• Title/Summary/Keyword: rRT-PCR

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MicroRNA Analysis in Normal Human Oral Keratinocytes and YD-38 Human Oral Cancer Cells

  • Kim, Hye-Ryun;Park, Eu-Teum;Cho, Kwang-Hee;Kim, Do-Kyung
    • International Journal of Oral Biology
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    • v.36 no.4
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    • pp.179-185
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    • 2011
  • MicroRNAs (miRNAs) are small non-coding RNAs that mediate gene expression at the post-transcriptional level by degrading or repressing targeted mRNAs. These molecules are about 21-25 nucleotides in length and exert their effects by binding to partially complementary sites in mRNAs, predominantly in the 3'-untranslated region (3'-UTR). Recent evidence has demonstrated that miRNAs can function as oncogenes or tumor suppressors through the modulation of multiple oncogenic cellular processes in cancer development, including initiation, cell proliferation, apoptosis, invasion and metastasis. In our present study, we examined the expression profile of miRNAs related to oral cancer cell growth inhibition using normal human oral keratinocytes (NHOK) and YD-38 human oral cancer cells. By miRNA microassay analysis, 40 and 31 miRNAs among the 1,769 examined were found to be up- and down-regulated in YD-38 cells compared with NHOK cells, respectively. Using qRT-PCR analysis, the expression levels of miR-30a and miR-1246 were found to be increased in YD-38 cells compared with NHOK cells, whereas miR-203 and miR-125a were observed to be decreased. Importantly, the overexpression of miR-203 and miR-125a significantly inhibited the growth of YD-38 cells. This finding and the microarray data indicate the involvement of specific miRNAs in the development and progression of oral cancer.

MicroRNA Expression Profiling in Cell and Mouse Models of Fabry Disease to Identify Biomarkers for Fabry Disease Nephropathy (파브리병의 바이오마커 발굴을 위한 파브리 마우스와 세포모델에서의 microRNA 발현 분석)

  • Jung, Namhee;Park, Saeyoung;Jeon, Yeo Jin;Choi, Yoonyoung;Jung, Sung-Chul
    • Journal of The Korean Society of Inherited Metabolic disease
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    • v.15 no.3
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    • pp.127-137
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    • 2015
  • Purpose: The main aim of this study was to compare and analyze expression profiles of microRNAs (miRNAs) to establish miRNA signature of Fabry nephropathy related epithelial mesenchymal transition (EMT). Methods: Expression profiles of miRNAs in kidney tissue samples and cell lines from normal and Fabry disease mouse model were examined by miRNA expression microarray analysis followed by quantitative real-time polymerase chain reaction analysis (qRT-PCR). Results: In the miRNA expression microarray analysis of Fabry mouse kidney tissues compared to wild type mouse, 5 and 3 miRNAs among 1,247 miRNAs examined were up- and down-regulated, respectively. Among them, miR-149-5p was down-regulated about 2-fold in Fabry kidney samples. The down-regulations of miR-149-5p were observed in kidney tissues of under 35 week-old-Fabry mice. However, this down-regulation was not observed in kidney tissues of 42 week-old Fabry mice. In SV40 MES 13 cells, mouse mesangial cells, treated with globotriaosylsphingosine (lyso-Gb3), miR-149-5p was also downregulated. The down-regulation of miR-149-5p induced up-regulation of its target genes related to EMT. Conclusion: The miRNA expression array and qRT-PCR results show that miR-149-5p expression was decreased in kidney tissues of Fabry mice compared to wild type mice under 35 weeks of age. Along with the observation of miR-149-5p expression in Fabry disease cell models, these results indicate that the down-regulated miR-149-5p were related to the biological response of mesangial cells to lyso-Gb3 and also have influence to the transcriptional up-regulation of its target genes. These results suggest miR-149-5p might play important roles in the Fabry nephropathy.

Anti-Oxidative and Anti-Inflammatory Effects of Green Tea Mixture and Dietary Fiber on Liver of High Fat Diet-Induced Obese Rats (고지방 식이로 유도된 비만 흰쥐의 간에서 식이섬유와 녹차혼합물의 항산화 및 항염증 효과)

