• Asian Pacific Journal of Cancer Prevention
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• 제13권7호
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• pp.3203-3207
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• 2012

Backgrounds: To investigate the inhibiting effects of multi-target anti-microRNA antisense oligonucleotide (MTg-AMOs) on proliferation and migration of human gastric cancer cells. Methods: Single anti-microRNA antisense oligonucleotides (AMOs) and MTg-AMOs for miR-221, 21, and 106a were designed and transfected into SGC7901, a gastric cancer cell line, to target the activity of these miRNAs. Their expression was analyzed using stem-loop RT-PCR and effects of MTg-AMOs on human gastric cancer cells were determined using the following two assay methods: CCK8 for cell proliferation and transwells for migration. Results: In the CCK-8 cell proliferation assay, $0.6{\mu}mol/L$ was selected as the preferred concentration of MTg-AMOs and incubation time was 72 hours. Under these experimental conditions, MTg-AMOs demonstrated better suppression of the expression of miR-221, miR-106a, miR-21 in gastric cancer cells than that of single AMOs (P = 0.014, 0.024; 0.038, respectively). Migration activity was also clearly decreased as compared to those in randomized and blank control groups ($28{\pm}4$ Vs $54{\pm}3$, P <0.01; $28{\pm}4$ Vs $59{\pm}4$, P < 0.01). Conclusions: MTg-AMOs can specifically inhibit the expression of multiple miRNAs, and effectively antagonize proliferation and migration of gastric cancer cells promoted by oncomirs.

• KSBB Journal
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• 제31권1호
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• pp.33-39
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• 2016

Developing the method for the selection of animal cell line producing therapeutic monoclonal antibody (mAb) is invaluable as its market is rapidly growing. Although the quality of produced mAb is as important as quantity, however there is no method developed for the selective screening of cell lines on the basis of both quantity and quality. From recent reports, the ratio of light and heavy chain mRNAs of mAb in the cell is a key parameter for the indication of product quality. Therefore, it is obvious that developing the novel method that can detect both light and heavy chain mRNAs in single live cell will provide unprecedented opportunities in bio-industry. Here, we have constructed oligonucleotide probes, molecular beacons for the detection of light or heavy chain mRNAs, respectively, in the live cells producing mAbs. Both beacons showed increased fluorescent intensity after transient transfection of plasmid expressing mAbs analyzed by fluorometer. Flow cytometric analysis clearly demonstrated that both molecular beacons can simultaneously detect the expression of light and heavy chain mRNAs of mAb in the same cell. The technique described in the thesis provides the new direction and concept for developing the method for the smart selection of cell lines producing recombinant proteins including therapeutic mAbs.

• Imaging Science in Dentistry
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• 제33권1호
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• pp.51-57
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• 2003

Purpose: To investigate the effects of irradiation on the phenotypic expression of the MC3T3-El osteoblastic cell line, particularly an the expression of type I collagen and alkaline phosphatase mRNA. Materials and Methods: Cells were irradiated with a single dose of 0.5, 1, 2, 4, and 8 Gy at a dose rate of 5.38 Gy/min using a cesium 137 irradiator. The specimens were then harvested and RNA extraction was carried out at 1 and 3 days after irradiation. The extracted RNA strands were reverse-transcribed and the resulting cDNA fragments were amplified by PCR. Results: The irradiated cells demonstrated a dose-dependent increase in type I collagen mRNA expression relative to the control group, with a maximum level of type I collagen mRNA expression occurring at 8 Gy. The degree of type I collagen mRNA expression increased significantly at 1 day after irradiation, but little differences were found between the control group and at the 3rd day. The amount of alkaline phosphatase mRNA expression increased significantly at land 3 days after irradiation in the 1 Gy exposed group compared with the control group. Conclusion: The amount of type I collagen and alkaline phosphatase mRNA expression increased significantly 1 day after irradiation when compared with the control group.

