• Title/Summary/Keyword: Quantitative real time RT-PCR

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Effects of Gintonin-enriched fraction on the gene expression of six lysophosphatidic receptor subtypes

  • Lee, Rami;Lee, Byung-Hwan;Choi, Sun-Hye;Cho, Yeon-Jin;Cho, Han-Sung;Kim, Hyoung-Chun;Rhim, Hyewhon;Cho, Ik-Hyun;Rhee, Man Hee;Nah, Seung-Yeol
    • Journal of Ginseng Research
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    • v.45 no.5
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    • pp.583-590
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    • 2021
  • Background: Gintonin, isolated from ginseng, acts as a ginseng-derived lysophosphatidic acid (LPA) receptor ligand and elicits the [Ca2+]i transient through six LPA receptor subtypes (LPARSs). However, the long-term effects of gintonin-enriched fraction (GEF) on the gene expression of six LPARSs remain unknown. We examined changes in the gene expression of six LPA receptors in the mouse whole brain, heart, lungs, liver, kidneys, spleen, small intestine, colon, and testis after long-term oral GEF administration. Methods: C57BL/6 mice were divided into two groups: control vehicle and GEF (100 mg/kg, p.o.). After 21-day saline or GEF treatment, total RNA was extracted from nine mouse organs. Quantitative-real-time PCR (qRT-PCR) and western blot were performed to quantify changes in the gene and protein expression of the six LPARSs, respectively. Results: qRT-PCR analysis before GEF treatment revealed that the LPA6 RS was predominant in all organs except the small intestine. The LPA2 RS was most abundant in the small intestine. Long-term GEF administration differentially regulated the six LPARSs. Upon GEF treatment, the LPA6 RS significantly increased in the liver, small intestine, colon, and testis but decreased in the whole brain, heart, lungs, and kidneys. Western blot analysis of the LPA6 RS confirmed the differential effects of GEF on LPA6 receptor protein levels in the whole brain, liver, small intestine, and testis. Conclusion: The LPA6 receptor was predominantly expressed in all nine organs examined; long-term oral GEF administration differentially regulated LPA3, LPA4, and LPA6 receptors in the whole brain, heart, lungs, liver, kidneys, small intestine, and testis.

Expression of the CXCL12/SDF-1 Chemokine Receptor CXCR7 in Human Brain Tumours

  • Tang, Tian;Xia, Qing-Jie;Chen, Jian-Bin;Xi, Ming-Rong;Lei, Ding
    • Asian Pacific Journal of Cancer Prevention
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    • v.13 no.10
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    • pp.5281-5286
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    • 2012
  • Purpose: Receptor 7 (CXCR7) has recently been characterized as a novel receptor for CXCL12/SDF-1 (stromal cell derived factor-1). Given the demonstrated importance of CXCL12/SDF-1 in angiogenesis and tumour metastasis, we hypothesized that CXCR7 may also play a role in tumour pathogenesis. Located in the limited space of the intracranial cavity, any brain tumours can be inherently serious and life-threatening. However, the expression of CXCR7 in pituitary adenoma, neurilemmoma or hemangioblastoma remains to be elucidated. Therefore, we aimed to determine the potential contribution of CXCR7 in the development of brain tumours. Methods: In this study we examined and quantified the mRNA expression of CXCR7 in four different human brain tumours - 27 patients with neurilemmoma (8 patients), pituitary adenoma (7 patients), hemangioblastoma (6 patients), or meningioma (6 patients) undergoing surgical resection in the West China Hospital of Sichuan University. There were 15 females and 12 males aged from 28 to 70 years old. Total RNA was isolated and mRNA was measured by quantitative real-time RT-PCR. One-way analysis of variance (ANOVA) was performed using SPSS 11.0 statistical software to compare the mRNA levels of CXCR7 among four groups. Results: We found that CXCR7 mRNA was detected in all tumour samples. Quantitative results showed that the levels of CXCR7 mRNA in brain tissues from patients with neurilemmoma or meningioma were significantly higher than those with pituitary adenoma or hemangioblastoma. Conclusions: The results suggest that the CXCR7 may play a role in progression, metastasis and angiogenesis of brain tumours.

