• Asian-Australasian Journal of Animal Sciences
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• v.31 no.8
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• pp.1103-1109
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• 2018

Objective: This study aimed to screen and identify the target genes of miR-375 in pig Sertoli (ST) cells and to elucidate the effect of miR-375 on the proliferation of ST cells. Methods: In this study, bioinformatics software was used to predict and verify miR-375 target genes. Quantitative polymerase chain reaction (PCR) was used to detect the relationship between miR-375 and its target genes in ST cells. Enzyme-linked immunosorbent assay (ELISA) of rearranged L-myc fusion (RLF) and hypoxia-induced gene domain protein 1A (HIGD1A) was performed on porcine ST cells, which were transfected with a miR-375 mimics and inhibitor to verify the results. Dual luciferase reporter gene assays were performed to assess the interactions among miR-375, RLF, and HIGD1A. The effect of miR-375 on the proliferation of ST cells was analyzed by CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS). Results: Five possible target genes of miR-375, including RLF, HIGD1A, colorectal cancer associated 2, POU class 3 homeobox 1, and WW domain binding protein 1 like, were found. The results of quantitative PCR suggested that mRNA expression of RLF and HIGD1A had a negative correlation with miR-375, indicating that RLF and HIGD1A are likely the target genes of miR-375. The ELISA results revealed that RLF and HIGD1A were negatively correlated with the miR-375 protein level. The luminescence results for the miR-375 group cotransfected with wild-type RLF and HIGD1A vector were significantly lower than those of the miR-375 group co-transfected with the blank vector or mutant RLF and HIGD1A vectors. The present findings suggest that RLF and HIGD1A are target genes of miR-375 and that miR-375 inhibits ST cell proliferation according to MTS analysis. Conclusion: It was speculated that miR-375 affects cell proliferation through its target genes, which play an important role in the development of testicular tissue.

• Journal of Korean Medicine for Obesity Research
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• v.19 no.2
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• pp.106-112
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• 2019

Objectives: The aim of this study was to observe the reduction of lipid accumulation by treatment with Akkermansia muciniphila extract on 3T3-L1 adipocytes. Methods: After treating pasteurized Akk. muciniphila strains in HT-29 colorectal cancer cell, the relative expression of interleukin (IL)-8, tumor necrosis factor-α, IL-6, and IL-1β mRNA was analyzed by real time polymerase chain reaction, respectively. 27 strains of Akk. muciniphila which have anti-inflammatory effects were selected. 3T3-L1 pre-adipocytes were treated with Akk. muciniphila for 24 hr and then measured the toxicity using water soluble tetrazolium salt assay. The cells were incubated for 4 days and then differentiated into adipocytes using the medium including adipogenic reagents for 10 days. The Akk. muciniphila was treated when the medium was exchanged for differentiation medium at 4^th day and insulin medium at 6^th day. To observe the lipid accumulation, the cells were stained with Oil red O dye and were measured using a spectrophotometer. Results: In the cytotoxicity test, the cell viability of 3T3-L1 pre-adipocytes was significantly increased compared to the control group which untreated with Akk. muciniphila, and there was no cytotoxicity of Akk. muciniphila at 1×10⁷ CFU/mL. The results on Oil red O staining and absorbance measurements were showed a significant decrease in lipid accumulation in the group which was treated with Akk. muciniphila compared to the control group. Conclusions: In our results, Akk. muciniphila has the inhibitory effect of lipid accumulation in 3T3-L1 adipocytes. This suggests that Akk. muciniphila could be help to improve obesity.

• Journal of Life Science
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• v.25 no.7
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• pp.773-779
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• 2015

This study examined extracts from five different kinds of lees and nuruk and their organic solvent fractions in terms of several biological functions, such as anti-adipogenic, anti-inflammatory, and anti-proliferative activities. The anti-adipogenic activity was investigated by treating mouse pre-adipocyte 3T3-L1 cells with one extract (YE) and four organic solvent fractions (YAc, PAc, RAc, and WPAc) during adipogenesis. Among the treated samples, the ethyl acetate fraction of W-Ju lees (WPAc) showed the strongest anti-adipogenic effect, which was confirmed with oil red O staining and down-regulation of pro-adipogenic genes such as PPAR-gamma and SCD-1. Treatment with WPAc also reduced the expression of PPAR-gamma in a time-dependent manner. The effects of five different extracts were examined on nitric oxide (NO) production in mouse RAW 264.7 cells to determine anti-inflammatory activity. The ethyl acetate fraction of B-Ju lees (PAc) significantly decreased NO production in LPS-stimulated RAW 264.7 cells and it also inhibited NO production in a dose-dependent manner. The PAc fraction also dramatically decreased the viability of human colorectal cancer HCT116 cells in a dose-dependent manner. In addition, PAc increased the expression of NAG-1 and ATF3 genes in a dose dependent manner. Overall, these results indicate that lees and nuruk have several biological functions, including anti-adipogenic, anti-inflammatory, and anti-proliferative activities.

