• Biomolecules & Therapeutics
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• 제22권3호
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• pp.207-212
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• 2014

Skin hyperpigmentation is one of the most common skin disorders caused by abnormal melanogenesis. The mechanism and key factors at play are not fully understood. Previous reports have indicated that cystamine (CTM) inhibits melanin synthesis, though its molecular mechanism in melanogenesis remains unclear. In the present study, we investigated the effect of CTM on melanin production using ELISA reader and the expression of proteins involved in melanogenesis by Western blotting, and examined the involvement of transglutaminase-2 (Tgase-2) in SK-MEL-2 human melanoma cells by gene silencing. In the results, CTM dose-dependently suppressed melanin production and dendrite extension in a-MSH-induced melanogenesis of SK-MEL-2 human melanoma cells. CTM also suppressed a-MSH-induced chemotactic migration as well as the expressions of melanogenesis factors TRP-1, TRP-2 and MITF in a-MSH-treated SK-MEL-2 cells. Meanwhile, gene silencing of Tgase-2 suppressed dendrite extension and the expressions of TRP-1 and TRP-2 in a-MSH-treated SK-MEL-2 cells. Overall, these findings suggested that CTM suppresses a-MSH-induced melanogenesis via Tgase-2 inhibition and that therefore, Tgase-2 might be a new target in hyperpigmentation disorder therapy.

• Biomolecules & Therapeutics
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• 제26권5호
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• pp.446-457
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• 2018

Adiponectin, a hormone predominantly originated from adipose tissue, has exhibited potent anti-inflammatory properties. Accumulating evidence suggests that autophagy induction plays a crucial role in anti-inflammatory responses by adiponectin. However, underlying molecular mechanisms are still largely unknown. Association of Bcl-2 with Beclin-1, an autophagy activating protein, prevents autophagy induction. We have previously shown that adiponectin-induced autophagy activation is mediated through inhibition of interaction between Bcl-2 and Beclin-1. In the present study, we examined the molecular mechanisms by which adiponectin modulates association of Bcl-2 and Beclin-1 in macrophages. Herein, we demonstrated that globular adiponectin (gAcrp) induced increase in the expression of AUF1 and ZFP36L1, which act as mRNA destabilizing proteins, both in RAW 264.7 macrophages and primary peritoneal macrophages. In addition, gene silencing of AUF1 and ZFP36L1 caused restoration of decrease in Bcl-2 expression and Bcl-2 mRNA half-life by gAcrp, indicating crucial roles of AUF1 and ZFP36L1 induction in Bcl-2 mRNA destabilization by gAcrp. Moreover, knock-down of AUF1 and ZFP36L1 enhanced interaction of Bcl-2 with Beclin-1, and subsequently prevented gAcrp-induced autophagy activation, suggesting that AUF1 and ZFP36L1 induction mediates gAcrp-induced autophagy activation via Bcl-2 mRNA destabilization. Furthermore, suppressive effects of gAcrp on LPS-stimulated inflammatory mediators expression were prevented by gene silencing of AUF1 and ZFP36L1 in macrophages. Taken together, these results suggest that AUF1 and ZFP36L1 induction critically contributes to autophagy induction by gAcrp and are promising targets for anti-inflammatory responses by gAcrp.

• Journal of Ginseng Research
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• 제40권1호
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• pp.47-54
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• 2016

Background: The roots of Panax ginseng contain noble tetracyclic triterpenoid saponins derived from dammarenediol-II. Dammarene-type ginsenosides are classified into the protopanaxadiol (PPD) and protopanaxatriol (PPT) groups based on their triterpene aglycone structures. Two cytochrome P450 (CYP) genes (CYP716A47 and CYP716A53v2) are critical for the production of PPD and PPT aglycones, respectively. CYP716A53v2 is a protopanaxadiol 6-hydroxylase that catalyzes PPT production from PPD in P. ginseng. Methods: We constructed transgenic P. ginseng lines overexpressing or silencing (via RNA interference) the CYP716A53v2 gene and analyzed changes in their ginsenoside profiles. Result: Overexpression of CYP716A53v2 led to increased accumulation of CYP716A53v2 mRNA in all transgenic roots compared to nontransgenic roots. Conversely, silencing of CYP716A53v2 mRNA in RNAi transgenic roots resulted in reduced CYP716A53v2 transcription. HPLC analysis revealed that transgenic roots overexpressing CYP716A53v2 contained higher levels of PPT-group ginsenosides ($Rg_1$, Re, and Rf) but lower levels of PPD-group ginsenosides (Rb1, Rc, $Rb_2$, and Rd). By contrast, RNAi transgenic roots contained lower levels of PPT-group compounds and higher levels of PPD-group compounds. Conclusion: The production of PPD- and PPT-group ginsenosides can be altered by changing the expression of CYP716A53v2 in transgenic P. ginseng. The biological activities of PPD-group ginsenosides are known to differ from those of the PPT group. Thus, increasing or decreasing the levels of PPT-group ginsenosides in transgenic P. ginseng may yield new medicinal uses for transgenic P. ginseng.

• Reproductive and Developmental Biology
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• 제31권1호
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• pp.21-28
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• 2007

During early embryo development, Oct-4 is an important transcription factor for the early differentiation the present study was first examined methylation status in distal enhancer and promoter region of Oct-4 during mouse pre-implantation embryo development. In oocyte and sperm, high methylation was observed in both distal and proximal of promoter in Oct-4. Following fertilization relatively high methylation level remained until 8-cell stage embryos, but decreased at the morula and blastocyst stage. Specific gene knock down of Oct-4 by siRNA injection into zygote induced higher methylation rates of both distal and proximal region of promoter of Oct-4. These results suggest a functional link between the DNA methylation status of distal and promoter resign in the Oct-4 gene and the gene sequence-specific transcriptional silencing by exogenous siRNA injection during mouse preimplantation embryos.

