• Title, Summary, Keyword: siRNA

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Designing Tyrosinase siRNAs by Multiple Prediction Algorithms and Evaluation of Their Anti-Melanogenic Effects

  • Kwon, Ok-Seon;Kwon, Soo-Jung;Kim, Jin Sang;Lee, Gunbong;Maeng, Han-Joo;Lee, Jeongmi;Hwang, Gwi Seo;Cha, Hyuk-Jin;Chun, Kwang-Hoon
    • Biomolecules & Therapeutics
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    • v.26 no.3
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    • pp.282-289
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    • 2018
  • Melanin is a pigment produced from tyrosine in melanocytes. Although melanin has a protective role against UVB radiation-induced damage, it is also associated with the development of melanoma and darker skin tone. Tyrosinase is a key enzyme in melanin synthesis, which regulates the rate-limiting step during conversion of tyrosine into DOPA and dopaquinone. To develop effective RNA interference therapeutics, we designed a melanin siRNA pool by applying multiple prediction programs to reduce human tyrosinase levels. First, 272 siRNAs passed the target accessibility evaluation using the RNAxs program. Then we selected 34 siRNA sequences with ${\Delta}G{\geq}-34.6kcal/mol$, i-Score value ${\geq}65$, and siRNA scales score ${\leq}30$. siRNAs were designed as 19-bp RNA duplexes with an asymmetric 3' overhang at the 3' end of the antisense strand. We tested if these siRNAs effectively reduced tyrosinase gene expression using qRT-PCR and found that 17 siRNA sequences were more effective than commercially available siRNA. Three siRNAs further tested showed an effective visual color change in MNT-1 human cells without cytotoxic effects, indicating these sequences are anti-melanogenic. Our study revealed that human tyrosinase siRNAs could be efficiently designed using multiple prediction algorithms.

Decreases in $Casz1$ mRNA by an siRNA Complex Do not Alter Blood Pressure in Mice

  • Ji, Su-Min;Shin, Young-Bin;Park, So-Yon;Lee, Hyeon-Ju;Oh, Berm-Seok
    • Genomics & Informatics
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    • v.10 no.1
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    • pp.40-43
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    • 2012
  • Recent genomewide association studies of large samples have identified genes that are associated with blood pressure. The Global Blood Pressure Genetics (Global BPgen) and Cohorts for Heart and Aging Research in Genome Epidemiology (CHARGE) consortiums identified 14 loci that govern blood pressure on a genomewide significance level, one of which is $CASZ1$ confirmed in both Europeans and Asians. $CASZ1$ is a zinc finger transcription factor that controls apoptosis and cell fate and suppresses neuroblastoma tumor growth by reprogramming gene expression, like a tumor suppressor. To validate the function of $CASZ1$ in blood pressure, we decreased $Casz1$ mRNA levels in mice by siRNA. $Casz1$ siRNA reduced mRNA levels by 59% in a mouse cell line. A polyethylenimine-mixed siRNA complex was injected into mouse tail veins, reducing $Casz1$ mRNA expression to 45% in the kidney. However, blood pressure in the treated mice was unaffected, despite a 55% reduction in $Casz1$ mRNA levels in the kidney on multiple siRNA injections daily. Even though $Casz1$ siRNA-treated mice did not experience any significant change in blood pressure, our study demonstrates the value of $in$ $vivo$ siRNA injection in analyzing the function of candidate genes identified by genomewide association studies.

Human Papillomavirus E6 Knockdown Restores Adenovirus Mediated-estrogen Response Element Linked p53 Gene Transfer in HeLa Cells

  • Kajitani, Koji;Ken-Ichi, Honda;Terada, Hiroyuki;Yasui, Tomoyo;Sumi, Toshiyuki;Koyama, Masayasu;Ishiko, Osamu
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.18
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    • pp.8239-8245
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    • 2016
  • The p53 gene is inactivated by the human papillomavirus (HPV) E6 protein in the majority of cervical cancers. Treatment of HeLa S3 cells with siRNA for HPV E6 permitted adenovirus-mediated transduction of a p53 gene linked to an upstream estrogen response element (ERE). Our previous study in non-siRNA treated HHUA cells, which are derived from an endometrial cancer and express estrogen receptor ${\beta}$, showed enhancing effects of an upstream ERE on adenovirus-mediated p53 gene transduction. In HeLa S3 cells treated with siRNA for HPV E6, adenovirus-mediated transduction was enhanced by an upstream ERE linked to a p53 gene carrying a proline variant at codon 72, but not for a p53 gene with arginine variant at codon 72. Expression levels of p53 mRNA and Coxsackie/adenovirus receptor (CAR) mRNA after adenovirus-mediated transfer of an ERE-linked p53 gene (proline variant at codon 72) were higher compared with those after non-ERE-linked p53 gene transfer in siRNA-treated HeLa S3 cells. Western blot analysis showed lower ${\beta}$-tubulin levels and comparatively higher p53/${\beta}$-tubulin or CAR/${\beta}$-tubulin ratios in siRNA-treated HeLa S3 cells after adenovirus-mediated ERE-linked p53 gene (proline variant at codon 72) transfer compared with those in non-siRNA-treated cells. Apoptosis, as measured by annexin V binding, was higher after adenovirus-mediated ERE-linked p53 gene (proline variant at codon 72) transfer compared with that after non-ERE-linked p53 gene transfer in siRNA-treated cells.

