• Asian Pacific Journal of Cancer Prevention
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• 제15권6호
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• pp.2753-2758
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• 2014

Background: GC-binding factor 2 (GCF2) is a transcriptional regulator that represses transcriptional activity of the epidermal growth factor receptor (EGFR) by binding to a specific GC-rich sequence in the EGFR gene promoter. In addition to this function, GCF2 has also been identified as a tumor-associated antigen and regarded as a potentially valuable serum biomarker for early human hepatocellular carcinoma (HCC) diagnosis. GCF2 is high expressed in most HCC tissues and cell lines including HepG2. This study focused on the influence of GCF2 on cell proliferation and apoptosis in HepG2 cells. Materials and Methods: GCF2 expression at both mRNA and protein levels in HepG2 cells was detected with reverse transcription (RT) PCR and Western blotting, respectively. RNA interference (RNAi) technology was used to knock down GCF2 mRNA and protein expression. Afterwards, cell viability was analyzed with a Cell Counting Kit-8 (CCK-8), and cell apoptosis and caspase 3 activity by flow cytometry and with a Caspase 3 Activity Kit, respectively. Results: Specific down-regulation of GCF2 expression caused cell growth inhibition, and increased apoptosis and caspase 3 activity in HepG2 cells. Conclusions: These primary results suggest that GCF2 may influence cell proliferation and apoptosis in HepG2 cells, and also provides a molecular basis for further investigation into the possible mechanism at proliferation and apoptosis in HCC.

• Asian Pacific Journal of Cancer Prevention
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• 제15권13호
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• pp.5437-5442
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• 2014

Esophageal squamous cell carcinoma (ESCC) is one of the most malignancies with a poor prognosis. The phospholipase $C{\varepsilon}$ gene (PLCE1) encodes a novel ras-related protein effector mediating the effects of R-Ras on the actin cytoskeleton and membrane protrusion. However, molecular mechanisms pertinent to ESCC are unclear. We therefore designed PLCE1-special small interfering RNA and transfected to esophageal squamous cell (EC) 9706 cells to investigat the effects of PLCE1 gene silencing on the cell cycle and apoptosis of ESCC and indicate its important role in the development of ESCC. Esophageal cancer tissue specimens and normal esophageal mucosa were obtained and assayed by immunohistochemical staining to confirm overexpression of PLCE1 in neoplasias. Fluorescence microscopy was used to examine transfection efficiency, while the result of PLCE1 silencing was examined by reverse transcription (RT-PCR). Flow cytometry and annexin V apoptosis assays were used to assess the cell cycle and apoptosis, respectively. Expression of cyclin D1 and caspase-3 was detected by Western-blotting. The level of PLCE1 protein in esophageal cancer tissue was significantly higher than that in normal tissue. After transfection, the expression of PLCE1 mRNA in EC 9706 was significantly reduced, compared with the control group. Furthermore, flow cytometry results suggested that the PLCE1 gene silencing arrested the cell cycle in the G0/G1 phase; apoptosis was significantly higher than in the negative control group and mock group. PLCE1 gene silencing by RNAi resulted in decreased expression of cyclin D1 and increased expression of caspase-3. Our study suggests that PLCE1 may be an oncogene and play an important role in esophageal carcinogenesis through regulating proteins which control cell cycling and apoptosis.

• Asian Pacific Journal of Cancer Prevention
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• 제14권12호
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• pp.7375-7379
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• 2013

The main aim of this study was to investigate the roles of GST-${\pi}$ and $pol{\beta}$ genes in the chemoresistance of esophageal carcinoma cells. Eukaryotic expression vectors containing each gene were constructed and transfected into EC9706 cells, and the biological effects of the two genes assessed based on a resistance index. We additionally investigated the in vitro and in vivo anti-resistance effects of GST-${\pi}$ and $pol{\beta}$ genes using recombinant lentiviruses carrying siRNAs against the two genes. Our results showed that upregulation of GST-${\pi}$ and $pol{\beta}$ genes suppresses chemosensitivity of esophageal carcinoma cells to cisplatin, while downregulation of these two genes with RNAi technology reverses this chemoresistance. Multi-site injection of recombinant lentivirus targeting the GST-${\pi}$ gene into transplanted cDDP tumors effectively reversed their chemoresistant phenotype. However, the same treatment against the $pol{\beta}$ gene did not lead to significant efficacy against chemoresistance.

