• Title, Summary, Keyword: Small interfering RNA

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Oct 3/4 siRNA가 마우스 수정란의 발달 및 유전자 발현에 미치는 영향

  • 최향순
    • Proceedings of the KSAR Conference
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    • pp.171-172
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    • 2004
  • Science에서는 2002년을 small RNA를 'molecule of the year'에 선정되었다. 이어서 2003년에는 전 과학분야를 대상으로 10대 중요 과학적 성과를 발표하였는데 그 중에 siRNA (small interfering RNA)는 RNAi(RNA interference) 현상을 유도할 수 있는 기술로 선정되었으며 그 이후로 많은 과학자들의 관심을 끌게 되었다. (중략)

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Small Interfering RNA-Mediated Suppression of Fas Modulate Apoptosis and Proliferation in Rat Intervertebral Disc Cells

  • Park, Jong-Beom;Park, Chanjoo
    • Asian Spine Journal
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    • v.11 no.5
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    • pp.686-693
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    • 2017
  • Study Design: In vitro cell culture model. Purpose: To investigate the effect of small interfering RNA (siRNA) on Fas expression, apoptosis, and proliferation in serum-deprived rat disc cells. Overview of Literature: Synthetic siRNA can trigger an RNA interference (RNAi) response in mammalian cells and precipitate the inhibition of specific gene expression. However, the potential utility of siRNA technology in downregulation of specific genes associated with disc cell apoptosis remains unclear. Methods: Rat disc cells were isolated and cultured in the presence of either 10% fetal bovine serum (FBS) (normal control) or 0% FBS (serum deprivation to induce apoptosis) for 48 hours. Fas expression, apoptosis, and proliferation were determined. Additionally, siRNA oligonucleotides against Fas (Fas siRNA) were transfected into rat disc cells to suppress Fas expression. Changes in Fas expression were assessed by reverse transcription-polymerase chain reaction and semiquantitatively analyzed using densitometry. The effect of Fas siRNA on apoptosis and proliferation of rat disc cells were also determined. Negative siRNA and transfection agent alone (Mock) were used as controls. Results: Serum deprivation increased apoptosis by 40.3% (p<0.001), decreased proliferation by 45.3% (p<0.001), and upregulated Fas expression. Additionally, Fas siRNA suppressed Fas expression in serum-deprived cultures, with 68.5% reduction at the mRNA level compared to the control cultures (p<0.001). Finally, Fas siRNA-mediated suppression of Fas expression significantly inhibited apoptosis by 9.3% and increased proliferation by 21% in serum-deprived cultures (p<0.05 for both). Conclusions: The observed dual positive effect of Fas siRNA might be a powerful therapeutic approach for disc degeneration by suppression of harmful gene expression.

VEGF siRNA Delivery by a Cancer-Specific Cell-Penetrating Peptide

  • Lee, Young Woong;Hwang, Young Eun;Lee, Ju Young;Sohn, Jung-Hoon;Sung, Bong Hyun;Kim, Sun Chang
    • Journal of Microbiology and Biotechnology
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    • v.28 no.3
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    • pp.367-374
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    • 2018
  • RNA interference provides an effective tool for developing antitumor therapies. Cell-penetrating peptides (CPPs) are delivery vectors widely used to efficiently transport small-interfering RNA (siRNA) to intracellular targets. In this study, we investigated the efficacy of the cancer-specific CPP carrier BR2 to specifically transport siRNA to cancer-target cells. Our results showed that BR2 formed a complex with anti-vascular endothelial growth factor siRNA (siVEGF) that exhibited the appropriate size and surface charge for in vivo treatment. Additionally, the BR2-VEGF siRNA complex exhibited significant serum stability and high levels of gene-silencing effects in vitro. Moreover, the transfection efficiency of the complex into a cancer cell line was higher than that observed in non-cancer cell lines, resulting in downregulated intracellular VEGF levels in HeLa cells and comprehensively improved antitumor efficacy in the absence of significant toxicity. These results indicated that BR2 has significant potential for the safe, efficient, and specific delivery of siRNA for diverse applications.