• Clinical and Experimental Vaccine Research
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• 제12권2호
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• pp.156-171
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• 2023

Purpose: The development of vaccines that confer protection against multiple avian influenza A (AIA) virus strains is necessary to prevent the emergence of highly infectious strains that may result in more severe outbreaks. Thus, this study applied reverse vaccinology approach in strategically constructing messenger RNA (mRNA) vaccine construct against avian influenza A (mVAIA) to induce cross-protection while targeting diverse AIA virulence factors. Materials and Methods: Immunoinformatics tools and databases were utilized to identify conserved experimentally validated AIA epitopes. CD8⁺ epitopes were docked with dominant chicken major histocompatibility complexes (MHCs) to evaluate complex formation. Conserved epitopes were adjoined in the optimized mVAIA sequence for efficient expression in Gallus gallus. Signal sequence for targeted secretory expression was included. Physicochemical properties, antigenicity, toxicity, and potential cross-reactivity were assessed. The tertiary structure of its protein sequence was modeled and validated in silico to investigate the accessibility of adjoined B-cell epitope. Potential immune responses were also simulated in C-ImmSim. Results: Eighteen experimentally validated epitopes were found conserved (Shannon index <2.0) in the study. These include one B-cell (SLLTEVETPIRNEWGCR) and 17 CD8⁺ epitopes, adjoined in a single mRNA construct. The CD8⁺ epitopes docked favorably with MHC peptidebinding groove, which were further supported by the acceptable ∆G_bind (-28.45 to -40.59 kJ/mol) and Kd (<1.00) values. The incorporated Sec/SPI (secretory/signal peptidase I) cleavage site was also recognized with a high probability (0.964814). Adjoined B-cell epitope was found within the disordered and accessible regions of the vaccine. Immune simulation results projected cytokine production, lymphocyte activation, and memory cell generation after the 1st dose of mVAIA. Conclusion: Results suggest that mVAIA possesses stability, safety, and immunogenicity. In vitro and in vivo confirmation in subsequent studies are anticipated.

• 한국수정란이식학회지
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• 제31권3호
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• pp.179-183
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• 2016

Even though klotho deficiency in mice exhibits multiple aging-like phenotypes, studies using large animal models such as pigs, which have many similarities to humans, have been limited due to the absence of cell lines or animal models. The objective of this study was to generate homozygous klotho knockout porcine cell lines and cloned embryos. A CRISPR sgRNA specific for the klotho gene was designed and sgRNA (targeting exon 3 of klotho) and Cas9 RNPs were transfected into porcine fibroblasts. The transfected fibroblasts were then used for single cell colony formation and 9 single cell-derived colonies were established. In a T7 endonuclease I mutation assay, 5 colonies (#3, #4, #5, #7 and #9) were confirmed as mutated. These 5 colonies were subsequently analyzed by deep sequencing for determination of homozygous mutated colonies and 4 (#3, #4, #5 and #9) from 5 colonies contained homozygous modifications. Somatic cell nuclear transfer was performed to generate homozygous klotho knockout cloned embryos by using one homozygous mutation colony (#9); the cleavage and blastocyst formation rates were 72.0% and 8.3%, respectively. Two cloned embryos derived from a homozygous klotho knockout cell line (#9) were subjected to deep sequencing and they showed the same mutation pattern as the donor cell line. In conclusion, we produced homozygous klotho knockout porcine embryos cloned from genome-edited porcine fibroblasts.

• 미생물학회지
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• 제42권4호
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• pp.277-285
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• 2006

