• Molecules and Cells
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• v.26 no.6
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• pp.611-615
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• 2008

DNA methylation is an epigenetic mechanism for gene silencing. In Arabidopsis, MET1 is the primary DNA methyltransferase that maintains CG DNA methylation. Plants having an overall reduction of MET1 activity, caused by a met1 mutation or a constitutively expressed MET1 antisense gene, display genome hypomethylation, inappropriate gene and transposon transcription, and developmental abnormalities. However, the effect of a transient reduction in MET1 activity caused by inhibiting MET1 expression in a restricted set of cells is not known. For this reason, we generated transgenic plants with a MET1 antisense gene fused to the DEMETER (DME) promoter (DME:MET1 a/s). Here we show that DME is expressed in leaf primordia, lateral root primoridia, in the region distal to the primary root apical meristem, which are regions that include proliferating cells. Endogenous MET1 expression was normal in organs where the DME:MET1 a/s was not expressed. Although DME promoter is active only in a small set of cells, these plants displayed global developmental abnormalities. Moreover, centromeric repeats were hypomethylated. The developmental defects were accumulated by the generations. Thus, not maintaining CG methylation in a small population of proliferating cells flanking the meristems causes global developmental and epigenetic abnormalities that cannot be rescued by restoring MET1 activity. These results suggest that during plant development there is little or no short-term molecular memory for reestablishing certain patterns of CG methylation that are maintained by MET1. Thus, continuous MET1 activity in dividing cells is essential for proper patterns of CG DNA methylation and development.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.4
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• pp.1599-1603
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• 2015

Background: Epigenetic silencing of tumor suppressor genes due to promoter hypermethylation is one of the frequent mechanisms observed in cancers. Hypermethylation of several tumor suppressor genes involved in cell cycle regulation has been reported in many types of tumors including oral squamous cell carcinomas. LATS1 (Large Tumor Suppressor, isoform 1) is a novel tumor suppressor gene that regulates cell cycle progression by forming complexes with the cyclin dependent kinase, CDK1. Promoter hypermethylation of the LATS1 gene has been observed in several carcinomas and also has been linked with prognosis. However, the methylation status of LATS1 in oral squamous cell carcinomas is not known. As oral cancer is one of the most prevalent forms of cancer in India, the present study was designed to investigate the methylation status of LATS1 promoter and associate it with histopathological findings in order to determine any associations of the genetic status with stage of differentiation. Materials and Methods: Tumor chromosomal DNA isolated from biopsy tissues of thirteen oral squamous cell carcinoma biopsy tissues were subjected to digestion with methylation sensitive HpaII enzyme followed by amplification with primers flanking CCGG motifs in promoter region of LATS1 gene. The PCR amplicons were subsequently subjected to agarose gel electrophoresis along with undigested amplification control. Results: HpaII enzyme based methylation sensitive PCR identified LATS1 promoter hypermethylation in seven out of thirteen oral squamous cell carcinoma samples. Conclusions: The identification of LATS1 promoter hypermethylation in seven oral squamous cell carcinoma samples (54%), which included one sample with epithelial dysplasia, two early invasive and one moderately differentiated lesions indicates that the hypermethylation of this gene may be one of the early event during carcinogenesis. To the best of our knowledge, this is the first study to have explored and identified positive association between LATS1 promoter hypermethylation with histopathological features in oral squamous cell carcinomas.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.2
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• pp.1067-1072
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• 2013