Cancer/Testis OIP5 and TAF7L Genes are Up-Regulated in Breast Cancer

  • Mobasheri, Maryam Beigom;Shirkoohi, Reza;Modarressi, Mohammad Hossein
    • Asian Pacific Journal of Cancer Prevention
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    • v.16 no.11
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    • pp.4623-4628
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    • 2015
  • Breast cancer still remains as the most frequent cancer with second mortality rate in women worldwide. There are no validated biomarkers for detection of the disease in early stages with effective power in diagnosis and therapeutic approaches. Cancer/testis antigens are recently promising tumor antigens and suitable candidates for targeted therapies and generating cancer vaccines. We conducted the present study to analyze transcript changes of two cancer/testis antigens, OIP5 and TAF7L, in breast tumors and cell lines in comparison with normal breast tissues by quantitative real time RT-PCR for the first time. Significant over-expression of OIP5 was observed in breast tumors and three out of six cell lines including MDA-MB-468, T47D and SKBR3. Not significant expression of TAF7L was evident in breast tumors but significant increase was noted in three out of six cell lines including MDA-MB-231, BT474 and T47D. OIP5 has ssignificant role in chromatin organization and cell cycle control during cell cycle exit and normal chromosome segregation during mitosis and TAF7L is a component of the transcription factor IID, which is involved in transcription initiation of most protein coding genes. TAF7Lis located at X chromosome and belongs to the CT-X gene family of cancer/testis antigens which contains about 50% of CT antigens, including those which have been used in cancer immunotherapy.

Hypoxia Induced Multidrug Resistance of Laryngeal Cancer Cells via Hypoxia-inducible Factor-1α

  • Li, Da-Wei;Dong, Pin;Wang, Fei;Chen, Xin-Wei;Xu, Cheng-Zhi;Zhou, Liang
    • Asian Pacific Journal of Cancer Prevention
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    • v.14 no.8
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    • pp.4853-4858
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    • 2013
  • Objectives: To investigate whether hypoxia has an effect on regulation of multidrug resistance (MDR) to chemotherapeutic drugs in laryngeal carcinoma cells and explore the role of hypoxia-inducible factor-$1{\alpha}$ (HIF-$1{\alpha}$). Methods: Laryngeal cancer cells were cultured under normoxic and hypoxic conditions. The sensitivity of the cells to multiple drugs and levels of apoptosis induced by paclitaxel were determined by MTT assay and annexin-V/propidium iodide staining analysis, respectively. HIF-$1{\alpha}$ expression was blocked by RNA interference. The expression of HIF-$1{\alpha}$ gene was detected by real-time quantitative RT-PCR and Western blotting. The value of fluorescence intensity of intracellular adriamycin accumulation and retention in cells was evaluated by flow cytometry. Results: The sensitivity to multiple chemotherapy agents and induction of apoptosis by paclitaxel could be reduced by hypoxia (P<0.05). A the same time, the adriamycin releasing index of cells was increased (P<0.05). However, resistance acquisition subject to hypoxia in vitro was suppressed by down-regulating HIF-$1{\alpha}$ expression. Conclusion: HIF-$1{\alpha}$ could be considered as a key regulator for mediating hypoxia-induced MDR in laryngeal cancer cells via inhibition of drug-induced apoptosis and decrease in intracellular drug accumulation.

Short-Hairpin RNA-Mediated MTA2 Silencing Inhibits Human Breast Cancer Cell Line MDA-MB231 Proliferation and Metastasis

  • Lu, Jun;Jin, Mu-Lan
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.14
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    • pp.5577-5582
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    • 2014
  • Objective: To observe the effects of metastasis-associated tumor gene family 2 (MTA2) depletion on human breast cancer cell proliferation and metastasis. Methods: A short-hairpin RNA targeting MTA2 was chemically synthesized and transfected into a lentivirus to construct Lv-shMTA2 for infection into the MDA-MB231 human breast cancer cell line. At 48 hours after infection cells were harvested and mRNA and protein levels of MTA2 were determined by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting, respectively. Cell viability and metastasis were assessed by CCK-8, wound-healing assay and Transwell assay, respectively. In addition, a xenograft model of human breast cancer was constructed to investigate cancerous cell growth and capacity for metastasis. Results: After infection with Lv-shMTA2, mRNA and protein levels of MTA2 was significantly reduced (p<0.05) and MDA-MB231 cell proliferation and metastasis were inhibited (p<0.05). In addition, mean tumor size was smaller than that in control group nude mice (p<0.05) and numbers of metastatic deposits in lung were lower than in control group mice (p<0.05). Depletion of MTA2 affected MMP-2 and apoptosis-related protein expression. Conclusions: For the first time to our knowledge we showed that MTA2 depletion could significantly inhibit human breast cancer cell growth and metastasis, implying that MTA2 might be involved in the progression of breast cancer. The role of MTA2 in breast cancer growth and metastasis might be linked with regulation of matrix metalloproteinase and apoptosis.