New Hairpin RNAi Vector with Brassica rapa ssp. pekinensis Intron for Gene Silencing in Plants

  • Lee, Gi-Ho;Lee, Gang-Seob;Park, Young-Doo
    • Horticultural Science & Technology
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    • v.35 no.3
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    • pp.323-332
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    • 2017
  • Homology-specific transcriptional and post-transcriptional silencing, an intrinsic mechanism of gene regulation in most eukaryotes, can be induced by anti-sense, co-suppression, or hairpin-based double-stranded RNA. Hairpin-based RNA interference (RNAi) has been applied to analyze gene function and genetically modify crops. However, RNAi vector construction usually requires high-cost cloning steps and large amounts of time, or involves methods that are protected by intellectual property rights. We describe a more effective method for generating intron-spliced RNAi constructs. To produce intron-spliced hairpin RNA, an RNAi cassette was ligated with the first intron and splicing sequences of the Brassica rapa ssp. pekinensis histone deacetylase 1 gene. This method requires a single ligation of the PCR-amplified target gene to SpeI-NcoI and SacI-BglII enzyme sites to create a gene-specific silencing construct. We named the resulting binary vector system pKHi and verified its functionality by constructing a vector to silence DIHYDROFLAVONOL 4-REDUCTASE (DFR), transforming it into tobacco plants, and confirming DFR gene-silencing via PCR, RT-qPCR, and analysis of the accumulation of small interfering RNAs. Reduction of anthocyanin biosynthesis was also confirmed by analyzing flower color of the transgenic tobacco plants. This study demonstrates that small interfering RNAs generated through the pKHi vector system can efficiently silence target genes and could be used in developing genetically modified crops.

Local Silencing of Connective Tissue Growth Factor by siRNA/Peptide Improves Dermal Collagen Arrangements

  • Cho Lee, Ae-Ri;Woo, Inhae
    • Tissue Engineering and Regenerative Medicine
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    • v.15 no.6
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    • pp.711-719
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    • 2018
  • BACKGROUND: Collagen organization within tissues has a critical role in wound regeneration. Collagen fibril diameter, arrangements and maturity between connective tissue growth factor (CTGF) small interfering RNA (siRNA) and mismatch scrambled siRNA-treated wound were compared to evaluate the efficacy of CTGF siRNA as a future implement for scar preventive medicine. METHODS: Nanocomplexes of CTGF small interfering RNA (CTGF siRNA) with cell penetrating peptides (KALA and $MPG^{{\Delta}NLS}$) were formulated and their effects on CTGF downregulation, collagen fibril diameter and arrangement were investigated. Various ratios of CTGF siRNA and peptide complexes were prepared and down-regulation were evaluated by immunoblot analysis. Control and CTGF siRNA modified cells-populated collagen lattices were prepared and rates of contraction measured. Collagen organization in rabbit ear 8 mm biopsy punch wound at 1 day to 8 wks post injury time were investigated by transmission electron microscopy and histology was investigated with Olympus System and TS-Auto software. CONCLUSION: CTGF expression was down-regulated to 40% of control by CTGF siRNA/KALA (1:24) complexes (p<0.01) and collagen lattice contraction was inhibited. However, down-regulated of CTGF by CTGF $siRNA/MPG^{{\Delta}NLS}$ complexes was not statistically significant. CTGF KALA-treated wound appeared with well formed-basket weave pattern of collagen fibrils with mean diameter of $128{\pm}22nm$ (n = 821). Mismatch siRNA/KALA-treated wound showed a high frequency of parallel small diameter fibrils (mean $90{\pm}20nm$, n = 563). CONCLUSION: Controlling over-expression of CTGF by peptide-mediated siRNA delivery could improve the collagen orientation and tissue remodeling in full thickness rabbit ear wound.

Altered expression of norepinephrine transporter and norepinephrine in human placenta cause pre-eclampsia through regulated trophoblast invasion

  • Na, Kyu-Hwan;Choi, Jong Ho;Kim, Chun-Hyung;Kim, Kwang-Soo;Kim, Gi Jin
    • Clinical and Experimental Reproductive Medicine
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    • v.40 no.1
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    • pp.12-22
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    • 2013
  • Objective: We investigated the norepinephrine transporter (NET) expression in normal and pre-eclamptic placentas and analyzed the invasion activity of trophoblastic cells based on norepinephrine (NE)-NET regulation. Methods: NET and NE expression levels were examined by western blot and enzyme-linked immunosorbent assay, respectively. Trophoblast invasion activity, depending on NE-NET regulation, was determined by NET-small interfering RNA (siRNA) and NET transfection into the human extravillous trophoblast cells with or without NE treatment and invasion rates were analyzed by zymography and an invasion assay. Results: NET mRNA was expressed at a low level in pre-eclamptic placentas compared with normal placentas and NE concentration in maternal plasma increased significantly in pre-eclamptic women compared to normal pregnant women (p<0.05). NET gene upregulation and NE treatment stimulated trophoblast cell invasion up to 2.5-fold (p<0.05) by stimulating matrix metalloproteinase-9 activity via the phosphoinositol-3-kinase/AKT signaling pathway, whereas NET-siRNA with NE treatment reduced invasion rates. Conclusion: NET expression is reduced by inadequate regulation of NE levels during placental development. This suggests that a complementary balance between NET and NE regulates trophoblast cell invasion activities during placental development.