Hiwi, a human homologue of the Piwi family, plays an important role in stem cell self-renewal and is overexpressed in various human tumors. This study aimed to determine whether an RNA interference-based strategy to suppress Hiwi expression could inhibit tumor growth in a xenograft mouse model. A rare population of $SSC^{lo}\;Alde^{br}$ cells was isolated and identified as lung cancer stem cells in our previous study. Plasmids containing U6 promoter-driven shRNAs against Hiwi or control plasmids were successfully established. The xenograft tumor model was generated by subcutaneously inoculating with lung cancer stem cell $SSC^{lo}\;Alde^{br}$ cells. After the tumor size reached about 8 mm in diameter, shRNA plasmids were injected into the mice via the tail vein three times a week for two weeks, then xenograft tumor growth was assessed. In nude mice, intravenously delivery of Hiwi shRNA plasmids significantly inhibited tumor growth compared to treatment with control scrambled shRNA plasmids or the vehicle PBS. No mice died during the experiment and no adverse events were observed in mice administered the plasmids. Moreover, delivery of Hiwi shRNA plasmids resulted in a significant suppressed expression of Hiwi and ALDH-1 in xenograft tumor samples, based on immunohistochemical analysis. Thus, shRNA-mediated Hiwi gene silencing in lung cancer stem cells by an effective in vivo gene delivery strategy appeared to be an effective therapeutic approach for lung cancer, and may provide some useful clues for RNAi gene therapy in solid cancers.

• 한국균학회소식:학술대회논문집
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• 2015.11a
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• pp.26-26
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• 2015

Phytophthora infestans is the causal agent of potato and tomato late blight, one of the most devastating plant diseases. P. infestans secretes effector proteins that are both modulators and targets of host plant immunity. Among these are the so-called RXLR effectors that function inside plant cells and are characterized by a conserved motif following the N-terminal signal peptide. In contrast, the effector activity is encoded by the C terminal region that follows the RXLR domain. Recently, I performed in planta functional profiling of different RXLR effector alleles. These genes were amplified from a variety of P. infestans isolates and cloned into a Potato virus X (PVX) vector for transient in planta expression. I assayed for R-gene specific induction of hypersensitive cell death. The findings included the discovery of new effector with avirulence activity towards the Solanum bulbocastanum Rpi-blb2 resistance gene. The Rpi-blb2 encodes a protein with a putative CC-NBS-LRR (a coiled-coil-nucleotide binding site and leucine-rich repeat) motif that confers Phytophthora late blight disease resistance. We examined the components required for Rpi-blb2-mediated resistance to P. infestans in Nicotiana benthamiana. Virus-induced gene silencing was used to repress candidate genes in N. benthamiana and to assay against P. infestans infections. NbSGT1 was required for disease resistance to P. infestans and hypersensitive responses (HRs) triggered by co-expression of AVRblb2 and Rpi-blb2 in N. benthamiana. RAR1 and HSP90 did not affect disease resistance or HRs in Rpi-blb2-transgenic plants. To elucidate the role of salicylic acid (SA) in Rpi-blb2-mediated resistance, we analyzed the response of NahG-transgenic plants following P. infestans infection. The increased susceptibility of Rpi-blb2-transgenic plants in the NahG background correlated with reduced SA and SA glucoside levels. Furthermore, Rpi-blb2-mediated HR cell death was associated with $H_2O_2$, but not SA, accumulation. SA affects basal defense and Rpi-blb2-mediated resistance against P. infestans. These findings provide evidence about the roles of SGT1 and SA signaling in Rpi-blb2-mediated resistance against P. infestans.

• The Plant Pathology Journal
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• v.40 no.1
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• pp.59-72
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• 2024

A comprehensive survey of mungbean-growing areas was conducted to observe leaf spot disease caused by Alternaria alternata. Alternaria leaf spot symptoms were observed on the leaves. Diversity of 50 genotypes of mungbean was assessed against A. alternata and data on pathological traits was subjected to cluster analysis. The results showed that genotypes of mungbean were grouped into four clusters based on resistance parameters under the influence of disease. The principal component biplot demonstrated that all the disease-related parameters (% disease incidence, % disease intensity, lesion area, and % of infection) were strongly correlated with each other. Alt a 1 gene that is precisely found in Alternaria species and is responsible for virulence and pathogenicity. Alt a 1 gene was amplified using gene specific primers. The isolated pathogen produced similar symptoms when inoculated on mungbean and tobacco. The sequence analysis of the internal transcribed spacer (ITS) region, a 600 bp fragment amplified using specific primers, ITS1 and ITS2 showed 100% identity with A. alternata. Potato virus X (PVX) -based silencing vector expressing Alt a 1 gene was constructed to control this pathogen through RNA interference in tobacco. Out of 50 inoculated plants, 9 showed delayed onset of disease. Furthermore, to confirm our findings at molecular level semi-quantitative reverse transcriptase polymerase chain reaction was used. Both phenotypic and molecular investigation indicated that RNAi induced through the VIGS vector was efficacious in resisting the pathogen in the model host, Tobacco (Nicotiana tabacum). To the best of our knowledge, this study has been reported for the first time.

