions in A. gossypii.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.1
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• pp.371-376
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• 2012

Background: Caveolin-1 (CAV-1), encoding the structural component of cellular caveolae, is a suggested tumor suppressor gene involved in cell signalling. Aberrant promoter methylation of CAV-1 is associated with inactivation of expression. We previously observed CAV-1 mutations in breast cancers and therefore devised this study to examine the hypermethylation status of the promoter region of CAV-1 with reference to breast cancer progression and development. Methods: Hypermethylation status of CAV-1 was analyzed by methylation specific PCR. Loss of expression of the CAV-1 gene was further evaluated by semi-quantitative rt-PCR. Results: 28/130 (21.5%) breast cancer cases showed promoter hypermethylation with reduced CAV-1 expression levels when compared with adjacent normal breast tissue. CAV-1 gene hypermethylation was significantly related to menopausal status, histopathological grade and age. Conclusion: The rationale of our study is that CAV-1 gene is transcriptionally repressed in breast cancer cells due to hypermethylation. Our results reveal that promoter hypermethylation and loss of expression of the CAV-1 gene is an important alternative mechanism for inactivation of CAV-1 leading to complete gene silencing.

• Reproductive and Developmental Biology
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• v.32 no.4
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• pp.229-235
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• 2008

Silencing of Dicer1 by siRNA did not inhibit development up to the blastocyst stage, but decreased expression of selected transcription factors, including Oct-4, Sox2 and Nanog, suggesting that Dicer1 gene expression is associated with differentiation processes at the blastocyst stage (Cui et al., 2007). In order to get insights into genes which may be linked with microRNA system, we compared gene expression profiles in Gapdh and Dicer1 siRNA-microinjected blastocysts using the Applied Biosystem microarray technology. Our data showed that 397 and 737 out of 16354 genes were up- and down-regulated, respectively, following siRNA microinjection (p<0.05), including 24 up- and 28 down-regulated transcription factors. Identification of genes that are preferentially expressed at particular Dicer1 knock down embryos provides insights into the complex gene regulatory networks that drive differentiation processes in embryos at blastocyst stage.

• BMB Reports
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• v.41 no.10
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• pp.739-744
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• 2008

Turnip yellow mosaic virus (TYMV) is a positive strand RNA virus that infects mainly Cruciferae plants. In this study, the TYMV genome was modified by inserting an extra subgenomic RNA promoter and a multiple cloning site. This modified TYMV was introduced into Nicotiana benthamiana using a Agrobacterium-mediated T-DNA transfer system (agroinfiltration). When a gene encoding $\beta$-glucuronidase or green fluorescent protein was expressed using this modified TYMV as a vector, replication of the recombinant viruses, especially the virus containing $\beta$-glucuronidase gene, was severely inhibited. The suppression of replication was reduced by co-expression of viral silencing suppressor genes, such as tombusviral p19, closteroviral p21 or potyviral HC-Pro. As expected, two subgenomic RNAs were produced from the recombinant TYMV, where the larger one contained the foreign gene. An RNase protection assay revealed that the recombinant subgenomic RNA was encapsidated as efficiently as the genuine subgenomic RNA.

• The Plant Pathology Journal
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• v.26 no.4
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• pp.402-408
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• 2010

Plant matrix metalloproteases (MMPs) are a family of apoplastic metalloproteases closely related to human matrilysins. Up-regulation of Nicotiana benthamiana matrix metalloprotease 1 (NMMP1) expression by treatment with pathogens, ethephon and aging indicates that the gene is related to plant defense and the aging process through ethylene signaling. NMMP1 expression was higher than in normal growth leaves following infection with an incompatible pathogen Pseudomonas syringae pv. tomato T1 or a compatible pathogen P. syringae pv. tabaci and in aged leaves. Transient overexpression of NMMP1 in N. benthamiana leaves lowered the growth of P. syringae pv. tabaci. However, NMMP1-silenced leaves showed increased growth of P. syringae pv. tabaci. These data strongly suggest that NMMP1 in N. benthamiana is a defense related gene, which is positively regulated by ethylene.

