• The Plant Pathology Journal
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• 제27권4호
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• pp.315-323
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• 2011

Soybean plants infected with Bean pod mottle virus (BPMV) develop acute symptoms that usually decrease in severity over time. In other plant-virus interactions, this type of symptom recovery has been associated with degradation of viral RNAs by RNA silencing, which is accompanied by the accumulation of virus-derived small interfering RNAs (siRNAs). In this study, changes in the accumulation of BPMV siRNAs were investigated in soybean plants infected with BPMV alone, or infected with both BPMV and Soybean mosaic virus (SMV) and in transgenic soybean plants expressing SMV helper component-protease (HC-Pro). In many potyviruses, HC-Pro is a potent suppressor of RNA silencing. In plants infected with BPMV alone, accumulation of siRNAs was positively correlated with symptom severity and accumulation of BPMV genomic RNAs. Plants infected with both BPMV and SMV and BPMV-infected transgenic soybean plants expressing SMV HC-Pro exhibited severe symptoms characteristic of BPMVSMV synergism, and showed enhanced accumulation of BPMV RNAs and siRNAs compared to plants infected with BPMV alone and nontransgenic plants. Likewise, SMV HC-Pro enhanced the accumulation of siRNAs produced from a silenced green fluorescent protein gene in transient expression assays, while the P19 silencing suppressor of Tomato bushy stunt virus did not. Consistent with the modes of action of HC-Pro in other systems, which have shown that HC-Pro suppresses RNA silencing by preventing the unwinding of duplex siRNAs and inhibiting siRNA methylation, these studies showed that SMV HC-Pro interfered with the activities of RNA-induced silencing complexes, but not the activities of Dicer-like enzymes in antiviral defenses.

• 한국생물공학회:학술대회논문집
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• 한국생물공학회 2005년도 생물공학의 동향(XVII)
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• pp.399-400
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• 2005

• Asian-Australasian Journal of Animal Sciences
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• 제24권11호
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• pp.1529-1540
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• 2011

Silencing of a specific gene using RNAi (RNA interference) is a valuable tool for functional analysis of a target gene. However, information on RNAi for analysis of gene function in farm animals is relatively nil. In the present study, we have validated the interfering effects of siRNA (small interfering RNA) using both quantitative and qualitative gene silencing in buffalo granulosa cells. Qualitative gene knockdown was validated using a fluorescent vector, enhanced green fluorescence protein (EGFP) and fluorescently labeled siRNA (Cy3) duplex. While quantitatively, siRNA targeted against the luciferase and CYP19 mRNA was used to validate the technique. CYP19 gene, a candidate fertility gene, was selected as a model to demonstrate the technique optimization. However, to sustain the expression of CYP19 gene in culture conditions using serum is difficult because granulosa cells have the tendency to luteinize in presence of serum. Therefore, serum free culture conditions were optimized for transfection and were found to be more suitable for the maintenance of CYP19 gene transcripts in comparison to culture conditions with serum. Decline in fluorescence intensity of green fluorescent protein (EGFP) was observed following co-transfection with plasmid generating siRNA targeted against EGFP gene. Quantitative decrease in luminescence was seen when co-transfected with siRNA against the luciferase gene. A significant suppressive effect on the mRNA levels of CYP19 gene at 100 nM siRNA concentration was observed. Also, measurement of estradiol levels using ELISA (enzyme-linked immunosorbent assay) showed a significant decline in comparison to control. In conclusion, the present study validated gene silencing using RNAi in cultured buffalo granulosa cells which can be used as an effective tool for functional analysis of target genes.