• Molecules and Cells
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• 제39권2호
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• pp.163-168
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• 2016

Caffeine has both positive and negative effects on physiological functions in a dose-dependent manner. C. elegans has been used as an animal model to investigate the effects of caffeine on development. Caffeine treatment at a high dose (30 mM) showed detrimental effects and caused early larval arrest. We performed a comparative proteomic analysis to investigate the mode of action of high-dose caffeine treatment in C. elegans and found that the stress response proteins, heat shock protein (HSP)-4 (endoplasmic reticulum [ER] chaperone), HSP-6 (mitochondrial chaperone), and HSP-16 (cytosolic chaperone), were induced and their expression was regulated at the transcriptional level. These findings suggest that high-dose caffeine intake causes a strong stress response and activates all three stress-response pathways in the worms, including the ER-, mitochondrial-, and cytosolic pathways. RNA interference of each hsp gene or in triple combination retarded growth. In addition, caffeine treatment stimulated a food-avoidance behavior (aversion phenotype), which was enhanced by RNAi depletion of the hsp-4 gene. Therefore, up-regulation of hsp genes after caffeine treatment appeared to be the major responses to alleviate stress and protect against developmental arrest.

• Journal of Photoscience
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• 제9권2호
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• pp.98-101
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• 2002

Euglena gracilis abruptly changes its swimming direction after a sudden increase or decrease in incident light intensity, that is, step-up or step-down photophobic responses, resulting in photoavoidance or photoaccumulation, respectively. To identify the photoreceptor molecules for these UV-A/blue-light type photobehaviors, we purified a flavoprotein from isolated putative photosencory organelles (PFBs) of Euglena. The purified flavoprotein, which noncovalently bound flavin adenine dinucleotide (FAD), seemed to be a heterotetramer of alpha- and beta-subunits. Predicted amino acid sequences of each of the subunits were similar to each other and contained two FAD-binding domains each followed by an adenylyl cyclase catalytic domain. The purified flavoprotein actually showed adenylyl cyclase activity, being drastically elevated by blue-light irradiation. Suppression of gene expression of the flavoprotein (Photoactivated Adenylyl Cyclase, PAC) by RNA interference (RNAi) caused loss of the step-up photophobic response, demonstrating that PAC actually mediates photoavoidance of Euglena.

• BMB Reports
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• 제56권12호
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• pp.657-662
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• 2023

Neurodegenerative diseases are characterized by distinct protein aggregates, such as those of α-synuclein and tau. Lysosomal defect is a key contributor to the accumulation and propagation of aberrant protein aggregates in these diseases. The discoveries of common proteinopathies in multiple forms of lysosomal storage diseases (LSDs) and the identification of some LSD genes as susceptible genes for those proteinopathies suggest causative links between LSDs and the proteinopathies. The present study hypothesized that defects in lysosomal genes will differentially affect the propagation of α-synuclein and tau proteins, thereby determining the progression of a specific proteinopathy. We established an imaging-based high-contents screening (HCS) system in Caenorhabditis elegans (C. elegans) model, by which the propagation of α-synuclein or tau is measured by fluorescence intensity. Using this system, we performed RNA interference (RNAi) screening to induce a wide range of lysosomal malfunction through knock down of 79 LSD genes, and to obtain the candidate genes with significant change in protein propagation. While some LSD genes commonly affected both α-synuclein and tau propagation, our study identified the distinct sets of LSD genes that differentially regulate the propagation of either α-synuclein or tau. The specificity and efficacy of these LSD genes were retained in the disease-related phenotypes, such as pharyngeal pumping behavior and life span. This study suggests that distinct lysosomal genes differentially regulate the propagation of α-synuclein and tau, and offer a steppingstone to understanding disease specificity.