• Tuberculosis and Respiratory Diseases
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• v.80 no.3
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• pp.247-254
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• 2017

Background: Airway epithelial cells are the first line of defense, against pathogens and environmental pollutants, in the lungs. Cellular stress by cadmium (Cd), resulting in airway inflammation, is assumed to be directly involved in tissue injury, linked to the development of lung cancer, and chronic obstructive pulmonary disease (COPD). We had earlier shown that ACN9 (chromosome 7q21), is a potential candidate gene for COPD, and identified significant interaction with smoking, based on genetic studies. However, the role of ACN9 in the inflammatory response, in the airway cells, has not yet been reported. Methods: We first checked the anatomical distribution of ACN9 in lung tissues, using mRNA in situ hybridization, and immunohistochemistry. Gene expression profiling in bronchial epithelial cells (BEAS-2B), was performed, after silencing ACN9. We further tested the roles of ACN9, in the intracellular mechanism, leading to Cd-induced production, of proinflammatory cytokines in BEAS-2B. Results: ACN9 was localized in lymphoid, and epithelial cells, of human lung tissues. ACN9 silencing, led to differential expression of 216 genes. Pathways of sensory perception to chemical stimuli, and cell surface receptor-linked signal transduction, were significantly enriched. ACN9 silencing, further increased the expression of proinflammatory cytokines, in BEAS-2B after Cd exposure. Conclusion: Our findings suggest, that ACN9 may have a role, in the inflammatory response in the airway.

• BMB Reports
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• v.40 no.4
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• pp.467-474
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• 2007

Autosomal recessive polycystic kidney disease (ARPKD) is one of the important genetic disorders in pediatric practice. Mutation of the polycystic kidney and hepatic disease gene 1 (PKHD1) was identified as the cause of ARPKD. The gene encodes a 67-exon transcript for a large protein of 4074 amino acids termed fibrocystin, but its function remains unknown. The neoplastic-like in cystic epithelial proliferation and the epidermal growth factor/epidermal growth factor receptor (EGF/EGFR) axis overactivity are known as the most important characteristics of ARPKD. Since the misregulation of $Ca^{2+}$ signaling may lead to aberrant structure and function of the collecting ducts in kidney of rat with ARPKD, present study aimed to investigate the further mechanisms of abnormal proliferation of cystic cells by inhibition of PKHD1 expression. For this, a stable PKHD1-silenced HEK-293T cell line was established. Then cell proliferation rates, intracellular $Ca^{2+}$ concentration and extracellular signal-regulated kinase 1/2 (ERK1/2) activity were assessed after treatment with EGF, a calcium channel blocker and agonist, verapamil and Bay K8644. It was found that PKHD1-silenced HEK-293T cell lines were hyperproliferative to EGF stimulation. Also PKHD1-silencing lowered the intracellular $Ca^{2+}$ and caused EGF-induced ERK1/2 overactivation in the cells. An increase of intracellular $Ca^{2+}$ in PKHD1-silenced cells repressed the EGF-dependent ERK1/2 activation and the hyperproliferative response to EGF stimulation. Thus, inhibition of PKHD1 can cause EGF-induced excessive proliferation through decreasing intracellular $Ca^{2+}$ resulting in EGF-induced ERK1/2 activation. Our results suggest that the loss of fibrocystin may lead to abnormal proliferation in kidney epithelial cells and cyst formation in ARPKD by modulation of intracellular $Ca^{2+}$.

