• The Korean Journal of Physiology and Pharmacology
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• 제17권6호
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• pp.511-516
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• 2013

Bladder cancer is the seventh most common cancer in men that smoke, and the incidence of disease increases with age. The mechanism of occurrence has not yet been established. Potassium channels have been linked with cell proliferation. Some two-pore domain $K^+$ channels (K2P), such as TASK3 and TREK1, have recently been shown to be overexpressed in cancer cells. Here we focused on the relationship between cell growth and the mechanosensitive K2P channel, TREK2, in the human bladder cancer cell line, 253J. We confirmed that TREK2 was expressed in bladder cancer cell lines by Western blot and quantitative real-time PCR. Using the patch-clamp technique, the mechanosensitive TREK2 channel was recorded in the presence of symmetrical 150 mM KCl solutions. In 253J cells, the TREK2 channel was activated by polyunsaturated fatty acids, intracellular acidosis at -60 mV and mechanical stretch at -40 mV or 40 mV. Furthermore, small interfering RNA (siRNA)-mediated TREK2 knockdown resulted in a slight depolarization from $-19.9mV{\pm}0.8$ (n=116) to $-8.5mV{\pm}1.4$ (n=74) and decreased proliferation of 253J cells, compared to negative control siRNA. 253J cells treated with TREK2 siRNA showed a significant increase in the expression of cell cycle boundary proteins p21 and p53 and also a remarkable decrease in protein expression of cyclins D1 and D3. Taken together, the TREK2 channel is present in bladder cancer cell lines and may, at least in part, contribute to cell cycle-dependent growth.

• The Plant Pathology Journal
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• 제40권1호
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• pp.73-82
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• 2024

Gardenia (Gardenia jasminoides) is a popular and economically vital plant known for its ornamental and medicinal properties. Despite its widespread cultivation, there has been no documentation of plant viruses on gardenia yet. In the present study, gardenia leaves exhibiting symptoms of plant viral diseases were sampled and sequenced by both metatranscriptome and small RNA sequencing. As a consequence, bean common mosaic virus (BCMV) was identified in gardenia for the first time and named BCMV-gardenia. The full genome sequence of BCMV-gardenia is 10,054 nucleotides (nt) in length (excluding the poly (A) at the 3' termini), encoding a large polyprotein of 3,222 amino acids. Sequence analysis showed that the N-termini of the polyprotein encoded by BCMV-gardenia is less conserved when compared to other BCMV isolates, whereas the C-termini is the most conserved. Maximum likelihood phylogenetic analysis showed that BCMVgardenia was clustered closely with other BCMV isolates identified outside the leguminous plants. Our results indicated that the majority of BCMV-gardenia virus-derived small interfering RNAs (vsiRNAs) were 21 nt and 22 nt, with 21 nt being more abundant. The first nucleotide at the 5' termini of vsiRNAs derived from BCMV-gardenia preferred U and A. The ratio of vsiRNAs derived from sense (51.1%) and antisense (48.9%) strands is approaching, and the distribution of vsiRNAs along the viral genome is generally even, with some hot spots forming in local regions. Our findings could provide new insights into the diversity, evolution, and host expansion of BCMV and contribute to the prevention and treatment of this virus.

• Asian Pacific Journal of Cancer Prevention
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• 제13권9호
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• pp.4435-4439
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• 2012

Purpose: This study aimed to explore the role of the Twist gene in the epithelial-mesenchymal transition of ovarian cancer. Methods: An RNA interference plasmid expressing a small interfering RNA (siRNA)-targeting Twist (Twist siRNA vector) was designed, constructed, and transfected into the human ovarian cancer cell line A2780. Transfection efficiency was assessed under a fluorescence microscope. Changes in the expression of Twist mRNA in A2780 after transfection with the pGenesil Twist shRNA plasmid were analyzed through RT-PCR. MTT assays and adhesion experiments were applied to determine changes in proliferation and adhesion ability of A2870 after transfection with the Twist shRNA plasmid. Changes in the expression of the E-cadherin and N-cadherin proteins in A2780 after transfection with the Twist shRNA plasmid were analyzed using Western blotting. Result: The restructuring plasmid pGenesil-Twist shRNA was constructed successfully. After 48 h of culture, 80% of the cells expressed high-intensity GFP fluorescence and stability. The expression of Twist decreased significantly after the transfection of the Twist shRNA plasmid (P<0.05). Proliferation of the transfected Twist shRNA cells showed no difference with that of the A2780-nontransfection or A2780-si-control groups (P>0.05) but the adhesion ability of A2780 decreased dramatically (P<0.05). Expression of the E-cadherin protein increased, whereas that of the N-cadherin protein decreased compared with that in the A2780-nontransfection or A2780-si-control groups (P<0.05). Conclusion: Twist is essential for epithelial-mesenchymal transition, invasion, and metastasis of ovarian cancer.

