담배가루이 RNAi 발현토마토에서 EPG를 이용한 담배가루이 섭식행동 비교

민지현,강찬영,정유빈,김정희,변일현,장현주,조민규,유용만,윤영남 한국응용곤충학회 2015 한국응용곤충학회 학술대회논문집 Vol.2015 No.10

담배가루이(Bemisia tabaci)는 토마토에 토마토황화잎말림바이러스병 (TYLCV)을 매개하는 중요해충이다. 본 연구에서는 담배가루이의 RNAi를 토마토 식물에 발현시키고, RNAi 발현토마토를 담배가루이 기주식물로 제공하여 EPG(Electrical Penetration Graph)를 사용하여 섭식행동을 분석하였다. EPG는 파종 후 4주째 되는 토마토에 담배가루이 RNAi를 접종삽입한 후 10일째 되는 RNAi 발현토마토를 이용하였으며, 담배가루이는 성충으로 우화 후 2-3일 되는 것을 사용하였다. 비교구 토마토는 무처리와 TRV RNA2, att site가 포함된 TRV RNA2가 삽입접종된 것으로, 3시간 동안 기록하였다. RNAi 발현토마토는 wh0001, wh0002, wh0030를 사용하였으며, 기록된 EPG 파형 분석을 통해 총 탐침횟수와 기간, 기주의 물관부, 체관부 섭식행동 등을 비교분석하였다. 그 결과, 비교구 대비 모든 RNAi 발현토마토에서 체관부 섭식은 낮게 나타났다. RNAi 발현토마토 wh0001은 첫 탐침까지 약 10배정도 빨랐지만, 기주에 구침을 넣지 않은 시간이 약 1.5배 증가하였다. RNAi 발현토마토 wh0002의 경우에는 첫 탐침까지 약 5배정도 오래 걸렸고, 총 탐침횟수도 가장 적었다. RNAi 발현토마토 wh0030은 물관부를 거의 섭식하지 않았으며, 구침을 기주에 접촉하지 않은 시간 또한 길었다. 따라서 모든 RNAi 발현토마토에서 비교구 토마토보다 섭식시간이 짧아지는 경향을 보여주고 있어, 현재 선발된 3종류의 RNAi는 섭식에 관련된 유전자를 방해하는 것으로 추정된다.

uPAR 및 uPA의 RNAi 형질감염이 타액선 종양세포주에 미치는 영향

오민구,이종헌 대한구강악안면병리학회 2020 대한구강악안면병리학회지 Vol.44 No.1

Despite existing chemotherapy and surgical resection strategies, salivary gland adenocarcinoma(AdCa NOS) is one of the major causes of mortality among malignant salivary gland tumors. New therapeutic measure are needed to improve the outcome for patients with AdCa. Overexpression of urokinase-type plasminogen activator receptor/urokinase-type plasminogen activator(uPAR-uPA) has been implicated in progression and metastasis of oral cancer. RNA interference(RNAi) which has emerged as an effective method to target specific genes for silencing has provided new opportunities for cancer therapy. But there has been rarely reported using RNAi-uPAR/uPA transfection in salivary gland AdCa. The purpose of this study were to examine the specific inhibition of uPAR/uPA mRNA and protein expression by RNAi transfection of uPAR/uPA through RT-PCR and Immunoslot blot, and to study tumor cell proliferation activity, adhesion, invasion and migration of SGT cell line in vitro compared to the controls. In adhesion assay, cells transfected with RNAi-uPAR/uPA inhibited markedly adhesion to vitronectin compared to parental cells. Angiogenic assays revealed a significant decrease in the angiogenic potential of SGT cells downregulated by both uPAR and uPA. In migration assay, suppressing uPAR and uPA inhibited the capacity of the cells to migrate compared to parental cells. In invasion assay, cells transfected with RNAi-uPAR/uPA showed the maximum decrease in invasion when compared to all other treatment conditions. RNAi expressing plasmids efficiently downregulated mRNA and protein expression of uPAR and uPA. Cell cycle analysis showed that the simultaneous downregulation of uPAR and uPA caused the accumulation of cells in the sub-G0/G1 phase in SGT cells. Immunoslot blot analysis revealed that downregulation of uPAR and uPA caused the prominent activation of caspase 8. It suggested that the RNAi targeting of the uPAR/uPA system could have a therapeutic potentiality for malignant salivary gland tumors.