  • Kim, Jong-Dai;Lee, Byung-Il;Jeon, Yun-Hui;Bak, Jong-Phil;Jin, Hai-Lan;Lim, Beong-Ou
    • Korean Journal of Medicinal Crop Science
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    • v.18 no.4
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    • pp.224-230
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    • 2010
  • This study was conducted to investigate anti-oxidative and inflammatory inhibition effects of green tea and dietary fiber mixture on liver of high fat diet-induced obese rats. 21 male rats were divided into 3 dietary groups and control group (A), high fat diet-induced group (B), and high fat (HF) diet-induced + EQ diet-$S^{(R)}$ diet group (C). Immunoblotting and RT-PCR analysis revealed protein expression, and anti-oxidant analysis revealed MDA (malondialdehyde), GSH (glutathione), and free DPPH radical. As a results, Body weight and food consumption were not significantly different between groups. The levels of MDA and GSH were lower in HF + $EQS^{(R)}$ group than in HF group. Also, the $EQS^{(R)}$ demonstrated to be more effective than HF group for a DPPH radicals scavenging activities. In addition, protein and mRNA level of TNF-$\alpha$ in HF + $EQS^{(R)}$ group showed relatively more potent pro-inflammatory activity inhibition compared to HF group. These results suggest that green tea mixture (EQ diet-$S^{(R)}$) provide positive effects on anti-oxidative and inflammatory inhibition effects on obese animal model or obesity related diseases.

Pro-inflammatory Cytokines and Their Receptors: Expression and Regulation in the Uterine Endometrium during the Estrous Cycle in Pigs

  • Yoo, Inkyu;Kim, Minjeong;Han, Jisoo;Jang, Hwanhee;Choi, Sun-Ho;Ka, Hakhyun
    • Journal of Embryo Transfer
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    • v.31 no.4
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    • pp.323-333
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    • 2016
  • Pro-inflammatory cytokines, interleukin-$1{\beta}$(IL1B), IL6, and tumor necrosis factor-alpha (TNF), are known to play important roles in regulating the endometrial function in the uterus during the estrous cycle and pregnancy in several species. However, the expression and function of these cytokines and their receptors in the uterine endometrium during the estrous cycle have not been studied in pigs. Thus, this study determined the expression and regulation of IL1B, IL6, TNF and their respective receptors, IL1R1, IL1RAP, IL6R, GP130, TNFRSF1A, and TNFRSF1B during the estrous cycle in pigs. To analyze levels of each gene expression in the uterine endometrium we obtained from endometrial tissues on Days 0, 3, 6, 9, 12, 15, and 18 of the estrous cycle. Real-time RT-PCR analysis showed that levels of IL1B, IL1RAP, IL6R, GP130, TNF, TNFRSF1A, and TNFRSF1B mRNAs were highest on Day 15 or 18 of the estrous cycle, which corresponds to the proestrus period. Levels of IL1R1 were highest on Day 0, while levels of IL6 were biphasic with high levels on Day 6 and Day 15. The abundance of IL1B, IL6, IL6R, and TNF mRNAs was decreased by progesterone, while levels of GP130 were increased by progesterone in endometrial tissue explants. These results showed that expression of pro-inflammatory cytokines and their receptors changed stage-specifically during the estrous cycle and regulated by progesterone in the uterine endometrium in pigs, suggesting that these pro-inflammatory cytokines may be involved in the regulation endometrial function during the estrous cycle in pigs.

Gene Expression Profile in the Liver Tissue of High Fat Diet-Induced Obese Mice

  • Minho Cha;Bongjoo Kang;Kim, Kyungseon;Woongseop Sim;Hyunhee Oh;Yoosik Yoon
    • Nutritional Sciences
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    • v.7 no.1
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    • pp.8-16
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    • 2004
  • The purpose of this study was to investigate the gene profiles that were up- or down-regulated in the livers of high-fat diet-induced obese mice and $db_-/db_-$ mice with deficient leptin receptor. C57/BL6 normal mice and $db_-/db_-$ mice, respectively, were divided into two groups and fed a standard or high-fat diet for four weeks. Liver weight was unchanged in the normal mice but the high-fat diet led to a 10% weight increase in the $db_-/db_-$mice. Adipose tissue mass increased by about 88% in the normal mice that were fed a high-fat diet and by about 17% in the $db_-/db_-$mice on the high-fat diet. In terms of serum lipids, total cholesterol significantly increased in mice on the high-fat diet. Microarray analysis was carried out using total RNA isolated from the livers of standard or high-fat diet-fed mice of the normal and $db_-/db_-$ strains. The change of gene expression was confirmed by RT-PCR. About 1.6% and 6.8% of total genes, respectively, showed different expression patterns in the normal mice fed the high-fat diet and $db_-/db_-$ mice. As a result of microarray, many genes involved in metabolism and signal pathways were shown to have different expression patterns. Expression of Mgst3 gene increased in the livers of normal and $db_-/db_-$ mice that were fed a high-fat diet. Wnt7b and Ptk9l were down-regulated in the livers of the normal mice and $db_-/db_-$ mice that were fed a high-fat diet. In conclusion, a high-fat diet induced obesity and affected gene expression involved in metabolism and signal pathway.

