• Title/Summary/Keyword: real-time qPCR

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Five miRNAs as Novel Diagnostic Biomarker Candidates for Primary Nasopharyngeal Carcinoma

  • Tang, Jin-Feng;Yu, Zhong-Hua;Liu, Tie;Lin, Zi-Ying;Wang, Ya-Hong;Yang, La-Wei;He, Hui-Juan;Cao, Jun;Huang, Hai-Li;Liu, Gang
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.18
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    • pp.7575-7581
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    • 2014
  • MicroRNAs (miRNAs) play an essential role in the development and progression of nasopharyngeal carcinomas (NPC). Despite advances in the field of cancer molecular biology and biomarker discovery, the development of clinically validated biomarkers for primary NPC has remained elusive. In this study, we investigated the expression and clinical significance of miRNAs as novel primary NPC diagnostic biomarkers. We used an array containing 2, 500 miRNAs to identify 22 significant miRNAs, and these candidate miRNAs were validated using 67 fresh NPC and 25 normal control tissues via quantitative real-time PCR (qRT-PCR). Expression and correlation analyses were performed with various statistical approaches, in addition to logistic regression and receiver operating characteristic curve analyses to evaluate diagnostic efficacy. qRT-PCR revealed five differentially expressed miRNAs (miR-93-5p, miR-135b-5p, miR-205-5p and miR-183-5p) in NPC tissue samples relative to control samples (p<0.05), with miR-135b-5p and miR-205-5p being of significant diagnostic value (p<0.01). Moreover, comparison of NPC patient clinicopathologic data revealed a negative correlation between miR-93-5p and miR-183-5p expression levels and lymph node status (p<0.05). These findings display an altered expression of many miRNAs in NPC tissues, thus providing information pertinent to pathophysiological and diagnostic research. Ultimately, miR-135b-5p and miR-205-5p may be implicated as novel NPC candidate biomarkers, while miR-93-5p, miR-650 and miR-183-5p may find application as relevant clinical pathology and diagnostic candidate biomarkers.

Analysis of Gene Expression Responses to a Salmonella Infection in Rugao Chicken Intestine Using GeneChips

  • Luan, D.Q.;Chang, G.B.;Sheng, Z.W.;Zhang, Y.;Zhou, W.;Li, Z.Z.;Liu, Y.;Chen, G.H.
    • Asian-Australasian Journal of Animal Sciences
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    • v.25 no.2
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    • pp.278-285
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    • 2012
  • Poultry products are an important source of Salmonella enterica. An effective way to reduce food poisoning due to Salmonella would be to breed chickens more resistant to infection. Unfortunately host responses to Salmonella are complex with many factors involved. To learn more about responses to Salmonella in young chickens of 2 wk old, a cDNA Microarray containing 13,319 probes was performed to compare gene expression profiles between two chicken groups under control and Salmonella infected conditions. Newly hatched chickens were orally infected with S. enterica serovar Enteritidis. Since the intestine is one of the important barriers the bacteria encounter after oral inoculation, intestine gene expression was investigated at 2 wk old. There were 588 differentially expressed genes detected, of which 276 were known genes, and of the total number 266 were up-regulated and 322 were down-regulated. Differences in gene expression between the two chicken groups were found in control as well as Salmonella infected conditions indicating a difference in the intestine development between the two chicken groups which might be linked to the difference in Salmonella susceptibility. The differential expressions of 4 genes were confirmed by quantitative real-time PCR and the results indicated that the expression changes of these genes were generally consistent with the results of GeneChips. The findings in this study have lead to the identification of novel genes and possible cellular pathways, which are host dependent.

Upregulation of STK15 in Esophageal Squamous Cell Carcinomas in a Mongolian Population

  • Chen, Guang-Lie;Hou, Gai-Ling;Sun, Fei;Jiang, Hong-Li;Xue, Jin-Feng;Li, Xiu-Shen;Xu, En-Hui;Gao, Wei-Shi;Cao, Jian-Ping
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.15
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    • pp.6021-6024
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    • 2014
  • Background: The STK15 gene located on chromosome 20q13.2 encodes a centrosome-associated kinase critical for regulated chromosome segregation and cytokinesis. Recent studies have demonstrated STK15 to be significantly associated with many tumors, with aberrant expression obseved in many human malignancies. The purpose of this study was to investigate expression of STK15 in esophageal squamous cell carcinomas (ESCCs) in a Mongolian population. Methods: Two non-synonymous single nucleotide polymorphisms in the coding region of STK15, rs2273535 (Phe31Ile) and rs1047972 (Val57Ile) were assessed in 380 ESCC patients and 380 healthy controls. We also detected STK15 mRNA expression in 39 esophageal squamous cell carcinomas and corresponding adjacent tissues by real time PCR. Results: rs2273535 showed a significant association with ESCC in our Mongolian population (rs227353, P allele = 0.0447, OR (95%CI) = 1.259 (1.005~1.578)). Real time PCR analysis of ESCC tissues showed that expression of STK15 mRNA in cancer tissues was higher than in normal tissues (p = 0.013). Conclusions: Our study showed that functional SNPs in the STK15 gene are associated with ESCC in a Mongolian population and up-regulation of STK15 mRNAoccurs in ESCC tumors compared adjacent normal tissues. STK15 may thus have an important role in the prognosis of ESCC and be a potential therapeutic target.