RNAi: 생명현상 본질 규명의 강력한 신(新)기술

정동길,조영숙,이원길 대한임상미생물학회 2007 Annals of clinical microbiology Vol.10 No.1

RNA interference (RNAi) is a gene-silencing technology by which small double-stranded RNAs are used to target the degradation of RNA with complementary sequence. RNAi is found in a wide variety of organisms Caenorhabditis elegans, insects, plants, microorganisms and animals). With RNAi, we have harnessed the gene function to be explored, revolutionized our ability to perform large-scale genetic screens, and even therapeutic potential.

Group I chitin deacetylases are essential for higher order organization of chitin fibers in beetle cuticle

Noh, Mi Young,Muthukrishnan, Subbaratnam,Kramer, Karl J.,Arakane, Yasuyuki American Society for Biochemistry and Molecular Bi 2018 The Journal of biological chemistry Vol.293 No.18

<P>Roles in the organization of the cuticle (exoskeleton) of two chitin deacetylases (CDAs) belonging to group I, TcCDA1 and TcCDA2, as well as two alternatively spliced forms of the latter, TcCDA2a and TcCDA2b, from the red flour beetle, Tribolium castaneum, were examined in different body parts using transmission EM and RNAi. Even though all TcCDAs are co-expressed in cuticle-forming cells from the hardened forewing (elytron) and ventral abdomen, as well as in the softer hindwing and dorsal abdomen, there are significant differences in the tissue specificity of expression of the alternatively spliced transcripts. Loss of either TcCDA1 or TcCDA2 protein by RNAi causes abnormalities in organization of chitinous horizontal laminae and vertical pore canals in all regions of the procuticle of both the hard and soft cuticles. Simultaneous RNAi for TcCDA1 and TcCDA2 produces the most serious abnormalities. RNAi of either TcCDA2a or TcCDA2b affects cuticle integrity to some extent. Following RNAi, there is accumulation of smaller disorganized fibers in both the horizontal laminae and pore canals, indicating that TcCDAs play a critical role in elongation/organization of smaller nanofibers into longer fibers, which is essential for structural integrity of both hard/thick and soft/thin cuticles. Immunolocalization of TcCDA1 and TcCDA2 proteins and effects of RNAi on their accumulation indicate that these two proteins function in concert exclusively in the assembly zone in a step involving the higher order organization of the procuticle.</P>

Double-stranded RNA in exosomes: Potential systemic RNA interference pathway in the Colorado potato beetle, Leptinotarsa decemlineata

Yoon June-Sun,Kim Kyungbo,Palli Subba Reddy 한국응용곤충학회 2020 Journal of Asia-Pacific Entomology Vol.23 No.4

Despite extensive research during the past decade elucidating the mechanism of RNA interference (RNAi) in insects, it is not clear how ingested or injected double-stranded RNA (dsRNA) triggers RNAi response in the whole body or even its progeny, which is referred to as systemic RNAi. In the present study, we aim to understand how the dsRNA delivered into cells causes systemic RNAi using Colorado potato beetle cells (Lepd-SL1). We first tested if dsRNA treatment induces systemic RNAi in Lepd-SL1 cells. Exposure of a new batch of Lepd-SL1 cells to the conditioned medium where Lepd-SL1 cells treated with dsRNA targeting inhibitor of apoptosis were grown for 6 h induced apoptosis in these new batch of cells. We hypothesized the exosomes in the conditioned medium are responsible for RNAi-inducing effect. To test this hypothesis, we isolated exosomes from the conditioned medium from Lepd-SL1 cells that had been treated with dsGFP (dsRNA targeting gene coding for green fluorescent protein) or dsLuc (dsRNA targeting gene coding for the luciferase) were grown. RNA present in the purified exosomes was analyzed to check if long dsRNA or siRNA is accumulated in them. The results from the electrophoretic mobility shift assay clearly showed that the long dsRNAs are present in the exosomes. By knockdown of candidate genes involved in endosome recycling and generation pathways, we found that Rab4 and Rab35 are involved in exosome production and transport.

