• BMB Reports
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• v.41 no.5
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• pp.376-381
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• 2008

The discovery of RNA silencing inhibition by virus encoded suppressors or low temperature leads to concerns about the stability of transgenic resistance. RNA-dependent RNA polymerase (RdRp) has been previously characterized to be essential for transgene-mediated RNA silencing. Here we showed that low temperature led to the inhibition of RNA silencing, the loss of viral resistance and the reduced expression of host RdRp homolog (NtRdRP1) in transgenic T4 progeny with untranslatable potato virus Y coat protein (PVY-CP) gene. Moreover, RNA silencing and the associated resistance were differently inhibited by potato virus X (PVX) and tobacco mosaic virus (TMV) infections. The increased expression of NtRdRP1 in both PVX and TMV infected plants indicated its general role in response to viral pathogens. Collectively, we propose that biotic and abiotic stress factors affect RNA silencing-mediated resistance in transgenic tobacco plants and that their effects target different steps of RNA silencing.

• Horticultural Science & Technology
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• v.34 no.2
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• pp.314-323
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• 2016

A transient ihpRNA-induced gene silencing system based on Agrobacterium-mediated injection infiltration has been established to evaluate candidate genes involved in proanthocyanidin (PAs) biosynthesis in persimmon (Diospyros kaki Thunb.). We chose DkPDS (phytoene desaturase) as a gene-silencing target to evaluate the newly developed transient gene silencing system. Our qRT-PCR analysis indicated that two ihpRNA constructs (pHG-PDS5' and pHG-PDS3') targeted DkPDS, which also led to significantly reduce expression of DkPDS in 'Mopanshi' persimmon leaves. To further confirm the reliability of the system, we successfully utilized it for DkLAR (leucoanthocyanidin reductase) gene silencing. The expression levels of DkLAR in 'Mopanshi' and 'Eshi 1' leaves were ca. 6-fold and ca. 5-fold lower than those in leaves harboring empty vector (pHG-GFP), respectively. DMACA (4-dimethylaminocinnamaldehyde) staining and the Folin-Ciocalteau assay showed that the accumulation of PAs was markedly inhibited in 'Mopanshi', 'Eshi 1' and 'Youhou' leaves. These results indicate that DkLAR plays an important role in the accumulation of PAs in persimmon. The transient ihpRNA-induced gene silencing method developed in this study is a highly efficient and useful tool for functional analysis of persimmon genes involved in PA biosynthesis.

• Journal of Plant Biotechnology
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• v.34 no.4
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• pp.307-312
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• 2007

Chlamydomonas reinhardtii was transformed with the coding sequence of the Tombus virus gene p19 to determine whether the gene functions as an RNAi suppressor in C. reinhardtii. Transformants were confirmed to have 1 to several copies of p19 gene in their chromosomes. When an RNAi strain of C. reinhardtii generated by transforming the inverted repeat (IR) sequence homologous to the 3'UTR region of the MAA7 gene was re-transformed with the gene p19, MAA7 transcript levels of transformants fluctuated and proliferation of trans-formants on the medium containing 5-FI was suppressed. Overall results suggest that p19-mediated silencing suppression works at a low level in C. reinhardtii because of difference in codon usage resulting in weak P19 expression unless p19-mediated silencing suppression in C. reinhardtii works in a different manner from higher plants.

• Asian Pacific Journal of Cancer Prevention
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• v.13 no.3
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• pp.1043-1048
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• 2012