• BMB Reports
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• 제40권3호
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• pp.404-411
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• 2007

Transformation of cantaloupes with the coat protein (cp) gene of papaya ringspot virus type W (PRSV-W), Thai isolate, was used to introduce virus resistance. Binary vectors containing either the full length coat protein coding region under control of the 35S CaMV promoter(pSA1175), or the inverted-repeat of a coat protein coding region (pSA1304), were constructed and used for Agrobacteriummediated transformation of cotyledonary explants of the cantaloupe cultivar Sun Lady. Four independent transgenic lines were obtained using pSA1304 and one using pSA1175. Integration of the PRSV-W cp gene into the genome of these transgenic lines was verified by PCR amplification, GUS assays and Southern blot hybridization. In vitro inoculation of these lines with PRSV-W revealed that whereas the line containing pSA1175 remained sensitive, the four lines containing pSA1304 were resistant. The presence of small RNA species, presumably siRNA, corresponding to regions of the viral cp gene in transgenic lines resistant to PRSV-W supports the involvement of post-transcriptional gene silencing in the establishment of resistance.

• Asian Pacific Journal of Cancer Prevention
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• 제13권5호
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• pp.1823-1827
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• 2012

The gene encoding the Nin one binding (NOB1) protein which plays an essential role in protein degradation has been investigated for possible tumor promoting functions. The present study was focused on NOB1 as a possible therapeutic target for breast cancer treatment. Lentivirus mediated NOB1 siRNA transfection was used to silence the NOB1 gene in two established breast cancer cell lines, MCF-7 and MDA-MB-231, successful transfection being confirmed by fluorescence imaging. NOB1 deletion caused significant decline in cell proliferation was observed in both cell lines as investigated by MTT assay. Furthermore the number and size of the colonies formed were also significantly reduced in the absence of NOB1. Moreover NOB1 gene knockdown arrested the cell cycle and inhibited cell cycle related protein expression. Collectively these results indicate that NOB1 plays an essential role in breast cancer cell proliferation and its gene expression could be a therapeutic target.

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

• Genomics & Informatics
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• 제5권3호
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• pp.107-112
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• 2007

MicroRNAs (miRNAs) are known to negatively control protein-coding genes by binding to messenger RNA (mRNA) in the cytoplasm. In innate immunity, the role of miRNA gene silencing is largely unknown. In this study, we performed microarray-based experiments using lipopolysaccharide (LPS)-stimulated macrophages derived from wild-type, MyD88 knockout (KO), TRIF KO, and MyD88/TRIF double KO mice. We employed a statistical approach to determine the importance of the commonality and specificity of miRNA binding sites among groups of temporally co-regulated genes. We demonstrate that both commonality and specificity are irrelevant to define a priori groups of co-down regulated genes. In addition, analyzing the various experimental conditions, we suggest that miRNA regulation may not only be a late-phase process (after transcription) but can also occur even early (1h) after stimulation in knockout conditions. This further indicates the existence of dynamic interactions between miRNA and signaling molecules/transcription factor regulation; this is another proof for the need of shifting from a 'hard-wired' paradigm of gene regulation to a dynamical one in which the gene co-regulation is established on a case-by-case basis.

• Bulletin of the Korean Chemical Society
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• 제33권5호
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• pp.1536-1540
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• 2012

Chicken primordial germ cells (cPGCs) are founder germ cells in embryonic stage of development that eventually give rise to sperms or oocytes. Currently cPGCs are only known cells enabling germline transmission in chicken and their cultivation protocols were recently established. Although genome modifications of chickens are now theoretically possible using cPGCs, there are still several hurdles to overcome to practically use cPGCs as mediators for chicken transgenesis. First, efficiency of gene delivery into cPGCs remains low with current methods. Second, there aregene silencing mechanisms against the expression of foreign genes in cPGCs. In this study, we successfully increased the efficiency of gene delivery in cPGCs by taking advantage of the TTAA-specific $piggybac$ transposon system. Moreover, a pipette-type electroporator significantly enhanced transfection efficiency up to 5-fold compared withcuvette-type methods. Taken together, the technological advances in our study will provide practical benefits for the application to fulfill genetic modifications of chicken genome.

• Asian Pacific Journal of Cancer Prevention
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• 제13권6호
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• pp.2445-2452
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• 2012

RNA interference has created a breakthrough in gene silencing technology and there is now much debate on the successful usage of RNAi based methods in treating a number of debilitating diseases. Cancer is often regarded as a result of mutations in genomic DNA resulting in faulty gene expression. The occurrence of cancer can also be influenced by epigenetic irregularities in the chromatin structure which leads to alterations and mutations in DNA resulting in cancer cell formation. A number of therapeutic approaches have been put forth to treat cancer. Anti cancer therapy often involves chemotherapy targeting all the cells in common, whereby both cancer cells as well as normal cells get affected. Hence RNAi technology has potential to be a better therapeutic agent as it is possible to deactivate molecular targets like specific mutant genes. This review highlights the successful use of RNAi inducers against different types of cancer, thereby paving the way for specific therapeutic medicines.