• Journal of Animal Science and Technology
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• v.53 no.3
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• pp.269-274
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• 2011

DNA methyltransferases (DNMTs) are closely associated with the epigenetic change and the gene silencing through the regulation of methylation status in animal genome. But, the role of DNMTs in transgene silencing has remained unclear. So, we examined whether the knockdown of DNMT influences the reactivation of transgene expression in the transgenic quails. In this study, we investigated the expression of DNMT3a, and DNMT3b in blastoderm, quail embryonic fibroblasts (QEFs) and limited embryonic tissues such as gonad, kidney, heart and liver of E6 transgenic quails (TQ2) by RT-PCR. We further analyzed the expression of DNMT3a at different stages of whole embryos during early embryonic development by qRT-PCR. DNMT3a expression was detected in all test samples; however, it showed the highest expression in E6 whole embryo. Embryonic fibroblasts collected from TQ2 quails were treated with two DNMT3a-targeted siRNAs (siDNMT3a-51 and siDNMT3a-88) for RNA interference assay, and changes in expression were then analyzed by qRT-PCR. The siDNMT3a-51 and siDNMT3a-88 reduced 53.34% and 64.64% of DNMT3a expression in TQ2 QEFs, respectively. Subsequently the treatment of each siRNA reactivated enhanced green fluorescent protein (EGFP) expression in TQ2 (224% and 114%). Our results might provide a clue for understanding the DNA methylation mechanism responsible for transgenic animal production and stable transgene expression.

• Asian Pacific Journal of Cancer Prevention
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• v.16 no.8
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• pp.3293-3299
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• 2015

Background: Arm protein lost in epithelial cancers, on chromosome X (ALEX) is a novel subgroup within the armadillo (ARM) family, which has one or two ARM repeat domains as opposed to more than six-thirteen repeats in the classical Armadillo family members. Materials and Methods: In the study, we explore the biological functions of ALEX1 in breast cancer cells. Overexpression of ALEX1 and silencing of ALEX1 were performed with SK-BR3 and MCF-7 cell lines. Cell proliferation and colony formation assays, along with flow cytometry, were carried out to evaluate the roles of ALEX1. Results: ALEX1 overexpression in SK-BR3 breast cancer cells inhibited proliferation and induced apoptosis. Furthermore, depletion of ALEX1 in MCF-7 breast cancer cells increased proliferation and inhibited apoptosis. Additional analyses demonstrated that the overexpression of ALEX1 activated the intrinsic apoptosis cascades through up-regulating the expression of Bax, cytosol cytochrome c, active caspase-9 and active caspase-3 and down-regulating the levels of Bcl-2 and mitochondria cytochrome c. Simultaneouly, silencing of ALEX1 inhibited intrinsic apoptosis cascades through down-regulating the expression of Bax, cytosol cytochrome c, active caspase-9, and active caspase-3 and up-regulating the level of Bcl-2 and mitochondria cytochrome c. Conclusions: Our data suggest that ALEX1 as a crucial tumor suppressor gene has been involved in cell proliferation and apoptosis in breast cancer, which may serve as a novel candidate therapeutic target.