본 연구에서는 신경인성 방광으로 요도 카테터를 유치하고 있는 환자의 카테터로부터 카테터 내 요로감염(Catheter-Associate Urinary Tract Infection; CA-UTI)에 관여하는 박테리아인 Escherichia coli, Pseudomonas aeruginosa 그리고 Staphylococcus aureus를 순수 분리, 동정하였다. 이 균주들을 대상으로 하여 quorum sensing mechanism을 규명하는 기초 연구로 각 균주의 quorum sensing 신호물질인 autoinducer (AIs)를 합성하는 유전자의 mRNA 발현을 확인하고, 정략분석 하였다. 각 세 균주를 단일과 세 균주의 혼합으로 24시간, 30일 동안 배양하며 일정 시간 간격으로 sample을 얻었다. 이 중 24시간 배양한 sample을 가지고 reverse transcription polymerase chain reaction (RT-PCR)을 수행하여 각 AIs 합성 유전자가 발현되는 최초 박테리아 밀도를 확인하였다. 단일배양에서 E. coli와 S. aureus의 AIs 합성 유전자(ygaG와 luxS)의 mRNA가 발현되는 최초 박테리아 밀도는 $2.4{\times}20^5$ CFU/ml, $5.4{\times}10^6$ CFU/ml 이었으며 P. aeruginosa의 rhlI와 lasI의 경우 $6.9{\times}10^4$ CFU/ml로 나타났다. 세 균주의 혼합배양에서 ygaG와 luxS의 mRNA가 발현되는 최초 박테리아 밀도는$7.3{\times}10^5$ CFU/ml, $1.6{\times}10^7$ CFU/ml이었으며 rhlI와 lasI의 경우 $2.1{\times}10^5$ CFU/ml로 나타났다. 또한 30일 배양한 sample의 RT-PCR 결과, 배양초기부터 각 AIs 합성 유전자들의 mRNA가 30일 동안 일정한 양만큼 지속적으로 발현됨을 확인하였다. Real-time RT-PCR을 이용한 AIs 합성 유전자의 mRNA 발현을 정량 분석한 결과 각 균주에서 단일배양보다 혼합배양시 AIs 합성 유전자의 발현이 더 많았다. 가장 많은 발현량의 차이를 보인 경우 E. coli ygaG의 mRNA 발현량은 단일배양보다 세 균주의 혼합배양시 최고 약 30배 이상이 증가하였고, P. aeruginosa rhlI의 경우 단일배양보다 혼합배양시 최고 약 40배, P. aeruginosa lasI의 경우 최고 약 250배 그리고 S. aureus luxS의 경우는 단일배양보다 혼합배양시 최고 약 5배 이상 mRNA 발현량이 증가하였다. 또한 세 균주의 4가지 유전자 중 P. aeruginosa의 rhlI와 lasI의 mRNA가 가장 많은 양으로 발현됨을 확인하였다.

• Imaging Science in Dentistry
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• 제38권3호
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• pp.125-132
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• 2008

Purpose : To investigate the effects of irradiation on transforming growth factor ${\beta}_1$ (TGF-${\beta}_1$) mRNA expression and calcific nodule formation in MC3T3-E1 osteoblastic cell line. Materials and Methods : Cells were cultured in alpha-minimum essential medium ($\alpha$-MEM) supplemented with 10% fetal bovine serum and antibiotics. When the cells reached the level of 70-80% confluence, culture media were changed with $\alpha$-MEM supplemented with 10% FBS, 5 mM $\beta$-glycerol phosphate, and $50\;{\mu}g/mL$ ascorbic acid. Thereafter the cells were irradiated with a single dose of 2, 4, 6, 8 Gy at a dose rate of 1.5 Gy/min. The expression pattern of TGF-${\beta}_1$ mRNA, calcium content and calcific nodule formation were examined on day 3, 7, 14, 21, 28, respectively, after the irradiation. Results : The amount of TGF-${\beta}_1$ mRNA expression decreased significantly on day 7 after irradiation of 4, 6, 8 Gy. It also decreased on day 14 after irradiation of 6, 8 Gy. and decreased on day 21 after irradiation of 8 Gy. The amount of calcium deposition decreased significantly on day 7 after irradiation of 4, 8 Gy (P < 0.01) and showed a decreased tendency on day 14, 21 after irradiation of 4, 6, 8 Gy. The number of calcific nodules was decreased on day 7 after irradiation of 4, 8 Gy. Conclusion: Irradiation with a single dose of 4, 6, 8 Gy influences negatively the bone formation at the molecular level by affecting the TGF-${\beta}_1$ mRNA expression that was associated with proliferation and the production of extracellular matrix in MC3T3-E1 osteoblastic cell line.