Quercetin Enhances Cisplatin Sensitivity of Human Osteosarcoma Cells by Modulating microRNA-217-KRAS Axis

  • Zhang, Xian;Guo, Qinggong;Chen, Jingtao;Chen, Zhaohui
    • Molecules and Cells
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    • v.38 no.7
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    • pp.638-642
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    • 2015
  • Quercetin can suppress osteosarcoma cell growth and metastasis. However, other effects of quercetin on osteosarcoma remain largely unknown. This research aims to evaluate the effects of quercetin in combination with cisplatin as treatment for osteosarcoma and investigate its regulatory mechanism. Cell viability and apoptosis in 143B cell line were determined after treatment with quercetin and/or cisplatin. RT-PCR and Western blot analysis were performed to determine the RNA or protein expression levels. Moreover, transwell assay was used to evaluate metastasis. Furthermore, rescue experiments were performed to investigate the potential regulatory mechanism of the treatment. Results showed that quercetin with concentration that was equal to or greater than $10{\mu}M$ inhibited 143B proliferation, while $5{\mu}M$ quercetin enhanced the cisplatin sensitivity of 143B cells. Expression of miR-217 was upregulated after quercetin and/or cisplatin treatment, while its target KRAS was downregulated both at mRNA and protein levels. MiR-217 knockdown led to the loss of enhanced cisplatin sensitivity while miR-217 overexpression showed the opposite effects, indicating that quercetin regulated cisplatin sensitivity by modulating the miR-217-KRAS axis. In conclusion, $5{\mu}M$ quercetin enhanced the cisplatin sensitivity by modulating the miR-217-KRAS axis. This finding suggests that quercetin may be administered with cisplatin to improve the treatment for osteosarcoma.

The effect of L-carnitine in the expression of matrix metalloproteinases by human dermal fibroblasts

  • Yoon, Eun-Jeong;Lee, Kyoung-Eun;Sim, Kwan-Sup;Lee, Bum-Chun;Pyo, Hyeong-Bae;Choe, Tae-boo
    • Proceedings of the SCSK Conference
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    • 2003.09b
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    • pp.12-25
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    • 2003
  • L-camitine ($\beta$ -hydroxy-${\gamma}$ -trimethyl-ammoniumbutyric acid) is a small water-soluble molecule important in mammalian fat metabolism. It is essential for the normal oxidation of fatty acids by the mitochondria, and is involved in the trans-esterification and excretion of acyl-CoA esters. In this paper, to investigate the relationship between aging and L-camitine, we investigated the effects of in vitro MMP inhibition and activity and expression of UVA-induced MMP 1 in human skin fibroblasts. Fluorometric assays of the proteolytic activities of MMP-l were performed using fluorescent collagen substrates. ELISA (enzyme linked immuno sorbent assay), gelatin-substrate zymography, and RT-PCR ELISA techniques were used for the effects of L-camitine on MMP expression and activity, MMP mRNA expression in UVA irradiated fibroblast. L-camitine inhibited the activities of MMP-l in a dose-dependent manner and the $IC_{50}$/ values calculated from semi-log plots were 2.45mM, and L-carnitine showed strong inhibition on MMP-2 (gelatinase) activity in UVA irradiated fibroblast by zymography. Also, UVA induced MMP expression was reduced 40% by treated with L-carnitine, and MMP-l mRNA expression was reduced dose-dependent manner. Therefore L-carnitine was able to significantly inhibition the MMP activity, regulation of MMP expression in protein and mRNA level. All these results suggest that L-carnitine may be useful as new anti-aging cofactor for protection against UVA induced MMP expression and activity.

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Anti-inflammatory Effects and Its Mechanisms of NANA (N-Acylneuraminic Acid) Isolated from Glycomacropeptide (유청단백질 Glycomacropeptide에서 분리한 NANA의 안전성 및 염증저하 메카니즘 구명 연구)