Identification and Functional Analysis of Mating Type Loci in the Pleurotus eryngii

  • Ryu, Jae San;Kim, Min-Keun;Park, Bokyung;Ali, Asjad;Joung, Wan-Kyu
    • 한국균학회소식:학술대회논문집
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    • 2015.11a
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    • pp.35-35
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    • 2015
  • Pleurotus eryngii has recently become a major cultivated mushroom; it uses tetrapolar heterothallism as a part of its reproductive process. Sexual development progresses only when the A and B mating types are compatible. Such mating incompatibility occasionally limits the efficiency of breeding programs in which crossing within loci-shared strains or backcrossing strategies are employed. Therefore, understanding the mating system in edible mushroom fungi will help provide a short cut in the development of new strains. We isolated and identified pheromone and receptor genes in the B3 locus of P. eryngii and performed a functional analysis of the genes in the mating process by transformation. A genomic DNA library was constructed to map the entire mating-type locus. The B3 locus was found to contain four pheromone precursor genes and four receptor genes. Remarkably, receptor PESTE3.3.1 has just 34 amino acid residues in its C-terminal cytoplasmic region; therefore, it seems likely to be a receptor-like gene. Real-time quantitative RT-PCR (real-time qRT-PCR) revealed that most pheromone and receptor genes showed significantly higher expression in monokaryotic cells than dikaryotic cells. The pheromone genes PEphb3.1 and PEphb3.3 and the receptor gene PESTE3.3.1 were transformed into P5 (A3B4). The transformants were mated with a tester strain (A4B4), and the progeny showed clamp connections and a normal fruiting body, which indicates the proposed role of these genes in mating and fruiting processes. This result also confirms that PESTE3.3.1 is a receptor gene. In this study, we identified pheromone and receptor genes in the B3 locus of P. eryngii and found that some of those genes appear to play a role in the mating and fruiting processes. These results might help elucidate the mechanism of fruiting differentiation and improve breeding efficiency.

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MiR-323-5p acts as a Tumor Suppressor by Targeting the Insulin-like Growth Factor 1 Receptor in Human Glioma Cells

  • Lian, Hai-Wei;Zhou, Yun;Jian, Zhi-Hong;Liu, Ren-Zhong
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.23
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    • pp.10181-10185
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    • 2015
  • Background: MicroRNAs, small noncoding RNA molecules, can regulate mammalian cell growth, apoptosis and differentiation by controlling the expression of target genes. The aim of this study was to investigate the function of miR-323-5p in the glioma cell line, U251. Materials and Methods: After over-expression of miR-323-5p using miR-323-5p mimics, cell growth, apoptosis and migration were tested by MTT, flow cytometry and cell wound healing assay, respectively. We also assessed the influence of miR-323-5p on the mRNA expression of IGF-1R by quantitative real-time reverse transcriptase PCR (qRT-PCR), and on the protein levels by Western blot analysi. In addition, dual-luciferase reporter assays were performed to determine the target site of miR-323-5p to IGF-1R 3'UTR. Results: Our findings showed that over-expression of miR-323-5p could promote apoptosis of U251 and inhibit the proliferation and migration of the glioma cells. Conclusions: This study demonstrated that increased expression of miR-323-5p might be related to glioma progression, which indicates a potential role of miR-323-5p for clinical therapy.

Molecular characterization and immunohistochemical localization of a mitogen-activated protein kinase, Accp38b, from Apis cerana cerana

  • Zhang, Liang;Meng, Fei;Li, Yuzhen;Kang, Mingjiang;Guo, Xingqi;Xu, Baohua
    • BMB Reports
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    • v.45 no.5
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    • pp.293-298
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    • 2012
  • The p38 mitogen-activated protein kinase (MAPK) is involved in various processes, including stress responses, development, and differentiation. However, little information on p38 MAPK in insects is available. In this study, a p38 MAPK gene, $Accp38b$, was isolated from $Apis$ $cerana$ $cerana$ and characterized. The quantitative real-time PCR (Q-PCR) analysis revealed that $Accp38b$ was induced by multiple stressors. Notably, the expression of $Accp38b$ was relatively higher in the pupae phase than in other developmental phases. During the pupae phase, Accp38b expression was higher in the thorax than in the head and abdomen and higher in the fat body than in the muscle and midgut. Immunohistochemisty showed significant positive staining of Accp38b in sections from the brain, eyes, fat body, and midgut of $A.$ $cerana$ $cerana$. These results suggest that Accp38b may play a crucial role in stress responses and have multiple aspects function during development.