• Journal of Nutrition and Health
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• v.52 no.2
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• pp.139-148
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• 2019

Purpose: Ulcerative colitis is a common inflammatory bowel disease. Prolonged colitis can be a risk factor for the development of colorectal cancer. Mulberry twig (MT, Sangzhi), a dry branch of Morus alba L., which is widely distributed throughout East Asia, has been shown to have anti-inflammatory activities in the cells. However, the effects of MT extracts on colitis in in vivo are limited. Therefore, in this study, we investigated the anti-inflammatory effects of MT extracts in the dextran sulfate sodium (DSS)-induced mouse colitis model. Methods: Six week-old, male ICR mice were divided into 3 groups: Control (n = 5), DSS (n = 7), and DSS+MT (n = 7) groups. Mice in the DSS and DSS+MT groups were administrated 3% DSS in drinking water for 5 days to induce colitis. At the same time, water extracts of MT (5 g/kg body weight/day) were orally administered to mice in the DSS+MT groups for 5 days. Results: The MT extracts significantly reduced the clinical and pathological characteristics of colitis. Disease activity index, mucosal thickness, and colonocyte proliferation were significantly reduced in the DSS+MT group compared with the DSS group. Furthermore, MT administration reduced the levels of plasma $TNF-{\alpha}$, IL-6, and the colonic myeloperoxidase activity as well as mRNA expression of $TNF-{\alpha}$, IL-6, Cox-2, and iNOS. Conclusion: Taken together, these results suggest that MT water extracts have potent anti-colitis activities in the mouse colitis model.

• Design & Manufacturing
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• v.15 no.2
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• pp.11-16
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• 2021

Recently, three-dimensional (3D) cell culture systems, which are superior to conventional two-dimensional (2D) vascular systems that mimic the in vivo environment, are being actively studied to reproduce drug responses and cell differentiation in organisms. Conventional two-dimensional cell culture methods (scaffold-based and non-scaffold-based) have a limited cell growth rate because the culture cannot supply the culture medium as consistently as microvessels. To solve this problem, we would like to propose a 3D culture system with an environment similar to living cells by continuously supplying the culture medium to the bottom of the 3D cell support. The 3D culture system is a structure in which microvascular structures are combined under a scaffold (agar, collagen, etc.) where cells can settle and grow. First, we have manufactured molds for the formation of four types of microvessel-mimicking chips: width / height ①100 ㎛ / 100 ㎛, ②100 ㎛ / 50 ㎛, ③ 150 ㎛ / 100 ㎛, and ④ 200 ㎛ / 100 ㎛. By injection molding, four types of microfluidic chips were made with GPPS (general purpose polystyrene), and a 100㎛-thick PDMS (polydimethylsiloxane) film was attached to the top of each microfluidic chip. As a result of observing the flow of the culture medium in the microchannel, it was confirmed that when the aspect ratio (height/width) of the microchannel is 1.5 or more, the fluid flows from the inlet to the outlet without a backflow phenomenon. In addition, the culture efficiency experiments of colorectal cancer cells (SW490) were performed in a 3D culture system in which PDMS films with different pore diameters (1/25/45 ㎛) were combined on a microfluidic chip. As a result, it was found that the cell growth rate increased up to 1.3 times and the cell death rate decreased by 71% as a result of the 3D culture system having a hole membrane with a diameter of 10 ㎛ or more compared to the conventional commercial. Based on the results of this study, it is possible to expand and build various 3D cell culture systems that can maximize cell culture efficiency by cell type by adjusting the shape of the microchannel, the size of the film hole, and the flow rate of the inlet.