  • Kim, Min-Ho;Kim, Jae-Hong;Lee, Yun-Kyoung;Kim, Wan-Sik;Kim, Hee-Kyoung
    • Journal of Dairy Science and Biotechnology
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    • v.29 no.2
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    • pp.17-23
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    • 2011
  • The focus of this study was to clarify the relation between the nitric oxide (NO) production and cytokine expression including tumor necrosis factor-${\alpha}$ (TNF) and interleukin-6 (IL-6), and also investigated the effect of G-NANA (N-acylneuraminic acid isolates from glycomacropeptide) or S-NANA (Synthetic N-acylneuraminic acid) on LPS stimuli from RAW264.7 cell. The NANA is the predominant sialic acid found in mammalian cells and G-NANA is isolation of GMP (GMP is a valuable bioactive peptide with a varying degree of glycosylation including sialic acid). The lipopolysaccharide (LPS) of Gram-negative bacteria induces the expression of cytokines and potent inducers of inflammatory cytokines such as TNF-${\alpha}$ and IL-6. In this experiment, upon stimulation with increasing concentrations of chitosan, the LPS-stimulated TNF-${\alpha}$ and IL-6 secretion was significantly recovered with in the incubation media of RAW264.7 cells. Consistently, RT-PCR with mRNA and immunoblot analysis with anti-cytokine antiserum including TNF-${\alpha}$ and IL-6 showed that the amount of TNF-${\alpha}$ and IL-6 secretion in the incubation media recovered with the concentration of chitosan. The LPS-stimulated NO secretion was significantly recovered with in the 6 and 12 h incubation media of RAW264.7 cells, too. The recovery effect of G-NANA on IL-6 and NO secretion may be induced via the stimulus of TNF-${\alpha}$ in RAW264.7 cell. These results once again suggest that G-NANA may have the anti-inflammatory effect via the stimulus of TNF-${\alpha}$ in the LPS-stimulated inflammation in RAW264.7 cells.

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Inhibitory Effects on Melanin Production in B16 Melanoma Cells of Sedum sarmentosum (B16 Melanoma 세포에서 돌나물 추출물의 멜라닌 생성 저해 효과)

  • Sim, Gwan-Sub;Kim, Jin-Hwa;Lee, Bum-Chun;Lee, Dong-Hwan;Lee, Geun-Soo;Pyo, Hyeong-Bae
    • YAKHAK HOEJI
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    • v.52 no.3
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    • pp.165-171
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    • 2008
  • To develop effective skin whitening agents, we tested natural herbal extracts for their melanogenic inhibitory activities. Sedum samentosum was selected for its inhibitory effect on melanogenesis in B16 melanoma cells. Ethanolic extract of S. samentosum (SSE) was evaluated for antioxidative effect and tyrosinase inhibitory activity of melanogenesis. We investigated the changes in protein level and mRNA level of tyrosinase, tyrosinase related protein (TRP)-1 and TRP-2 by using western blotting and RT-PCR, respectively. SSE showed scavenging activities of free radicals and reactive oxygen species (ROS) with the $IC_{50}$ of 342.7 $\mug/ml$ against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical and 64.69 $\mug/ml$ against superoxide radicals in the xanthine/xanthine oxidase system, respectively. SSE treatment suppressed the biosynthesis of melanin up to 46% and reduced tyrosinase activity up to 51% at 100 $\mug/ml$ in B16 melanoma cells. The tyrosinase activity and tyrosinase expression in B16 melanoma cells were reduced in a dose-dependent manner by SSE. Also, SSE was able to significantly inhibit tyrosinase and TRP-1 expression in mRNA level. These results suggest that SSE inhibited melanin production which may be dependent on tyrosinase activity and expression in B16 melanoma cells, and an effective whitening agent for the skin.

The Inhibition of MicroRNA-139-5p Promoted Osteoporosis of Bone Marrow-Derived Mesenchymal Stem Cells by Targeting Wnt/Beta-Catenin Signaling Pathway by NOTCH1

  • Feng, Yimiao;Wan, Pengbo;Yin, Linling;Lou, Xintian
    • Journal of Microbiology and Biotechnology
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    • v.30 no.3
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    • pp.448-458
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    • 2020
  • We investigated the therapeutic effects of microRNA-139-5p in relation to osteoporosis of bone marrow-derived mesenchymal stem cell (BMSCs) and its underlying mechanisms. In this study we used a dexamethasone-induced in vivo model of osteoporosis and BMSCs were used for the in vitro model. Real-time quantitative polymerase chain reaction (RT-PCR) and gene chip were used to analyze the expression of microRNA-139-5p. In an osteoporosis rat model, the expression of microRNA-139-5p was increased, compared with normal group. Down-regulation of microRNA-139-5p promotes cell proliferation and osteogenic differentiation in BMSCs. Especially, up-regulation of microRNA-139-5p reduced cell proliferation and osteogenic differentiation in BMSCs. Overexpression of miR-139-5p induced Wnt/β-catenin and down-regulated NOTCH1 signaling in BMSCs. Down-regulation of miR-139-5p suppressed Wnt/β-catenin and induced NOTCH1 signaling in BMSCs. The inhibition of NOTCH1 reduced the effects of anti-miR-139-5p on cell proliferation and osteogenic differentiation in BMSCs. Activation of Wnt/β-catenin also inhibited the effects of anti-miR-139-5p on cell proliferation and osteogenic differentiation in BMSCs. Taken together, our results suggested that the inhibition of microRNA-139-5p promotes osteogenic differentiation of BMSCs via targeting Wnt/β-catenin signaling pathway by NOTCH1.