• Molecules and Cells
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• 제26권3호
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• pp.308-313
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• 2008

In animals, microRNAs (miRNAs) and small interfering RNAs (siRNAs) repress expression of protein coding genes by assembling distinct RNA-induced silencing complexes (RISCs). It has previously been shown that passenger-strand cleavage is the predominant mechanism when siRNA duplexes are loaded into Argonaute2 (Ago2)-containing RISC, while an unwinding bypass mechanism is favored for miRNA duplexes with mismatches. Here I present experimental data indicating that some mammalian miRNAs are assembled into Ago2-containing RISC by cleaving their corresponding miRNA star strands. This phenomenon may depend on the secondary structure near the scissile phosphate of the miRNA duplex. In addition, I show that ATP is not required for star-strand cleavage in this process. Taken together, the data here provide insight into the miRNA-loading mechanisms in mammals.

• 치위생과학회지
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• 제13권3호
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• pp.321-329
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• 2013

과산화수소는 치아미백에 널리 사용되는 물질로 과다 사용시 치수세포에 손상을 일으킬 수 있다. 본 연구의 목적은 활성산소인 과산화수소에 의해 유도되는 상아모세포의 단계별 분화와 LOX isoforms과의 관계를 밝히고자 하였다. 치수세포에 분화유도 배지와 과산화수소를 시간과 농도별로 처리한 후 LOX 유전자 발현은 RT-PCR로 측정하였고, LOX enzyme activity는 고감도 형광분석으로 확인하였다. 또한 가장 많은 발현억제를 보인 LOX와 LOXL을 선택하여 siRNA 처리 후 분화표지자의 발현변화와 LOX enzyme activity를 확인하였다. 1. 과산화수소 처리에 따라 LOX, LOXL, LOXL3 mRNA 발현은 농도와 시간 의존적으로 감소하였으나 LOXL2와 LOXL4 mRNA는 변화가 없었다. 2. 과산화수소 처리된 LOX enzyme activity는 0.3 mM과 24시간에서 가장 많은 증가를 보였다. 3. ALP, OPN, OCN의 mRNA 발현은 LOX와 LOXL siRNAs 모두에서 억제되었고, DMP1과 DSPP는 LOX siRNA에서 더 많은 억제 효과를 보였다. 하지만, 분화단계별(초기, 중기, 말기) 차이는 보이지 않았다. 4. LOX와 LOXL siRNAs를 처리하여 LOX enzyme activity를 측정한 결과 LOX siRNA를 처리한 실험군에서 더 많은 억제효과를 보였다. 이러한 결과는 상아모세포 성장과 분화과정에 낮은 농도의 과산화수소가 분화를 유도하고 여기에 LOX가 관련됨을 알 수 있었다. 결론적으로, 과산화수소는 LOX 유전자 발현조절을 통해 치수세포의 성장과 분화에서 중추적인 역할을 할 것이라고 생각된다.

• Journal of Microbiology and Biotechnology
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• 제24권9호
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• pp.1291-1299
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• 2014

Recently, multifunctional nanomaterials have been developed as nanotherapeutic agents for cellular imaging and targeted cancer treatment because of their ease of synthesis and low cytotoxicity. In this study, we developed a multifunctional, two-component nanorod consisting of gold (Au) and nickel (Ni) blocks that enables dual-fluorescence imaging and the targeted delivery of small interfering RNA (siRNA) to improve cancer treatment. Fluorescein isothiocyanate-labeled luteinizing hormone-releasing hormone (LHRH) peptides were attached to the surface of a Ni block via a histidine-tagged LHRH interaction to specifically bind to a breast cancer cell line, MCF-7. The Au block was modified with TAMRA-labeled thiolated siRNA in order to knock down the vascular endothelial growth factor protein to inhibit cancer growth. These two-component nanorods actively targeted and internalized into MCF-7 cells to induce apoptosis through RNA interference. This study demonstrates the feasibility of using two-component nanorods as a potential theranostic in breast cancer treatment, with capabilities in dual imaging and targeted gene delivery.

