• Bioinformatics and Biosystems
• /
• 제2권1호
• /
• pp.17-23
• /
• 2007

Short interfering RNA(siRNA)는 특별한 gene의 발현을 막는데 사용될 수 있고 그 gene의 기능과 치료의 적용에 많은 가능성을 가지고 있지만, 효과적인 siRNA를 디자인하는 방법은 아직까지 명확하지 않다. 효과적인 siRNA는 서열적인 경향을 가지고 있는데 낮은 G/C content, Sense strand의 3' 끝에 적은 안정성과 1번 위치에는 G/C, 19번 위치에는 A/U의 존재 여부를 들 수 있다. 이러한 특성 말고도 최근에는 mRNA의 2차구조가 RNAi 작용에 중요한 역할을 하게 되는데 복잡한 구조(hairpin, multi loop)를 가지고 수소결합을 많이 하여 안정한 상태에 있는 부분은 siRNA의 기능을 크게 줄어들게 한다. 또한, siRNA가 특정한 mRNA에 작동하도록 BLAST 검색을 하여 부작용의 가능성을 배제한다.

• Asian Pacific Journal of Cancer Prevention
• /
• 제15권12호
• /
• pp.4773-4780
• /
• 2014

Background: To investigate the effects of small interference RNA (siRNA) targeting BCR/ABL mRNA on proliferation and apoptosis in the K562 human chronic myeloid leukemia (CML) cell line and to provide a theoretical rationale and experimental evidence for its potential clinical application for anti-CML treatment. Materials and Methods: The gene sequence for BCR/ABL mRNA was found from the GeneBank. The target gene site on the BCR/ABL mRNA were selected according to Max-Planck-Institute (MPI) and rational siRNA design rules, the secondary structure of the candidate targeted mRNA was predicted, the relevant thermodynamic parameters were analyzed, and the targeted gene sequences were compared with BLAST to eliminate any sequences with significant homology. Inhibition of proliferation was evaluated by MTT assay and colony-formation inhibiting test. Apoptosis was determined by flow cytometry (FCM) and the morphology of apoptotic cells was identified by Giemsa-Wright staining. Western blotting was used to analyze the expression of BCR/ABL fusion protein in K562 cells after siRNA treatment. Results: The mRNA local secondary structure calculated by RNA structure software, and the optimal design of specific siRNA were contributed by bioinformatics rules. Five sequences of BCR/ABL siRNAs were designed and synthesized in vitro. Three sequences, siRNA1384, siRNA1276 and siRNA1786, which showed the most effective inhibition of K562 cell growth, were identified among the five candidate siRNAs, with a cell proliferative inhibitory rate nearly 50% after exposure to 12.5nmol/L~50nmol/L siRNA1384 for 24,48 and 72 hours. The 50% inhibitory concentrations ($IC_{50}$) of siRNA1384, siRNA1276 and siRNA1786 for 24hours were 46.6 nmol/L, 59.3 nmol/L and 62.6 nmol/L, respectively, and 65.668 nmol/L, 76.6 nmol/L, 74.4 nmol/L for 72 hours. The colony-formation inhibiting test also indicated that, compared with control, cell growth of siRNA treated group was inhibited. FCM results showed that the rate of cell apoptosis increased 24 hours after transfecting siRNA. The results of annexinV/PI staining indicated that the rate of apoptosis imcreased (1.53%, 15.3%, 64.5%, 57.5% and 21.5%) following treamtne with siRNAs (siRNA34, siRNA372, siRNA1384, siRNA1276 and siRNA1786). Morphological analysis showed td typical morphologic changes of apoptosis such as shrunken, fragmentation nucleus as well as "apoptotic bodies" after K562 cell exposure to siRNA. Western blot analysis showed that BCR/ABL protein was reduced sharply after a single dose of 50nmol/L siRNA transfection. Conclusions: Proliferation of K562 cells was remarkbly inhibited by siRNAs (siRNA1384, siRNA1276 and siRNA1786) in a concentration-dependent manner in vitro, with effective induction of apoptosis at a concentration of 50 nmol/L. One anti-leukemia mechanism in K562 cells appeared that BCR/ABL targeted protein was highly down-regulated. The siRNAs (siRNA1384, siRNA1276 and siRNA1786) may prove valuable in the treatment of CML.

• Asian Pacific Journal of Cancer Prevention
• /
• 제13권12호
• /
• pp.5975-5979
• /
• 2012

Background: To investigate the influence of telomerase activity, apoptosis, radiosensitivity of cervical cancer after siRNA-mediated knockdown of telomerase RNA and evaluate in vivo growth with gene interference. Methods: We studied siRNA-targeting-telomerase RNA transfection into the Hela cell line. Expression of hTERC mRNA was detected by RT-PCR and telomerase activity was measured by the TRAP assay. Growth inhibition was determined by MTT assay and radiosensitivity of the cervical cancer cells was examined by colony formation assay. In addtion, effects of hTERC inhibition in vivo were studied by injection of siRNA-transfected Hela cells into nude mice. Results: The hTERC siRNA effectively downregulated the expression of hTERC mRNA and also reduced the telomerase activity to 30% of the untreated control vlaue. The viability of hTERC siRNA transfected Hela cells was reduced by 44.7% after transfection. After radiation treatment, the radiosensitivity of Hela cells with hTERC knockdown was increased. In vivo, the tumors developing from the hTERC siRNA-transfected cells were of reduced size, indicating that the hTERT siRNA also depressed the tumorigenic potential of the Hela cells. Conclusions: Our results supported the concept of siRNA-mediated inhibition of telomerase mRNA which could inhibit the expression of hTERC and telomerase activity. Furthermore, radiosensitivity was upregulated after knockdown the hTERC in vivo and in vitro.