• 한국발생생물학회지:발생과생식
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• 제12권2호
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• pp.159-168
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• 2008

Nanog is a newly identified member of the homeobox family of DNA binding transcription factors that functions to maintain the undifferentiated state of stem cells. However, molecular mechanisms underlying the function of Nanog remain largely unknown. To elucidate the regulatory roles of Nanog involved in maintenance of P19 embryonal carcinoma (EC) stem cells, we transfected three small interfering RNA (siRNA) duplexes targeted against different regions of the Nanog gene into P19 cells. The Nanog siRNA-100 duplexes effectively decreased the expression of Nanog up to 30.7% compared to other two Nanog siRNAs, the Nanog siRNA-400 (67.9 %) and -793 (53.0%). When examined by RT-PCR and real-time PCR, the expression of markers for pluripotency such as Fgf4, Oct3/4, Rex1, Sox1 and Yes was downregulated at 48 h after transfection with Nanog siRNA-100. Furthermore, expression of the ectodermal markers, Fgf5 and Isl1 was reduced by Nanog knockdown. By contrast, the expression of other markers for pluripotency such as Cripto, Sox2 and Zfp57 was not affected by Nanog knockdown at this time. On the other hand, the expression of Lif/Stat3 pathway molecules and of the endoderm markers including Dab2, Gata4, Gata6 and the germ cell nuclear factor was not changed by Nanog knockdown. The results of this study demonstrated that the knockdown of Nanog expression by RNA interference in P19 cells was sufficient to modulate the expression of pluripotent markers involved in the self-renewal of EC stem cells. These results provide the valuable information on potential downstream targets of Nanog and add to our understanding of the function of Nanog in P19 EC stem cells.

• Genomics & Informatics
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• 제9권3호
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• pp.102-113
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• 2011

C-terminal SRC kinase (CSK) is a ubiquitously expressed, cytosolic enzyme that phosphorylates and inactivates several SRC family protein tyrosine kinases. Recent genomewide association studies have implicated CSK in the regulation of blood pressure. The current study aim is to determine the blood pressure association of the genes regulated by CSK down-regulation. The CSK mRNA expression was downregulated in vascular smooth muscle cells using small interfering RNA (siRNA). CSK mRNA levels fell by 90% in cells that were treated with CSK siRNA; the RNA from these cells was examined by microarray using the Illumina HumanRef-8 v3 platform, which comprises 24,526 reference mRNA probes. On treatment with CSK siRNA, 19 genes were downregulated by more than 2-fold and 13 genes were upregulated by more than 2-fold. Three (CANX, SLC30A7, and HMOX1) of them revealed more than 3 fold differential expression. Interestingly, the HMOX1 SNPs were associated with diastolic blood pressure in the 7551 Koreans using Korea Association REsource data, and the result was supported by the other reports that HMOX1 linked to blood vessel maintenance. Among the remaining 29 differentially expressed genes, seven (SSBP1, CDH2, YWHAE, ME2, PFTK1, G3BP2, and TUFT1) revealed association with both systolic and diastolic blood pressures. The CDH2 gene was linked to blood pressures. Conclusively, we identified 32 differentially expressed genes which were regulated by CSK reduction, and two (HOMX1 and CDH2) of them might influence the blood pressure regulation through CSK pathway.

• Journal of Plant Biotechnology
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• 제46권3호
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• pp.205-216
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• 2019

Since the RNA interference (RNAi) had been discovered in many organisms, small non-coding RNA-mediated gene silencing technology, including RNAi have been widely applied to analysis of gene function, as well as crop improvement. Despite the usefulness of RNAi technology, RNAi transgenic crops have various potential environmental risks, including off-target and non-target effects. In this study, we developed methods that can be effectively applied to environmental risk assessment of RNAi transgenic crops and verified these methods in 35S::dsRNAi_eGFP rice transgenic plant we generated. Off-target genes, which can be non-specifically suppressed by the expression of dsRNAi_eGFP, were predicted by using the published web tool, pssRNAit, and verified by comparing their expressions between wild-type (WT) and 35S::dsRNAi_eGFP transgenic rice. Also, we verified the non-target effects of the 35S:: dsRNAi_eGFP plant by evaluating horizontal and vertical transfer of small interfering RNAs (siRNAs) produced in the 35S::dsRNAi_eGFP plant into neighboring WT rice and rhizosphere microorganisms, respectively. Our results suggested that the methods we developed, could be widely applied to various RNAi transgenic crops for their environmental risk assessment.