• Journal of Microbiology and Biotechnology
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• 제19권8호
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• pp.781-786
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• 2009

Small interfering synthetic double-stranded RNA (siRNA) was applied to suppress the expression of the human cytotoxic-T-Iymphocyte antigen 4-immunoglobulin (hCTLA4Ig) gene transformed in transgenic rice cell cultures. The sequence of the 21-nucleotide siRNA was deliberately designed and synthesized with overhangs to inactivate the expression of hCTLA4Ig. The chemically synthesized siRNA duplex was combined with polyethyleneimine (PEl) at a mass ratio of 1:10 (0.33 ${\mu}g$ siRNA:3.3 ${\mu}g$ PEl) to produce complexes. The siRNA complexes (siRNA+PEI) were labeled with Cy3 in order to subsequently confirm the delivery by fluorescent microscopy. In addition, the cells were treated with sonoporation at 40 kHz and 419W for 90 s to improve the delivery. The siRNA complexes alone inhibited the expression of hCTLA4Ig to 45% compared with control. The siRNA complexes delivered with sonoporation downregulated the production of hCTLA4Ig to 73%. Therefore, we concluded that the delivery of siRNA complexes into plant cells could be enhanced successfully by sonoporation.

• 대한약침학회지
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• 제15권3호
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• pp.31-38
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• 2012

Sophorae Radix (SR) plays a role in a number of physiologic and pharmacologic functions in many organs. Objective: The aim of this study was to clarify the potential role for transient receptor potential melastatin 7 (TRPM7) channels in SR-inhibited growth and survival of AGS and MCF-7 cells, the most common human gastric and breast adenocarcinoma cell lines. Methods: The AGS and the MCF-7 cells were treated with varying concentrations of SR. Analyses of the caspase-3 and - 9 activity, the mitochondrial depolarization and the poly (ADPribose) polymerase (PARP) cleavage were conducted to determine if AGS and MCF-7 cell death occured by apoptosis. TRPM7 channel blockers ($Gd^{3+}$ or 2-APB) and small interfering RNA (siRNA) were used in this study to confirm the role of TRPM7 channels. Furthermore, TRPM7 channels were overexpressed in human embryonic kidney (HEK) 293 cells to identify the role of TRPM7 channels in AGS and MCF-7 cell growth and survival. Results: The addition of SR to a culture medium inhibited AGS and MCF-7 cell growth and survival. Experimental results showed that the caspase-3 and -9 activity, the mitochondrial depolarization, and the degree of PARP cleavage was increased. TRPM7 channel blockade, either by $Gd^{3+}$ or 2-APB or by suppressing TRPM7 expression with small interfering RNA, blocked the SR-induced inhibition of cell growth and survival. Furthermore, TRPM7 channel overexpression in HEK 293 cells exacerbated SR-induced cell death. Conclusions: These findings indicate that SR inhibits the growth and survival of gastric and breast cancer cells due to a blockade of the TRPM7 channel activity. Therefore, TRPM7 channels may play an important role in the survival of patients with gastric and breast cancer.

• Journal of Veterinary Science
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• 제23권2호
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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.

• BMB Reports
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• 제42권3호
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• pp.178-183
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• 2009

Tyrosinase (TYR) plays a critical role in cellular melanogenesis and, thus, has been the major target of pharmacological approaches for the control of skin pigmentation. This study examined an alternative molecular approach using TYR-small interfering RNA (siRNA) to control melanogenesis in the human melanocytes. Both the mRNA and protein levels of TYR were significantly lowered by TYR-siRNA treatment, whereas TYR-related protein 1 and TYR-related protein 2 displayed no such changes. TYR-siRNA treatment inhibited the cellular melanin synthesis from the externally supplied TYR substrate L-tyrosine. TYR-siRNA also suppressed melanin synthesis and decreased the viability of cells exposed to ultraviolet radiation, supporting a critical role of melanin in protection against ultraviolet radiation. These results suggest that molecular approaches using siRNA targeted to the enzymes of melanogenic pathway may provide a novel strategy for the control of cell pigmentation.