Differential Expression of PPARγ, FASN, and ACADM Genes in Various Adipose Tissues and Longissimus dorsi Muscle from Yanbian Yellow Cattle and Yan Yellow Cattle

  • Ji, Shuang;Yang, Runjun;Lu, Chunyan;Qiu, Zhengyan;Yan, Changguo;Zhao, Zhihui
    • Asian-Australasian Journal of Animal Sciences
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    • v.27 no.1
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    • pp.10-18
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    • 2014
  • The objective of this study was to investigate the correlation between cattle breeds and deposit of adipose tissues in different positions and the gene expressions of peroxisome proliferator-activated receptor gamma ($PPAR{\gamma}$), fatty acid synthase (FASN), and Acyl-CoA dehydrogenase (ACADM), which are associated with lipid metabolism and are valuable for understanding the physiology in fat depot and meat quality. Yanbian yellow cattle and Yan yellow cattle reared under the same conditions display different fat proportions in the carcass. To understand this difference, the expression of $PPAR{\gamma}$, FASN, and ACADM in different adipose tissues and longissimus dorsi muscle (LD) in these two breeds were analyzed using the Real-time quantitative polymerase chain reaction method (qRT-PCR). The result showed that $PPAR{\gamma}$ gene expression was significantly higher in adipose tissue than in LD in both breeds. $PPAR{\gamma}$ expression was also higher in abdominal fat, in perirenal fat than in the subcutaneous fat (p<0.05) in Yanbian yellow cattle, and was significantly higher in subcutaneous fat in Yan yellow cattle than that in Yanbian yellow cattle. On the other hand, FASN mRNA expression levels in subcutaneous fat and abdominal fat in Yan yellow cattle were significantly higher than that in Yanbian yellow cattle. Interestingly, ACADM gene shows greater fold changes in LD than in adipose tissues in Yan yellow cattle. Furthermore, the expressions of these three genes in lung, colon, kidney, liver and heart of Yanbian yellow cattle and Yan yellow cattle were also investigated. The results showed that the highest expression levels of $PPAR{\gamma}$ and FASN genes were detected in the lung in both breeds. The expression of ACADM gene in kidney and liver were higher than that in other organs in Yanbian yellow cattle, the comparison was not statistically significant in Yan yellow cattle.