• Journal of Korean Neurosurgical Society
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• v.63 no.6
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• pp.707-716
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• 2020

Objective : The function of B7H3, a member of the B7 family of proteins, in neuroblastoma (NB) remains poorly characterized. Here we examine the expression pattern of B7H3 in clinical NB specimens and characterize the phenotype of B7H3 knock-down in NB cell line. Methods : Immunohistochemical (IHC) staining was carried out to assess the expression of B7H3 in clinical NB specimens. Survival association was analyzed using five Gene Expression Omnibus (GEO) datasets (GSE85047, GSE45480, GSE62564, GSE16476, GSE49710). Clonogenic survival and flow cytometry were performed after B7H3 knockdown to assess the cellular proliferation and cell survival in vitro. Impact of B7H3 silencing on NB growth was examined in vivo using the SH-SY5Y xenograft model. Results : On IHC staining, B7H3 was widely expressed in clinical NB specimens. Analysis of the transcriptional profiles of five GEO datasets clinically annotated NB specimens revealed that decreased B7H3 expression was associated with improved overall survival. B7H3 knockdown suppressed the proliferation of the SH-SY5Y NB model in vitro and in vivo. Cell cycle analysis revealed that B7H3 silencing induced G1/S arrest. This arrest was associated with the suppression of E2F1 expression and induction of Rb expression. Conclusion : Our results demonstrate that B7H3 expression correlate with clinical survival in NB patients. Preliminary studies suggest that B7H3 may mediate the G1/S transition.

• The Plant Pathology Journal
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• v.36 no.3
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• pp.280-288
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• 2020

RNA interference (RNAi) has attracted attention as a promising approach to control plant viruses in their insect vectors. In the present study, to suppress replication of the rice stripe virus (RSV) in its vector, Laodelphax striatellus, using RNAi, dsRNAs against L. striatellus genes that are strongly upregulated upon RSV infection were delivered through a rice leaf-mediated method. RNAi-based silencing of peroxiredoxin, cathepsin B, and cytochrome P450 resulted in significant down regulation of the NS3 gene of RSV, achieving a transcriptional reduction greater than 73.6% at a concentration of 100 ng/μl and, possibly compromising viral replication. L. striatellus genes might play crucial roles in the transmission of RSV; transcriptional silencing of these genes could suppress viral replication in L. striatellus. These results suggest effective RNAi-based approaches for controlling RSV and provide insight into RSV-L. striatellus interactions.

• Parasites, Hosts and Diseases
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• v.62 no.2
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• pp.226-237
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• 2024

Ticks, blood-sucking ectoparasites, spread diseases to humans and animals. Haemaphysalis longicornis is a significant vector for tick-borne diseases in medical and veterinary contexts. Identifying protective antigens in H. longicornis for an anti-tick vaccine is a key tick control strategy. Enolase, a multifunctional protein, significantly converts D-2-phosphoglycerate and phosphoenolpyruvate in glycolysis and gluconeogenesis in cell cytoplasm. This study cloned a complete open reading frame (ORF) of enolase from the H. longicornis tick and characterized its transcriptional and silencing effect. We amplified the full-length cDNA of the enolase gene using rapid amplification of cDNA ends. The complete cDNA, with an ORF of 1,297 nucleotides, encoded a 432-amino acid polypeptide. Enolase of the Jeju strain H. longicornis exhibited the highest sequence similarity with H. flava (98%), followed by Dermacentor silvarum (82%). The enolase motifs identified included N-terminal and C-terminal regions, magnesium binding sites, and several phosphorylation sites. Reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated that enolase mRNA transcripts were expressed across all developmental stages of ticks and organs such as salivary gland and midgut. RT-PCR showed higher transcript levels in syn-ganglia, suggesting that synganglion nerves influence enolase's role in tick salivary glands. We injected enolase double-stranded RNA into adult unfed female ticks, after which they were subsequently fed with normal unfed males until they spontaneously dropped off. RNA interference significantly (P<0.05) reduced feeding and reproduction, along with abnormalities in eggs (no embryos) and hatching. These findings suggest enolase is a promising target for future tick control strategies.