• BMB Reports
• /
• 제48권3호
• /
• pp.147-152
• /
• 2015

Small interfering RNA (siRNA) functions through pairing with specific mRNA sequences and results in the mRNA's degradation. It is a potential therapeutic approach for many diseases caused by altered gene expression. The delivery of siRNA is still a major problem due to its rapid degradation in the circulation. Various strategies have been proposed to help with the cellular uptake of siRNA and short or small hairpin RNA (shRNA). Here, we reviewed recently published data regarding local applications of siRNA. Compared with systemic delivery methods, local delivery of siRNA/shRNA has many advantages, such as targeting the specific tissues or organs, mimicking a gene knockout effect, or developing certain diseases models. The eye, brain, and tumor tissues are 'hot' target tissues/organs for local siRNA delivery. The siRNA can be delivered locally, in naked form, with chemical modifications, or in formulations with viral or non-viral vectors, such as liposomes and nanoparticles. This review provides a comprehensive overview of RNAi local administration and potential future applications in clinical treatment.

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

• Molecules and Cells
• /
• 제24권2호
• /
• pp.247-254
• /
• 2007

Improvement in the pharmacokinetic properties of short interfering RNAs (siRNAs) is a prerequisite for the therapeutic application of RNA interference technology. When injected into mice as unmodified siRNAs complexed to DOTAP/Chol-based cationic liposomes, all 12 tested siRNA duplexes caused a strong induction of cytokines including interferon ${\alpha}$, indicating that the immune activation by siRNA duplexes is independent of sequence context. When modified by various combinations of 2'-OMe, 2'-F, and phosphorothioate substitutions, introduction of as little as three 2'-OMe substitutions into the sense strand was sufficient to suppress immune activation by siRNA duplexes, whereas the same modifications were much less efficient at inhibiting the immune response of single stranded siRNAs. It is unlikely that Toll-like receptor 3 (TLR3) signaling is involved in immune stimulation by siRNA/liposome complexes since potent immune activation by ds siRNAs was induced in TLR3 knockout mice. Together, our results indicate that chemical modification of siRNA provides an effective means to avoid unwanted immune activation by therapeutic siRNAs. This improvement in the in vivo properties of siRNAs should greatly facilitate successful development of siRNA therapeutics.

• Tissue Engineering and Regenerative Medicine
• /
• 제15권6호
• /
• pp.711-719
• /
• 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.

• Journal of Pharmaceutical Investigation
• /
• 제35권5호
• /
• pp.343-348
• /
• 2005

Recently, siRNA has been emerging as new therapeutic agents for various diseases such as cancers and infectious diseases. However, the evaluation for delivery systems for siRNA has not been fully done. In this study, we designed and delivered siRNA of oncogenic E6 and E7 proteins to several cell lines and tested the delivery efficiencies of various cationic nonviral delivery vectors. Of cationic delivery systems tested in this study, lipid-based Lipofectamine revealed higher delivery efficiency of siRNA to cervical cancer cell line, SiHa, compared to other delivery systems. Notably, the polyethylenimine, which showed the comparable delivery efficiencies in plasmid DNA, did not show significant delivery of siRNA in cervical cancer cells. These results indicate that the mechanisms involved in siRNA delivery might be different from those in plasmid DNA delivery, and that cationic lipid-based delivery vehicles deliver siRNA with higher efficiency to intracellular target sites.

• 생명과학회지
• /
• 제29권6호
• /
• pp.653-661
• /
• 2019

본 연구는 siRNA 기반 치료제등의 핵산치료제 개발에 있어서 필수적인 약물의 생체내 흡수, 분포, 대사, 배설에 대한 동태의 확인을 위해 stem-loop RT-qPCR 법을 이용하여 보다 더 정확한 시험법을 확립하고자 하였다. siRNA에 특이적인 primer와 probe를 선별하여 siRNA 정량검출 시험법을 최적화하였다. siRNA 표준시료를 이용하여 최적화된 시험법을 적용하였을 때 siRNA 표준시료에 대한 Cp 값(y)간의 선형분석 결과, 기울기 평균 -3.3, 결정계수 $R^2$>0.99으로 확인되어 siRNA 표준시료와 Cp 값 간의 회귀성이 매우 높아 정량 분석이 가능한 시험법임을 확인하였고, 같은 표준시료를 이용한 stem-loop RT-qPCR의 검출한계(LOD)는 10 fM, 최소정량한계(LLOQ)는 100 fM이었다. 확립된 시험법의 신뢰성을 확인하기 위해 시험자를 다르게 하고, 시험법을 3회 반복하여 각각 진행한 결과, siRNA 표준시료에 대한 Cp 값(y)간의 선형분석 결과 기울기와 결정계수 $R^2$의 재현성(slope ${\pm}-3.2$, 결정계수 $R^2$>0.99)을 확인하였고, 표준 곡선으로부터 환산된 siRNA 표준시료의 회수율(recovery ${\pm}20%$)과 완건성이 우수함을 확인하였다. 확립된 stem-loop RT-qPCR을 생체내 존재하는 약물 검증에 적용할 수 있는지 확인하기 위하여 시험동물에 siRNA를 주입 후 시간별 혈액을 채취하여 확립된 시험법으로 시험을 진행하였고 약물 동태학적 분석을 통해 siRNA치료제의 혈액내의 안정성을 확인하였다. 따라서 본연구에서 개발된 stem-loop RT-qPCR 분석법은 정확성, 정밀성 및 민감도가 높은 분석법으로 핵산치료제 개발 연구의 다양한 생체시료 분석 연구에 적용할 수 있을 것으로 기대한다.

• Journal of Veterinary Science
• /
• 제23권2호
• /
• pp.24.1-24.10
• /
• 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.