생쥐 초기 배아에서 Aquaporin 8과 9의 발현에 관한 연구

  • 신현상;계명찬;강수만;이성은;이지원;강한승;김문규
    • Proceedings of the Korean Society of Developmental Biology Conference
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    • 2003.10a
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    • pp.68-68
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    • 2003
  • Aquaporin은 막관통 통로 단백질(transmembrane channel protein)로서, 삼투압의 농도구배에 따라 세포막을 가로질러 물분자를 이동시키는 기능을 하고 있다. 포유류 초기배아에서 포배강 형성은 영양외배엽세포에서 $Na^+ / K^+$ATPase에 의한 이온 농도 구배가 형성되면 auqaporin에 의해 물이 포배강으로 유입되면서 이루어진다. 본 연구에서는 생쥐 초기배아에서 반정량적인 역전사 중합효소 연쇄반응 방법(semi-quantitative RT-PCR)과 실시간 역전사 중합효소 연쇄반응 방법(real-time RT-PCR)을 통하여 AQP8과 9의 mRNA발현을 조사하고 다중 면역형광현미경 방법(confocal immunofluorescence microscopy)을 통해 단백질 발현양상을 분석하였다. AQP8 mRNA는 상실기까지 발현되지 않다가 포배기에 이르러 발현되었고 AQP9 mRNA는 수정란에서부터 발현되어 포배기에는 유의할 정도로 증가하였다. 따라서 AQP8 mRNA는 배아유전자가 활성화되어 나타나는 것이고 AQP9 mRNA는 모계유전자 기원임을 알 수 있었다. AQP8 단백질은 상실배 단계까지 발현되지 않다가 포배시기에 영양외배엽세포사이의 접합면에 발현되었고 AQP9 단백질은 상실배 시기에 할구 사이의 인접 부위에서 강하게 발현되었다가 포배시기에는 세포간의 접합면에 약하게 발현하는 경향을 나타내었다. 실시간 역전사 중합효소 연쇄반응 방법으로 조사한 결과 포배에서 물과 글리세롤을 통과시키는 AQP9는 mRNA의 발현양이 AQP8보다 약 4배 정도 많았다. 또한 포배기에 이르러서야 물만을 통과시키는 AQP8의 발현이 나타나는 것을 보아 포배강 형성시 외부에서 영양외배엽을 통해 포배강으로 유입되는 물의 이동(trans- trophectodermal water movements)에 AQP9보다 AQP8이 더 중요하게 관여할 것으로 사료된다., K, Pb, Cd, Cr, Co, Cu, Ni)을 측정하였다. 실험 조건1의 결과로서 각 국의 유아용 일회용 기저귀의 중금속 함량은 거의 유사한 경향을 나타내었으며 Cr, Zn, Pb, Ni, Mn, Mg, Li, K는 detection limit(2 ppm) 이하였고, Cd, Fe, Co, Cu, Ca, Al, Sr는 검출되었지만 기준치 이하였다. 실험 조건2의 결과로서 측정 항목(Cr, Sb, Cd, Pb, Ni, Co, Cu)중 Cr, Cd, Ni, Cu는 detection limit(0.1 ppm) 이하였고, Sb, Pb, Co는 검출되었지만 기준치 이하였다.았다. 4%의 경우에는 8$0^{\circ}C$이하로 온도를 낮추는 것이 좋은 상태를 나타내었다. 이와 같은 결과는 일반적으로 화학적 레팅을 4%, 7%에서한 선행결과와 상당히 다른 결과이다.염 농도가 증가할수록 감소 현상을 보였다.X>, 75BG30은 8.6$\mu\textrm{m}$, 75BG40은 7.02$\mu\textrm{m}$로 나타났다. 따라서 경화제 양에 관계없이 10$\mu\textrm{m}$ 이하로 나타나, 경화제 10$m\ell$만으로 미세한 크기를 얻을 수 있음을 알 수 있다. 젤리 강도 변화에 따른 차이는 300BF는 78.09$\mu\textrm{m}$ 300BG는 56.32$\mu\textrm{m}$로, 75BF나 75BG에 비하여 현저히 증가하여, 젤라틴의 젤리 강도는 캡슐 제조 조건의 주요한 변수임을 알 수 있다.추출물 투여시 혈당강하 및 혈중콜레스테롤 강하가 나타났으며, 상엽복합추출물 투여와 운동을 병행시 이러한 감소 효과가 더 뚜렷하게 나타났다.교육의 적임자로 보는 시각이 비교적 높았고 약 1/2정도는 영양교육에 참여하겠다는 의지를 가지고 있을 뿐만 아니라 실제로 영양지도를

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Modulated Gene Expression of Toxoplasma gondii Infected Retinal Pigment Epithelial Cell Line (ARPE-19) via PI3K/Akt or mTOR Signal Pathway

  • Zhou, Wei;Quan, Juan-Hua;Gao, Fei-Fei;Ismail, Hassan Ahmed Hassan Ahmed;Lee, Young-Ha;Cha, Guang-Ho
    • Parasites, Hosts and Diseases
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    • v.56 no.2
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    • pp.135-145
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    • 2018
  • Due to the critical location and physiological activities of the retinal pigment epithelial (RPE) cell, it is constantly subjected to contact with various infectious agents and inflammatory mediators. However, little is known about the signaling events in RPE involved in Toxoplasma gondii infection and development. The aim of the study is to screen the host mRNA transcriptional change of 3 inflammation-related gene categories, PI3K/Akt pathway regulatory components, blood vessel development factors and ROS regulators, to prove that PI3K/Akt or mTOR signaling pathway play an essential role in regulating the selected inflammation-related genes. The selected genes include PH domain and leucine- rich-repeat protein phosphatases (PHLPP), casein kinase2 (CK2), vascular endothelial growth factor (VEGF), pigment epithelium-derived factor (PEDF), glutamate-cysteine ligase (GCL), glutathione S-transferase (GST), and NAD(P)H: quinone oxidoreductase (NQO1). Using reverse transcription polymerase chain reaction (RT-PCR) and quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR), we found that T. gondii up-regulates PHLPP2, $CK2{\beta}$, VEGF, GCL, GST and NQO1 gene expression levels, but down-regulates PHLPP1 and PEDF mRNA transcription levels. PI3K inhibition and mTOR inhibition by specific inhibitors showed that most of these host gene expression patterns were due to activation of PI3K/Akt or mTOR pathways with some exceptional cases. Taken together, our results reveal a new molecular mechanism of these gene expression change dependent on PI3K/Akt or mTOR pathways and highlight more systematical insight of how an intracellular T. gondii can manipulate host genes to avoid host defense.