siRNAs Derived from Cymbidium Mosaic Virus and Odontoglossum Ringspot Virus Down-modulated the Expression Levels of Endogenous Genes in Phalaenopsis equestris

  • Lan, Han-hong;Wang, Cui-mei;Chen, Shuang-shuang;Zheng, Jian-ying
    • The Plant Pathology Journal
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    • v.35 no.5
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    • pp.508-520
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    • 2019
  • Interplay between Cymbidium mosaic virus (CymMV)/Odontoglossum ringspot virus (ORSV) and its host plant Phalaenopsis equestris remain largely unknown, which led to deficiency of effective measures to control disease of P. equestris caused by infecting viruses. In this study, for the first time, we characterized viral small interfering RNAs (vsiRNAs) profiles in P. equestris co-infected with CymMV and ORSV through small RNA sequencing technology. CymMV and ORSV small interfering RNAs (siRNAs) demonstrated several general and specific/new characteristics. vsiRNAs, with A/U bias at the first nucleotide, were predominantly 21-nt long and they were derived predominantly (90%) from viral positive-strand RNA. 21-nt siRNA duplexes with 0-nt overhangs were the most abundant 21-nt duplexes, followed by 2-nt overhangs and then 1-nt overhangs 21-nt duplexes in infected P. equestris. Continuous but heterogeneous distribution and secondary structures prediction implied that vsiRNAs originate predominantly by direct Dicer-like enzymes cleavage of imperfect duplexes in the most folded regions of the positive strand of both viruses RNA molecular. Furthermore, we totally predicted 54 target genes by vsiRNAs with psRNATarget server, including disease/stress response-related genes, RNA interference core components, cytoskeleton-related genes, photosynthesis or energy supply related genes. Gene Ontology classification showed that a majority of the predicted targets were related to cellular components and cellular processes and performed a certain function. All target genes were down-regulated with different degree by vsiRNAs as shown by real-time reverse transcription polymerase chain reaction. Taken together, CymMV and ORSV siRNAs played important roles in interplay with P. equestris by down modulating the expression levels of endogenous genes in host plant.

Transient Expression of Homologous Hairpin RNA Interferes with Broad bean wilt virus 2 Infection in Nicotiana benthamiana

  • Yoon, Ju-Yeon;Ryu, Ki Hyun;Choi, Seung-Kook;Choi, Gug Sun;Kwon, Soon Bae;Park, Jin Woo;Palukaitis, Peter
    • Research in Plant Disease
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    • v.18 no.4
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    • pp.268-276
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    • 2012
  • Broad bean wilt virus 2 (BBWV2), genus Fabavirus, subfamily Comovirinae, family Secoviridae, causes damage in many economically important horticultural and ornamental crops. Sequence alignments showed several conserved sequences in 5' non-coding regions (5' NCRs) of RNA 1 and RNA 2 in all BBWV2 strains characterized so far. Based on this observation, we generated a hpRNA construct (pIR-BBWV2) harboring an inverted repeat containing a 210 bp cDNA fragment homologous to 5' NCR portion of BBWV2 RNA 1 to investigate the silencing potential for its ability to interfere with a rapidly replicating BBWV2. Agrobacterium-mediated transient expression of the IR-BBWV2 had a detrimental effect on BBWV2 infection, showing no distinct symptoms in non-inoculated leaves of the agroinfiltrated Nicotiana benthamiana plants. BBWV2 genomic RNAs were not detected by RT-PCR from tissues of both the inoculated leaves and upper leaves of the agroinfiltrated plants. Accumulation of virus-derived small interfering RNAs was detected in the inoculated leaf tissues of N. benthamiana plants elicited by transient expression of IR-BBWV2 indicating that RNA silencing is responsible for the resistance to BBWV2.