Effects of Down-regulation of HDAC6 Expression on Proliferation, Cell Cycling and Migration of Esophageal Squamous Cell Carcinoma Cells and Related Molecular Mechanisms

  • Li, Ning;Tie, Xiao-Jing;Liu, Pei-Jie;Zhang, Yan;Ren, Hong-Zheng;Gao, Xin;Xu, Zhi-Qiao
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.2
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    • pp.685-689
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    • 2013
  • Objective: To study the effects of down-regulation of HDAC6 expression on proliferation, cell cycling and migration of esophageal squamous cell carcinoma (ESCC) cells and related molecular mechanisms. Methods: ESCC cell line EC9706 cells were randomly divided into untreated (with no transfection), control siRNA (transfected with control siRNA) and HDAC6 siRNA (transfected with HDAC6 small interfering RNA) groups. Effects of HDAC6 siRNA interference on expression of HDAC6 mRNA and protein in EC9706 cells were investigated by semi-quantitative RT-PCR, Western blotting and immunocytochemistry methods. Effects of down-regulation of HDAC6 expression on cell proliferation, cell cycle, and cell migration were studied using a CCK-8 kit, flow cytometry and Boyden chambers, respectively. Changes of mRNA and protein expression levels of cell cycle related factor (p21) and cell migration related factor (E-cadherin) were investigated by semi-quantitative RT-PCR and Western blotting methods. Results: After transfection of HDAC6 siRNA, the expression of HDAC6 mRNA and protein in EC9706 cells was significantly downregulated. In the HDAC6 siRNA group, cell proliferation was markedly inhibited, the percentage of cells in G0/G1 phase evidently increased and the percentage of cells in S phase decreased, and the number of migrating cells significantly and obviously decreased. The mRNA and protein expression levels of p21 and E-cadherin in the HDAC6 siRNA group were significantly higher than those in the untreated group and the control siRNA group, respectively. Conclusions: HDAC6 siRNA can effectively downregulate the expression of HDAC6 mRNA and protein in EC9706 cells. Down-regulation of HDAC6 expression can obviously inhibit cell proliferation, arrest cell cycling in the G0/G1 phase and reduce cell migration. The latter two functions may be closely related with the elevation of mRNA and protein expression of p21 and E-cadherin.

siMacro: A Fast and Easy Data Processing Tool for Cell-Based Genomewide siRNA Screens

  • Singh, Nitin Kumar;Seo, Bo Yeun;Vidyasagar, Mathukumalli;White, Michael A.;Kim, Hyun Seok
    • Genomics & Informatics
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    • v.11 no.1
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    • pp.55-57
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    • 2013
  • Growing numbers of studies employ cell line-based systematic short interfering RNA (siRNA) screens to study gene functions and to identify drug targets. As multiple sources of variations that are unique to siRNA screens exist, there is a growing demand for a computational tool that generates normalized values and standardized scores. However, only a few tools have been available so far with limited usability. Here, we present siMacro, a fast and easy-to-use Microsoft Office Excel-based tool with a graphic user interface, designed to process single-condition or two-condition synthetic screen datasets. siMacro normalizes position and batch effects, censors outlier samples, and calculates Z-scores and robust Z-scores, with a spreadsheet output of >120,000 samples in under 1 minute.