• Asian Pacific Journal of Cancer Prevention
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• 제13권8호
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• pp.4147-4151
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• 2012

The current study was conducted to explore the inhibitory effects of a small interfering RNA (siRNA) on 3-phosphoinositide-dependent protein kinase 1 (PDK1) expression in esophageal cancer 9706 (EC9706) cells and the influence on their biological behavior. After transfection of a synthesized PDK1 siRNA, PDK1 mRNA and protein expression and the phosphorylation level of the downstream Akt protein were assessed using RT-PCR and Western blot analysis. Proliferation, apoptosis, cell invasion and in vivo tumor formation capacity were also investigated using MTT, flow cytometry, Transwell invasion trials, and nude mouse tumor transplantion, respectively. PDK1 siRNA effectively suppressed PDK1 mRNA and protein expression, and down-regulated the phosphorylation level of the Akt protein in the EC9706 cells (P < 0.05). It also inhibited cell proliferation and invasion, and promoted apoptosis; such effects were particularly obvious at 48 h and 72 h after transfection (P < 0.05). Growth of transplanted tumors was inhibited in nude mice, with decreased PDK1 expression in tumor tissues. PDK1 may be closely correlated with proliferation, apoptosis and invasion of esophageal cancer cells and thus may serve as an effective target for gene therapy.

• Parasites, Hosts and Diseases
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• 제52권2호
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• pp.131-135
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• 2014

Acanthamoeba cysts are resistant to unfavorable physiological conditions and various disinfectants. Acanthamoeba cysts have 2 walls containing various sugar moieties, and in particular, one third of the inner wall is composed of cellulose. In this study, it has been shown that down-regulation of cellulose synthase by small interfering RNA (siRNA) significantly inhibits the formation of mature Acanthamoeba castellanii cysts. Calcofluor white staining and transmission electron microscopy revealed that siRNA transfected amoeba failed to form an inner wall during encystation and thus are likely to be more vulnerable. In addition, the expression of xylose isomerase, which is involved in cyst wall formation, was not altered in cellulose synthase down-regulated amoeba, indicating that cellulose synthase is a crucial factor for inner wall formation by Acanthamoeba during encystation.

• BMB Reports
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• 제45권1호
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• pp.20-25
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• 2012

The present study aimed to investigate the function of translationally controlled tumor protein (TCTP) in the regulation of Oct4 in mouse embryonic carcinoma P19 cells and mouse J1 embryonic stem (ES) cells. The mRNA level of endogenous TCTP in somatic cells was 2-4 folds higher than that in pluripotent P19 and J1 ES cells. Overexpression of TCTP in mouse pluripotent cells not only reduced the level of Oct4 transcription, but also decreased the pluripotency of stem cells. The N-terminal end of TCTP (amino acids 1-60) played an important role in suppressing the Oct4 promoter. Moreover, overexpression of TCTP in P19 cells suppressed the Oct4 promoter activity in a dose- and a time-dependent manner. In addition, knockdown of TCTP by small interfering RNA increased the expression of Oct4. Our study indicates that TCTP downregulates the Oct4 expression by binding the Sf1 site of Oct4 promoter in mouse pluripotent cells.

• Animal cells and systems
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• 제16권5호
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• pp.376-384
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• 2012

Although ginsenosides have a variety of physiologic or pharmacologic functions in various regions, there are only a few reports on the effects of transient receptor potential melastatin 7 (TRPM7) channels. Here, we showed evidence suggesting that TRPM7 channels play an important role in ginseng total saponin (GTS)-mediated cellular injury. The combination techniques of electrophysiology, pharmacological analysis, small interfering RNA (siRNA) method and cell death assays were used. GTS depolarized the resting membrane potentials and decreased the amplitude of pacemaker potentials in cultured interstitial cells of Cajal (ICCs) in gastrointestinal (GI) tract. The TRPM7-like currents in single ICCs and the overexpressing TRPM7 in HEK293 cells were inhibited by GTS. However, GTS had no effect on $Ca^{2+}$-activated $Cl^-$ conductance. GTS inhibited the survival of human gastric (AGS) and brea (MCF-7) adenocarcinoma cells. Also, GTS inhibited the TRPM7-like currents in AGS and MCF-7 cells. The GTS-mediated cytotoxicity was inhibited by TRPM7-specific siRNA. In addition, we showed that overexpression of TRPM7 channels in HEK293 cells was inhibited by GTS. Thus, TRPM7 channels are involved in GTS-mediated cell death in AGS and MCF-7 cells, and these channels may represent a novel target for physiological disorders where GTS plays an important role.