Characterization of the small hive beetle transcriptome focused on the insecticide target site and RNA interference genes

Kim, Kyungmun,Kim, Sang Hyeon,Yoon, Kyungjae Andrew,Cho, Yun Sang,Yoo, Mi-Sun,Lee, Si Hyeock 한국응용곤충학회 2018 Journal of Asia-Pacific Entomology Vol.2 No.1

<P><B>Abstract</B></P> <P>The small hive beetle (SHB), <I>Aethina tumida</I>, is an invasive pest species in most Northern Hemisphere countries, including Korea. SHB causes serious damage to apiaries by destroying overwintering honey bee colonies. To obtain basic information for efficient management of SHB, genes encoding conventional insecticide targets, specifically the voltage-sensitive sodium channel α-subunit (VSSC) and acetylcholinesterase (AChE), and RNA interference (RNAi)-related components were annotated and characterized following analysis of transcriptomes of adults and larvae. A single VSSC gene was identified but no apparent mutations associated with pyrethroid resistance were detected. Genes encoding two AChEs (AtAChE1 and AtAChE2) were identified from the SHB transcriptome. No apparent mutations associated with resistance to organophosphorus and carbamate insecticides were identified in the AtAChE1 gene, whereas the S238G mutation, originally identified from the Colorado potato beetle, was detected in the AtAChE2 gene. Native polyacrylamide electrophoresis in conjunction with western blotting revealed that AtAChE1 was the main catalytic enzyme and therefore a toxicologically more relevant target. AtAChE1 was determined to exist in both membrane-anchored and soluble forms. The main components of RNA interference (RNAi) were identified, suggesting that RNAi is likely functional in SHB and an RNAi-based approach is a feasible alternative control measure.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Transcriptome of small hive beetle (<I>Aethina tumida</I>) was analyzed and annotated. </LI> <LI> Insecticide target (<I>Atvssc</I> and <I>Atace</I>) and RNAi-related genes were characterized. </LI> <LI> Of two <I>Atace</I> genes (<I>Atace1</I> and <I>Atace2</I>)<I>, Atace1</I> was determined to encode the major enzyme. </LI> <LI> No apparent mutations associated with insecticide resistance were detected in either <I>Atvssc</I> or <I>Atace1</I>. </LI> <LI> Two additional RNAi-related components (Hen1 and loqs) were newly identified. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

RNAi-based functional genomics in Tribolium castaneum and possible application for controlling insect pests

노미영,Richard W. BEEMAN,Yasuyuki Arakane 한국곤충학회 2012 Entomological Research Vol.42 No.1

RNA interference or RNAi has been observed in eukaryotes and plays a critical role not only in messenger RNA degradation but also in fine-tuning of gene activation, transcription and translation. In insects, double-stranded RNA (dsRNA)-mediated gene silencing is a simple and powerful reverse genetics tool for analyzing gene functions in both model and non-model organisms. The red flour beetle, Tribolium castaneum, is an excellent genetic model for pest insect species. Since the completion of the genome sequence of T. castaneum, numerous studies of gene function have been reported. Here we review recent RNAi-based functional genomics research and systemic RNAi in T. castaneum, and we discuss potential applications of RNAi technology to pest insect control.