• Research in Plant Disease
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• v.21 no.3
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• pp.186-192
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• 2015

Cucumber mosaic virus possesses 2b gene known as a suppressor of post-transcriptional gene silencing (PTGS). To investigate its function and effect in plant, transgenic Nicotiana benethamiana expressing 2b gene was developed and analyzed in phenotypic characteristics and differential gene expression (DEG) comparing with wild-type. Eight lines of transgenic plants ($T_0$) were obtained with difficulty and showed severe deformed phenotypes in leaves, flowers, petioles and etc. Moreover, transgenic plants were hardly able to set seeds, but small amounts of seeds were barely produced in some of transgene-hemizygous plants. DEG analysis showed that transgenic plant ectopically accumulated diverse RNA transcripts at higher levels than wild-type probably due to the disturbance in RNA metabolism, especially of RNA decay, caused by 2b-mediated inhibition of PTGS. These ectopic accumulations of RNAs disrupt protein and RNA homeostasis and then subsequently lead to abnormal phenotypes of transgenic plants.

• Journal of Veterinary Science
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• v.23 no.2
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• pp.24.1-24.10
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• 2022

Background: Small interfering RNA technology has been considered a prospective alternative antiviral treatment using gene silencing against influenza viruses with high mutations rates. On the other hand, there are no reports on its effectiveness against the highly pathogenic avian influenza H5N1 virus isolated from Indonesia. Objectives: The main objective of this study was to improve the siRNA design based on the nucleoprotein gene (siRNA-NP) for the Indonesian H5N1 virus. Methods: The effectiveness of these siRNA-NPs (NP672, NP1433, and NP1469) was analyzed in vitro in Marbin-Darby canine kidney cells. Results: The siRNA-NP672 caused the largest decrease in viral production and gene expression at 24, 48, and 72 h post-infection compared to the other siRNA-NPs. Moreover, three serial passages of the H5N1 virus in the presence of siRNA-NP672 did not induce any mutations within the nucleoprotein gene. Conclusions: These findings suggest that siRNA-NP672 can provide better protection against the Indonesian strain of the H5N1 virus.

• Asian Pacific Journal of Cancer Prevention
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• v.15 no.11
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• pp.4555-4561
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• 2014

Background: Silencing due to methylation of suppressor of cytokine signaling-3 (SOCS-3), a negative regulator gene for the JAK/STAT signaling pathway has been reported to play important roles in leukemogenesis. Imatinib mesylate is a tyrosine kinase inhibitor that specifically targets the BCR-ABL protein and induces hematological remission in patients with chronic myeloid leukemia (CML). Unfortunately, the majority of CML patients treated with imatinib develop resistance under prolonged therapy. We here investigated the methylation profile of SOCS-3 gene and its downstream effects in a BCR-ABL positive CML cells resistant to imatinib. Materials and Methods: BCR-ABL positive CML cells resistant to imatinib (K562-R) were developed by overexposure of K562 cell lines to the drug. Cytotoxicity was determined by MTS assays and $IC_{50}$ values calculated. Apoptosis assays were performed using annexin V-FITC binding assays and analyzed by flow cytometry. Methylation profiles were investigated using methylation specific PCR and sequencing analysis of SOCS-1 and SOCS-3 genes. Gene expression was assessed by quantitative real-time PCR, and protein expression and phosphorylation of STAT1, 2 and 3 were examined by Western blotting. Results: The $IC_{50}$ for imatinib on K562 was 362nM compared to 3,952nM for K562-R (p=0.001). Percentage of apoptotic cells in K562 increased upto 50% by increasing the concentration of imatinib, in contrast to only 20% in K562-R (p<0.001). A change from non-methylation of the SOCS-3 gene in K562 to complete methylation in K562-R was observed. Gene expression revealed down-regulation of both SOCS-1 and SOCS-3 genes in resistant cells. STAT3 was phosphorylated in K562-R but not K562. Conclusions: Development of cells resistant to imatinib is feasible by overexposure of the drug to the cells. Activation of STAT3 protein leads to uncontrolled cell proliferation in imatinib resistant BCR-ABL due to DNA methylation of the SOCS-3 gene. Thus SOCS-3 provides a suitable candidate for mechanisms underlying the development of imatinib resistant in CML patients.