• Asian Pacific Journal of Cancer Prevention
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• 제15권5호
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• pp.2291-2295
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• 2014

Although nucleolar protein nucleostemin (NS) is essential for cell proliferation and early embryogenesis and expression has been observed in some types of human cancer and stem cells, the molecular mechanisms involved in mediation of cell proliferation and cell cycling remains largely elusive. The aim of the present study was to evaluate NS as a potential target for gene therapy of human breast carcinoma by investigating NS gene expression and its effects on SKBR-3 cell proliferation and apoptosis. NS mRNA and protein were both found to be highly expressed in all detected cancer cell lines. The apoptotic rate of the pcDNA3.1-NS-Silencer group ($12.1-15.4{\pm}3.8%$) was significantly higher than those of pcDNA3.1-NS ($7.2-12.0{\pm}1.7%$) and non-transfection groups ($4.1-6.5{\pm}1.8%$, P<0.01). MTT assays showed the knockdown of NS expression reduced the proliferation rate of SKBR-3 cells significantly. Matrigel invasion and wound healing assays indicated that the number of invading cells was significantly decreased in the pcDNA3.1-NS-siRNA group (P<0.01), but there were no significant difference between non-transfected and over-expression groups (P>0.05). Moreover, RNAi-mediated NS down-regulation induced SKBR-3 cell G1 phase arrest, inhibited cell proliferation, and promoted p53 pathway-mediated cell apoptosis in SKBR-3 cells. NS might thus be an important regulator in the G2/M check point of cell cycle, blocking SKBR-3 cell progression through the G1/S phase. On the whole, these results suggest NS might be a tumor suppressor and important therapeutic target in human cancers.

• Asian Pacific Journal of Cancer Prevention
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• 제13권10호
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• pp.5137-5142
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• 2012

UHRF2 is a member of the ubiquitin plant homeo domain RING finger family, which has been proven to be frequently up-regulated in colorectal cancer cells and play a role as an oncogene in breast cancer cells. However, the role of UHRF2 in glioma cells remains unclear. In this study, we performed real-time quantitative PCR on 32 pathologically confirmed glioma samples (grade I, 4 cases; grade II, 11 cases; grade III, 10 cases; and grade IV, 7 cases; according to the 2007 WHO classification system) and four glioma cell lines (A172, U251, U373, and U87). The expression of UHRF2 mRNA was significantly lower in the grade III and grade IV groups compared with the noncancerous brain tissue group, whereas its expression was high in A172, U251, and U373 glioma cell lines. An in vitro assay was performed to investigate the functions of UHRF2. Using a lentivirus-based RNA interference (RNAi) approach, we down-regulated UHRF2 expression in the U251 glioma cell line. This down-regulation led to the inhibition of cell proliferation, an increase in cell apoptosis, and a change of cell cycle distribution, in which S stage cells decreased and G2/M stage cells increased. Our results suggest that UHRF2 may be closely related to tumorigenesis and the development of gliomas.

• 한국육종학회지
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• 제50권4호
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• pp.463-471
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• 2018

들깨는 우리나라에서 재배되어온 대표적인 유지작물로 지질의 함량 중 알파리놀렌산의 함량이 60% 전후로 불포화도가 높아서 산패가 쉽게 일어나는 문제가 있다. 알파리놀렌산은 소포체 유래의 ${\Delta}15$ desaturase (FAD3)와 엽록체 유래의 ${\Delta}15$ desaturase (FAD7)에 의해서 합성된다. 엽록체 유래의 FAD7 유전자 발현의 손상없이 종자의 알파리놀렌산 함량을 낮추기 위해 소포체 유래 FAD3 유전자를 RNAi기법을 이용하여 발현을 억제하였다. 재배종인 엽실들깨의 배축을 이용하여 아그로박테리움 매개 형질전환법으로 제초제(바스타) 저항성을 가진 형질전환체 17개체를 획득하였다. 형질전환체는 0.3% (v/v) 바스타제초제를 이용하여 선발하였으며 Northern blot으로 FAD3 유전자의 발현이 억제되는 것을 확인하였다. 온실에서 수확한 12개체 종자 지방산 함량을 분석한 결과 알파리놀렌산 함량이 10-20% 2개체, 30-40% 7개체, 대조구와 비슷한 60%대 3개체를 획득하였다. 형질전환체의 $T_2$ 종자의 분리비와 지방산 조성을 분석한 결과 동형접합체 계통에서 6-10% 알파리놀렌산 함량을 보였으며 이형접합계통은 20-26% 알파리놀렌산 함량을 나타내어 동형으로 고정시 FAD3 유전자 발현이 상당히 강력히 억제됨을 확인할 수 있었다. 들기름의 지방산 중 알파리놀렌산 함량의 감소는 들깨의 산패를 방지하고 감마리놀렌산 등의 고가의 건강기능성 지방산 생산에 활용할 수 있을 것으로 기대된다.