Downstream Genes Regulated by Bcl2l10 RNAi in the Mouse Oocytes

김은아,김경화,이현서,이수연,김은영,서유미,배지현,이경아 한국발생생물학회 2011 발생과 생식 Vol.15 No.1

Previously, we have shown that Bcl2l10 as a member of Bcl-2 family, key regulators of the apoptotic process, is dominantly expressed in oocytes of ovary but several member of the Bcl-2 family are not expressed in oocytes. Recent our studies had been processed about roles and regulatory mechanisms of Bcl2l10 in oocytes. Microinjection of Bcl2l10 RNAi into the cytoplasm of germinal vesicle oocytes resulted in metaphase I (MI) arrest and exhibited abnormalities in their spindles and chromosome configurations (Yoon et al., 2009). The present study was conducted to elucidate the downstream genes regulated by Bcl2l10 and signaling networks in Bcl2l10 RNAi microinjected oocytes by using microarray analysis. Surprisingly, we found that a large proportion of genes regulated by Bcl2l10 RNAi were involved in the cell cycle and actin skeletal system regulation as important upstream genes of Bcl2l10. Among the transcripts with highly significant fold change of genes changed by more than 2-fold, Tpx2 and Cep192 are respectively 16.1- and 8.2-fold down regulated by Bcl2l10 RNAi. Tpx2 and Cep192 are known as cofactors that control Aurora A kinase activity and localization. Therefore, Bcl2l10 may have important roles during oocyte meiosis as functional upstream regulator of Tpx2 and Cep192.

Downstream Genes Regulated by Bcl2l10 RNAi in the Mouse Oocytes

Kim Eun-Ah,Kim Kyeoung-Hwa,Lee Hyun-Seo,Lee Su-Yeon,Kim Eun-Young,Seo You-Mi,Bae Jee-Hyeon,Lee Kyung-Ah 한국발생생물학회 2011 발생과 생식 Vol.15 No.1

Previously, we have shown that Bcl2l10 as a member of Bcl-2 family, key regulators of the apoptotic process, is dominantly expressed in oocytes of ovary but several member of the Bcl-2 family are not expressed in oocytes. Recent our studies had been processed about roles and regulatory mechanisms of Bcl2l10 in oocytes. Microinjection of Bcl2l10 RNAi into the cytoplasm of germinal vesicle oocytes resulted in metaphase I (MI) arrest and exhibited abnormalities in their spindles and chromosome configurations (Yoon et al., 2009). The present study was conducted to elucidate the downstream genes regulated by Bcl2l10 and signaling networks in Bcl2l10 RNAi microinjected oocytes by using microarray analysis. Surprisingly, we found that a large proportion of genes regulated by Bcl2l10 RNAi were involved in the cell cycle and actin skeletal system regulation as important upstream genes of Bcl2l10. Among the transcripts with highly significant fold changes more than 2-fold, Tpx2 and Cep192 are 16.1- and 8.2-fold down regulated respectively by Bcl2l10 RNAi. Tpx2 and Cep192 are known as cofactors that control Aurora A kinase activity and localization. Therefore, we concluded that Bcl2l10 may have important roles during oocyte meiosis as functional upstream regulator of Tpx2 and Cep192.

Utility of RNAi-mediated prnp gene silencing in neuroblastoma cells permanently infected by prions: Potentials and limitations

Kim, Y.,Han, B.,Titlow, W.,Mays, C.E.,Kwon, M.,Ryou, C. Elsevier/North-Holland 2009 Antiviral research Vol.84 No.2

Prion diseases are incurable, transmissible neurodegenerative disorders in humans and animals. Because the disease-associated isoform of prion protein, PrP<SUP>Sc</SUP>, is conformationally converted from cellular prion protein, PrP<SUP>C</SUP>, knockdown of PrP<SUP>C</SUP> expression by RNA interference (RNAi) implicates therapy for prion diseases. In this study, introduction of small interfering (si) and small hairpin (sh) RNAs targeting the prion protein gene (prnp) transcripts triggered specific gene silencing and reduced the PrP<SUP>C</SUP> level in both prion-free and -infected neuroblastoma cell lines. Furthermore, this approach suppressed PrP<SUP>Sc</SUP> formation and ultimately eliminated PrP<SUP>Sc</SUP> from prion-infected cell lines. However, prolonged culture of cured cells resulted in reappearance of PrP<SUP>Sc</SUP> in the cell population, presumably by de novo PrP<SUP>Sc</SUP> formation from residual PrP<SUP>C</SUP> uncontrolled by RNAi and PrP<SUP>Sc</SUP> remained under the detection limit. Protein misfolding cyclic amplification assays further confirmed that lysate of cured cells was sufficient to support PrP<SUP>Sc</SUP> propagation. Our data not only suggest a potential treatment option but also implicate a caveat for using an RNAi approach for prion diseases. These findings provide critical information required to advance RNAi-based prevention and therapy for prion diseases of humans and animals.