MiR-150-5p Suppresses Colorectal Cancer Cell Migration and Invasion through Targeting MUC4

  • Wang, Wei-Hua;Chen, Jie;Zhao, Feng;Zhang, Bu-Rong;Yu, Hong-Sheng;Jin, Hai-Ying;Dai, Jin-Hua
    • Asian Pacific Journal of Cancer Prevention
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    • v.15 no.15
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    • pp.6269-6273
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    • 2014
  • Growing evidence suggests that miR-150-5p has an important role in regulating genesis of various types of cancer. However, the roles and the underlying mechanisms of miR-150-5p in development of colorectal cancer (CRC) remain largely unknown. Transwell chambers were used to analyze effects on cell migration and invasion by miR-150-5p. Quantitative real-time PCR (qRT-PCR), Western blotting and dual-luciferase 3' UTR reporter assay were carried out to identify the target genes of miR-150-5p. In our research, miR-150-5p suppressed CRC cell migration and invasion, and MUC4 was identified as a direct target gene. Its effects were partly blocked by re-expression of MUC4. In conclusiomn, miR-150-5p may suppress CRC metastasis through directly targeting MUC4, highlighting its potential as a novel agent for the treatment of CRC metastasis.

Comparison of Alpha-Factor Preprosequence and a Classical Mammalian Signal Peptide for Secretion of Recombinant Xylanase xynB from Yeast Pichia pastoris

  • He, Zuyong;Huang, Yuankai;Qin, Yufeng;Liu, Zhiguo;Mo, Delin;Cong, Peiqing;Chen, Yaosheng
    • Journal of Microbiology and Biotechnology
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    • v.22 no.4
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    • pp.479-483
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    • 2012
  • The secretory efficiency of recombinant xylanase xynB from yeast Pichia pastoris between the ${\alpha}$-factor preprosequence and a classical mammalian signal peptide derived from bovine ${\beta}$-casein was compared. The results showed that although the bovine ${\beta}$-casein signal peptide could direct high-level secretion of recombinant xylanase, it was relatively less efficient than the ${\alpha}$-factor preprosequence. In contrast, the bovine ${\beta}$-casein signal peptide caused remarkably more recombinant xylanase trapped intracellularly. Real-time RT-PCR analysis indicated that the difference in the secretory level between the two signal sequences was not due to the difference in the transcriptional efficiency.

Production of Cellulases by Rhizopus stolonifer from Glucose-Containing Media Based on the Regulation of Transcriptional Regulator CRE

  • Zhang, Yingying;Tang, Bin;Du, Guocheng
    • Journal of Microbiology and Biotechnology
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    • v.27 no.3
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    • pp.514-523
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    • 2017
  • Carbon catabolite repression is a crucial regulation mechanism in microorganisms, but its characteristic in Rhizopus is still unclear. We extracted a carbon regulation gene, cre, that encoded a carbon catabolite repressor protein (CRE) from Rhizopus stolonifer TP-02, and studied the regulation of CRE by real-time qPCR. CRE responded to glucose in a certain range, where it could significantly regulate part of the cellulase genes (eg, bg, and cbh2) without cbh1. In the comparison of the response of cre and four cellulase genes to carboxymethylcellulose sodium and a simple carbon source (lactose), the effect of CRE was only related to the concentration of reducing sugars. By regulating the reducing sugars to range from 0.4% to 0.6%, a glucose-containing medium with lactose as the inducer could effectively induce cellulases without the repression of CRE. This regulation method could potentially reduce the cost of enzymes produced in industries and provide a possible solution to achieve the largescale synthesis of cellulases.

Design and Performance Evaluation using Computational Fluid Dynamics (CFD) Analysis of Wetcyclones for the Collection of Airborne Bacteria (공기 중 박테리아 포집을 위한 습식 사이클론의 CFD 해석을 이용한 설계 및 성능 평가)

  • Hyun Sik Ko;Jungwoo Park;Jiwoo Jung;Jungho Hwang
    • Particle and aerosol research
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    • v.19 no.3
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    • pp.77-87
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    • 2023
  • We present the development of a wetcyclone sampler designed for the sampling of airborne bacteria. The wetcyclone design involves a combination of two traditional cyclone shapes and computational fluid dynamics (CFD) analysis to validate its effectiveness in terms of pressure drop and collection efficiency. The wetcyclone exhibits a collection efficiency of over 90% for bacteria, specifically targeting Staphylococcus aureus. Additionally, the wetcyclone enables continuous bioaerosol sampling using a liquid medium (deionized water), demonstrating a concentration ratio exceeding >105 and a stable microbial recovery rate of 81.9%. The application of real-time quantitative polymerase chain reaction (qPCR) and the colony counting method ensures precise measurement of the concentration ratio and microbial recovery rate.