Dual-Target Gene Silencing by Using Long, Synthetic siRNA Duplexes without Triggering Antiviral Responses

  • Chang, Chan Il;Kang, Hye Suk;Ban, Changill;Kim, Soyoun;Lee, Dong-ki
    • Molecules and Cells
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    • v.27 no.6
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    • pp.689-695
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    • 2009
  • Chemically synthesized small interfering RNAs (siRNAs) can specifically knock-down expression of target genes via RNA interference (RNAi) pathway. To date, the length of synthetic siRNA duplex has been strictly maintained less than 30 bp, because an early study suggested that double-stranded RNAs (dsRNAs) longer than 30 bp could not trigger specific gene silencing due to the induction of non-specific antiviral interferon responses. Contrary to the current belief, here we show that synthetic dsRNA as long as 38 bp can result in specific target gene silencing without non-specific antiviral responses. Using this longer duplex structure, we have generated dsRNAs, which can simultaneously knock-down expression of two target genes (termed as dual-target siRNAs or dsiRNAs). Our results thus demonstrate the structural flexibility of gene silencing siRNAs, and provide a starting point to construct multifunctional RNA structures. The dsiRNAs could be utilized to develop a novel therapeutic gene silencing strategy against diseases with multiple gene alternations such as viral infection and cancer.

Validation of Gene Silencing Using RNA Interference in Buffalo Granulosa Cells

  • Monga, Rachna;Datta, Tirtha Kumar;Singh, Dheer
    • Asian-Australasian Journal of Animal Sciences
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    • v.24 no.11
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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.

Rationally designed siRNAs without miRNA-like off-target repression

  • Seok, Heeyoung;Jang, Eun-Sook;Chi, Sung Wook
    • BMB Reports
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    • v.49 no.3
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    • pp.135-136
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    • 2016
  • Small interfering RNAs (siRNAs) have been developed to intentionally repress a specific gene expression by directing RNA-induced silencing complex (RISC), mimicking the endogenous gene silencer, microRNAs (miRNAs). Although siRNA is designed to be perfectly complementary to an intended target mRNA, it also suppresses hundreds of off-targets by the way that miRNAs recognize targets. Until now, there is no efficient way to avoid such off-target repression, although the mode of miRNA-like interaction has been proposed. Rationally based on the model called "transitional nucleation" which pre-requires base-pairs from position 2 to the pivot (position 6) with targets, we developed a simple chemical modification which completely eliminates miRNA-like off-target repression (0%), achieved by substituting a nucleotide in pivot with abasic spacers (dSpacer or C3 spacer), which potentially destabilize the transitional nucleation. Furthermore, by alleviating steric hindrance in the complex with Argonaute (Ago), abasic pivot substitution also preserves near-perfect on-target activity (∼80-100%). Abasic pivot substitution offers a general means of harnessing target specificity of siRNAs to experimental and clinical applications where misleading and deleterious phenotypes from off-target repression must be considered.

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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Oncomodulin/Truncated Protamine-Mediated Nogo-66 Receptor Small Interference RNA Delivery Promotes Axon Regeneration in Retinal Ganglion Cells

  • Cui, Zhili;Kang, Jun;Hu, Dan;Zhou, Jian;Wang, Yusheng
    • Molecules and Cells
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    • v.37 no.8
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    • pp.613-619
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    • 2014
  • The optic nerve often suffers regenerative failure after injury, leading to serious visual impairment such as glaucoma. The main inhibitory factors, including Nogo-A, oligodendrocyte myelin glycoprotein, and myelin-associated glycoprotein, exert their inhibitory effects on axonal growth through the same receptor, the Nogo-66 receptor (NgR). Oncomodulin (OM), a calcium-binding protein with a molecular weight of an ~12 kDa, which is secreted from activated macrophages, has been demonstrated to have high and specific affinity for retinal ganglion cells (RGC) and promote greater axonal regeneration than other known polypeptide growth factors. Protamine has been reported to effectively deliver small interference RNA (siRNA) into cells. Accordingly, a fusion protein of OM and truncated protamine (tp) may be used as a vehicle for the delivery of NgR siRNA into RGC for gene therapy. To test this hypothesis, we constructed OM and tp fusion protein (OM/tp) expression vectors. Using the indirect immunofluorescence labeling method, OM/tp fusion proteins were found to have a high affinity for RGC. The gel shift assay showed that the OM/tp fusion proteins retained the capacity to bind to DNA. Using OM/tp fusion proteins as a delivery tool, the siRNA of NgR was effectively transfected into cells and significantly down-regulated NgR expression levels. More importantly, OM/tp-NgR siRNA dramatically promoted axonal growth of RGC compared with the application of OM/tp recombinant protein or NgR siRNA alone in vitro. In addition, OM/tp-NgR siRNA highly elevated intracellular cyclic adenosine monophosphate (cAMP) levels and inhibited activation of the Ras homolog gene family, member A (RhoA). Taken together, our data demonstrated that the recombinant OM/tp fusion proteins retained the functions of both OM and tp, and that OM/tp-NgR siRNA might potentially be used for the treatment of optic nerve injury.