The COPS3 gene has stimulating effect on cell proliferation and progression of osteosarcomas and related cells. However, the features of COPS3 and its potential application as a therapeutic target in other cancers has not yet been studied. In this study, therefore, the effect of COPS3 silencing via COPS3 siRNA on lung cancer cell proliferation was examined. Expression levels of COPS3 gene in COPS3 siRNA infected cells and control siRNA infected cells were compared with real time PCR and Western blot analysis. Cell proliferation levels were comprehensively analyzed by MTT, BrdU incorporationy, and colony formation assays. For mechanistic assessment the effects of COPS3 silencing on cell cycle and apoptosis were analyzed using flow cytometry. Results showed that successful silencing of the COPS3 gene at both translational and transcriptional levels significantly reduced the proliferation and colony formation by lung cancer cells (p<0.01). Flow cytometry showed cell cycle arrest in the G0/G1 phase after COPS3 silencing, and more importantly, apoptosis was induced as a result of COPS3 knockdown, which negatively affected cell survival. Therefore, these results provide another piece of important evidence that the COPS3 gene expressed in lung cancer cells may play a critical role in stimulating proliferation. Down-regulation of COPS3 could significantly inhibit lung cancer cell growth, which was most likely mediated via induction of cell cycle arrest in G0/G1 phase and apoptosis.

• Horticultural Science & Technology
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• v.30 no.6
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• pp.734-742
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• 2012

To compare RNA interference-mediated gene silencing technique and T-DNA integration for gene function analysis in Chinese cabbage, BrSAMS-knockout (KO) line and BrSAMS-knockdown (KD) line were used. The KO line had lost the function of a Brassica rapa S-adenosylmethionine synthetase (BrSAMS) gene by T-DNA insertion and the KD line had shown down-regulated BrSAMS genes' expression by dsRNA cleavage. From microarray results of the KO and KD lines, genes linked to SAMS such as sterol, sucrose, homogalacturonan biosynthesis and glutaredoxin-related protein, serine/threonine protein kinase, and gibberellin-responsive protein showed distinct differences in their expression levels. Even though one BrSAMS gene in the KO line was broken by T-DNA insertion, gene expression pattern of that line did not show remarkable differences compared to wild type control. However, the KD line obtained by RNAi technique showed prominent difference in its gene expression. Besides, change of polyamine and ethylene synthesis genes directly associated with BrSAMS was displayed much more in the KD line. In the microarray analysis of the KO line, BrSAMS function could not be clearly defined because of BrSAMS redundancy due to the genome triplication events in Brassicaceae. In conclusion, we supposed that gene knock-down method by RNAi silencing is more effective than knock-out method by T-DNA insertion for gene function analysis of polyploidy crops such as Chinese cabbage.

• Plant Biotechnology Reports
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• v.5 no.3
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• pp.273-281
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• 2011

Diverse epigenetic phenotypes are frequently found during research on transgenic plants. To understand the factors underlying such diversity, hundreds of independent 35S-GFP transgenic N. benthamiana plants were analyzed. The diverse GFP-expression phenotypes of the transgenic plants were classified into three major types based on the GFP expression patterns and their response to 35S-GFP agroinfiltration: steady-green, silenced and non-uniform phenotype. The non-uniform phenotype was further sub-divided into five minor phenotypes: variegated, red-dropped, on-silencing, partitioned and misty, according to the distribution of GFP expression on the leaves. Many of transgenic plants continuously generated diverse phenotypes over several generations despite the transgene identity. Such epigenetic GFP phenotyping was found to be the result of spontaneous transgene silencing mediated by either or both of post-transcriptional gene silencing (PTGS) and transcriptional gene silencing (TGS). This finding was verified by the detection of 21- and 24-nt small interfering RNA (siRNA) molecules, and DNA methylation in the transgenic plants that showed repeated epigenetic variation. Agroinfiltration demonstrated that irregular distribution of GFP on a leaf was the result of erratic transgene silencing, and the technique also proved to be a rapid and effective method for selecting fully silenced plants within 3 days. Furthermore, two novel phenotypes described are potential materials for in-depth investigations into the genes and mechanisms responsible for spontaneous transgene silencing.