• International Journal of Oral Biology
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• 제39권2호
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• pp.107-114
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• 2014

Taste is an important sense in survival and growth of animals. The growth and maintenance of taste buds, the receptor organs of taste sense, are under the regulation of various neurotrophic factors. But the distribution aspect of neurotrophic factors and their receptors in distinct taste cell types are not clearly known. The present research was designed to characterize mRNA expression pattern of neurotrophic factors and their receptors in distinct type of taste cells. In male 45-60 day-old Sprague-Dawley rats, epithelial tissues with and without circumvallate and folliate papillaes were dissected and homogenized, and mRNA expressions for neurotrophic factors and their receptors were determined by RT-PCR. The mRNA expressions of brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3), receptor tyrosine kinase B (TrkB), exclusion of nerve growth factor (NGF), neurotrophin-4/5 (NT4/5), receptor tyrosine kinase A (TrkA), receptor tyrosine kinase C (TrkC), and p75NGFR were observed in some population of taste cell. In support of this result and to characterize which types of taste cells express NT3, BDNF, or TrkB, we examined mRNA expressions of NT3, BDNF, or TrkB in the $PLC{\beta}2$ (a marker of Type II cell)-and/or SNAP25 (a marker of Type III cell)-positive taste cells by a single taste cell RT-PCR and found that the ratio of positively stained cell numbers were 17.4, 6.5, 84.1, 70.3, and 1.4 % for $PLC{\beta}2$, SNAP25, NT3, BDNF, and TrkB, respectively. In addition, all of $PLC{\beta}2$-and SNAP25-positive taste cells expressed NT3 mRNA, except for one taste bud cell. The ratios of NT3 mRNA expressions were 100% and 91.7% in the SNAP25-and $PLC{\beta}2$-positive taste cells, respectively. However, two TrkB-positive taste cells co-expressed neither $PLC{\beta}2$ nor SNAP 25. The results suggest that the most of type II or type III cells express BDNF and NT3 mRNA, but the expression is shown to be less in type I taste cells.

• Asian Pacific Journal of Cancer Prevention
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• 제16권17호
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• pp.7589-7594
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• 2015

Background: MicroRNAs (miRNAs) are small non-coding RNA molecules, implicated in several activities like initiation, progression and prognosis of various cancers. Single nucleotide polymorphisms (SNPs) in miRNA genes can lead to alteration in mRNA expression, resulting in diverse functional consequences. The aim of our study was to investigate the association of miR-149C>T and miR-196a2C>T SNPs with susceptibility to development of oral squamous cell carcinoma (OSCC) in South Indian subjects. Materials and Methods: 100 OSCC patients and 102 healthy controls from the general population were recruited for the study. Genetic analysis was performed by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP) as per a standard protocol. Results: The genotype frequencies in miR-196a2 polymorphism, of TT, CT and CC in the OSCC patients were 69%,10% and 22% respectively while for control group it was 80%, 15% and 5% respectively. The CC genotype of miR196a2 polymorphism was significantly associated with oral squamous cell carcinoma. The genotype frequencies in miR-149 polymorphisms of CC, CT and TT in the oral squamous cell carcinoma (OSCC) patients were 72%, 22% and 6% respectively and for control group 88%, 12% and 0% respectively. CT and TT genotypes of miR149 polymorphism were found to be significantly associated with OSCC (p = 0.05 and 0.07). Conclusions: Our study suggests that miR-196a2C>T and miR-149C>T polymorphisms may play crucial roles in the development of OSCC in South Indian subjects.

• BMB Reports
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• 제45권4호
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• pp.233-238
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• 2012

We present a top down separation platform for yeast ribosomal proteins using affinity chromatography and capillary electrophoresis which is designed to allow deposition of proteins onto a substrate. FLAG tagged ribosomes were affinity purified, and rRNA acid precipitation was performed on the ribosomes followed by capillary electrophoresis to separate the ribosomal proteins. Over 26 peaks were detected with excellent reproducibility (<0.5% RSD migration time). This is the first reported separation of eukaryotic ribosomal proteins using capillary electrophoresis. The two stages in this workflow, affinity chromatography and capillary electrophoresis, share the advantages that they are fast, flexible and have small sample requirements in comparison to more commonly used techniques. This method is a remarkably quick route from cell to separation that has the potential to be coupled to high throughput readout platforms for studies of the ribosomal proteome.