Effects of Castration on Androgen Receptor, IGF-I Ea, MGF and Myostatin Gene Expression in Skeletal Muscles of Male Pigs

  • Yao, Yuchang;Cai, Zhaowei;Zhang, Lifan;Zhao, Chunjiang;Wu, Keliang;Xu, Ningying;Liu, Gang;Wu, Changxin
    • Asian-Australasian Journal of Animal Sciences
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    • v.22 no.8
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    • pp.1069-1077
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    • 2009
  • Castration of male pig produces significant negative effects on skeletal muscle development. The androgen receptor (AR), two splice variants of insulin-like growth factor-I (IGF-I Ea and MGF) and the myostatin gene may play important roles in this process. In the present study, the expression of AR, IGF-I Ea, MGF and myostatin genes in three skeletal muscles, the brachialis, longissimus and semitendinosus, were studied using real-time quantitative RT-PCR. Our experimental design used 14 pairs of male Landrace sire${\times}$Yorkshire dam piglets. The two piglets in each pair were full sibs, one of which was castrated at 21 d of age; the other remained intact. The study group was divided into subgroups of equal size. Animals in the first subgroup were slaughtered at 147 d and those of the second at 210 d of age. Carcass weight and lean meat yield were similar between boars and barrows at 147 d of age (p>0.05), whereas barrows had lower carcass weight and less lean meat yield at 210 d of age (p<0.05). Castration caused down-regulation of AR gene expression at both 147 and 210 d of age (p<0.05). The two splice variants of the IGF-I gene from porcine skeletal muscle were cloned using RT-PCR, and it was found that MGF differs from IGF-I Ea in having a 52-base insert in the last coding exon of the mRNA. Both splice variants were down-regulated by castration only at 210 d of age (p<0.05). No differences in expression of the myostatin gene were observed between boars and barrows at either 147 or 210 d of age (p>0.05). These results suggest that the downregulation of AR, IGF-I Ea and MGF gene expression following castration helps to explain the negative effect of castration on skeletal muscle development.

Sedum sarmentosum Enhances Hyaluronan Synthesis in Transformed Human Keratinocytes and Increases Water Content in Human Skin (돌나물추출물에 의한 사람 각질형성세포에서의 Hyaluronan Synthesis 촉진과 인체 피부의 보습력 증진)

  • Sim, Gwan-Sub;Kim, Jin-Hwa;Lee, Dong-Hwan;Na, Young;Lee, Geun-Soo;Pyo, Hyeong-Bae
    • Journal of the Society of Cosmetic Scientists of Korea
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    • v.33 no.1 s.60
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    • pp.17-22
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    • 2007
  • In this study, we investigated the effects of Sedum sarmentosum extract on the expression of hyaluronan synthase (HAS) genes and hyaluronan (HA) production in HaCaT keratinocytes. We also assessed water content (electrical capacitance) and transepidermal water loss (TEWL) in human skin after topical treatment with Sedum sarmentosum extract. Sedum sarmentosum extract increased expression of HAS-2 and HAS-3 genes in HaCaT cells, when assayed by real-time reverse-transcriptase polymerase chain reactions (RT-PCR). Sedum sarmentosum extract induced HA production in HaCaT cells, when determined by enzyme-linked immunosorbent assay (ELISA). Finally, treatment of Sedum sarmentosum extract on human skin increased the skin hydration and decreased TEWL when measured using Corneometer and Tewameter. Our study suggests that Sedum sarmentosum extract should be a very useful cosmetic ingredient, as a skin moisturizer.