• BMB Reports
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• v.50 no.4
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• pp.226-231
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• 2017

At the post-transcriptional and translational levels, microRNA (miRNA) represses protein-coding genes via seed pairing to the 3' untranslated regions (UTRs) of mRNA. Although working models of miRNA-mediated gene silencing are successfully established using miRNA transfections and knockouts, the regulatory interaction between miRNA and long non-coding RNA (lncRNA) remain unknown. In particular, how the mRNA-resembling lncRNAs with 5' cap, 3' poly(A)-tail, or coding features, are regulated by miRNA is yet to be examined. We therefore investigated the functional interaction between miRNAs and lncRNAs with/without those features, in miRNA-transfected early zebrafish embryos. We observed that the greatest determinants of the miRNA-mediated silencing of lncRNAs were the 5' cap and 3' poly(A)-tails in lncRNAs, at both the post-transcriptional and translational levels. The lncRNAs confirmed to contain 5' cap, 3' poly(A)-tail, and the canonical miRNA target sites, were observed to be repressed in the level of both RNA and ribosome-protected fragment, while those with the miRNA target sites and without 5' cap and 3' poly(A)-tail, were not robustly repressed by miRNA introduction, thus suggesting a role as a miRNA-decoy.

• Molecules and Cells
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• v.20 no.2
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• pp.167-172
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• 2005

Ceruloplasmin (Cp) is a copper protein with important functions in iron homeostasis and in inflammation. Cp mRNA expression is induced by interferon (IFN)-${\gamma}$ in U937 monocytic cells, but synthesis of Cp protein is halted after about 12 h by transcript-specific translational silencing. The silencing mechanism requires binding of a 4-component cytosolic inhibitor complex, IFN-gamma-activated inhibitor of translation (GAIT), to a defined structural element (GAIT element) in the Cp 3'-UTR. Translational silencing of Cp mRNA requires the essential proteins of mRNA circularization, suggesting that the translational inhibition requires end-to-end mRNA closure. These studies describe a new mechanism of translational control, and may shed light on the role that macrophage-derived Cp plays at the intersection of iron homeostasis and inflammation.

• Asian Pacific Journal of Cancer Prevention
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• v.14 no.9
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• pp.5391-5396
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• 2013

Aim: To investigate the role of Golgi phosphoprotein 3 (GOLPH3) in tumour growth and metastasis of esophageal squamous cancer. Methods: A lentiviral shRNA-vector was utilized to stably knockdown GOLPH3 in Eca-109 esophageal squamous cancer cells. mRNA transcription and protein expression of GOLPH3 were examined by real-time quantitative PCR and Western blotting, respectively. Cell proliferation activity was assessed by MTT assay and invasion and migration potentials by matrigel invasion and transwell motility assays. Results: Stable knockdown in the GOLPH3 cell line was established. PD-A gene expression was significantly suppressed by lentivirus-mediated RNAi, which resulted in reducing the capacity for cell proliferation, migration, invasion and adhesion in vitro. In vivo, GOLPH3 depletion resulted in inhibition of tumour growth, with stable decrease in the expression of GOLPH3 in tumor xenografts. Conclusions: Our findings suggest that lentivirus mediated silencing of the GOLPH3 gene has a significant anti-tumour effect on esophageal squamous cancer in vitro and in vivo. In addition, the results indicate that GOLPH3 might be an effective molecular target for gene therapy in esophageal squamous cancer.

• Journal of Plant Biotechnology
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• v.45 no.3
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• pp.183-189
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• 2018

The plant hormone auxin regulates the overall metabolic processes essential for plant growth and development. Auxin signaling is mediated by early auxin response genes, which are classified into three major families: AUXIN/INDOLE ACETIC ACID (AUX/IAA), GRETCHEN HAGEN3 (GH3) and SMALL AUIN UP RNA (SAUR). The SAUR gene family is the largest family among early auxin response genes and encodes the small and highly unstable gene products. The functional roles of SAUR genes have remained unclear for many years. The traditional genetic and molecular studies on the SAUR functions have been hampered by their likely genetic redundancy and tandem arrays of highly related genes in the plant genome, together with the molecular characteristics of SAUR. However, recent studies have suggested possible roles of SAUR in a variety of tissues and developmental stages in accordance with the novel approaches such as gain-of-function and RNA silencing techniques. In this review, the recent research progress on the functional roles and regulatory mechanisms of SAUR and a set of possible future works are discussed.