• Journal of Korean Neurosurgical Society
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• v.65 no.6
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• pp.790-800
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• 2022

Objective : EID3 (EP300-interacting inhibitor of differentiation) was identified as a novel member of EID family and plays a pivotal role in colorectal cancer development. However, its role in glioma remained elusive. In current study, we identified EID3 as a novel oncogenic molecule in human glioma and is critical for glioma cell survival, proliferation and invasion. Methods : A total of five patients with glioma were recruited in present study and fresh glioma samples were removed from patients. Four weeks old male non-obese diabetic severe combined immune deficiency (NOD/SCID) mice were used as transplant recipient models. The subcutaneous tumor size was calculated and recorded every week with vernier caliper. EID3 and AMP-activated protein kinase α1 (AMPKα1) expression levels were confirmed by real-time polymerase chain reaction and Western blot assays. Colony formation assays were performed to evaluate cell proliferation. Methyl thiazolyl tetrazolium (MTT) assays were performed for cell viability assessment. Trypan blue staining approach was applied for cell death assessment. Cell Apoptosis DNA ELISA Detection Kit was used for apoptosis assessment. Results : EID3 was preferentially expressed in glioma tissues/cells, while undetectable in astrocytes, neuronal cells, or normal brain tissues. EID3 knocking down significantly hindered glioma cell proliferation and invasion, as well as induced reduction of cell viability, apoptosis and cell death. EID3 knocking down also greatly inhibited tumor growth in SCID mice. Knocking down of AMPKα1 could effectively rescue glioma cells from apoptosis and cell death caused by EID3 absence, indicating that AMPKα1 acted as a key downstream regulator of EID3 and mediated suppression effects caused by EID3 knocking down inhibition. These findings were confirmed in glioma cells generated patient-derived xenograft models. AMPKα1 protein levels were affected by MG132 treatment in glioma, which suggested EID3 might down regulate AMPKα1 through protein degradation. Conclusion : Collectively, our study demonstrated that EID3 promoted glioma cell proliferation and survival by inhibiting AMPKα1 expression. Targeting EID3 might represent a promising strategy for treating glioma.

• Animal Bioscience
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• v.37 no.2
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• pp.193-202
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• 2024

Objective: Oxidative stress (OS) is a pathological process arising from the excessive production of free radicals in the body. It has the potential to alter animal gene expression and cause damage to the jejunum. However, there have been few reports of changes in the expression of long noncoding RNAs (lncRNAs) in the jejunum in piglets under OS. The purpose of this research was to examine how lncRNAs in piglet jejunum change under OS. Methods: The abdominal cavities of piglets were injected with diquat (DQ) to produce OS. Raw reads were downloaded from the SRA database. RNA-seq was utilized to study the expression of lncRNAs in piglets under OS. Additionally, six randomly selected lncRNAs were verified using quantitative real-time polymerase chain reaction (qRT-PCR) to examine the mechanism of oxidative damage. Results: A total of 79 lncRNAs were differentially expressed (DE) in the treatment group compared to the negative control group. The target genes of DE lncRNAs were enriched in gene ontology (GO) terms and Kyoto encyclopedia of genes and genomes (KEGG) signaling pathways. Chemical carcinogenesis-reactive oxygen species, the Foxo signaling pathway, colorectal cancer, and the AMPK signaling pathway were all linked to OS. Conclusion: Our results demonstrated that DQ-induced OS causes differential expression of lncRNAs, laying the groundwork for future research into the processes involved in the jejunum's response to OS.

• Radiation Oncology Journal
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• v.26 no.3
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• pp.166-172
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• 2008

Purpose: The measurement of radiosensitivity of individuals is useful in radiation therapy. Unfortunately, the measurement of radiation survival using a clonogenic assay, which is the established standard, can be difficult and time consuming. The aim of this study is to compare radiosensitivity results obtained from the MTT and clonogenic assays, and to evaluate whether the MTT assay can be used on clinical specimens. Materials and Methods: HCT-8, LoVo, CT-26, and WiDr were the colon cancer cell lines used for this study. The clonogenic assay was performed to obtain the cell survival curves and surviving fractions at a dose of 2 Gy ($SF_2$) as the standard technique for radiosensitivity. Also, the MTT assay was performed for each of the cell lines (in vitro). To simulate clinical specimens, the cell lines were inoculated into nude mice, removed when the tumors reached 1 cm in diameter, and chopped. Next, the tumors were subjected to the same process involved with the MTT assay in vitro. The inhibition rates (IR) of 10 Gy or 20 Gy of irradiation for in vitro and ex vivo were calculated based on the optical density of the MTT assay, respectively. Results: According to $SF_2$ and the cell survival curve, the HCT-8 and WiDr cell lines were more resistant to radiation than LoVo and CT-26 (p<0.05). The IR was measured by in vitro. The MTT assay IR was 17.3%, 21%, 30% and 56.5% for the WiDr, HCT-8, LoVo and CT-26 cell lines, respectively. In addition, the IR measured ex vivo by the MTT assay was 23.5%, 26%, 38% and 53% in the HCT-8, WiDr, LoVo and CT-26 tumors, respectively. Conclusion: The radiosensitivity measured by the MTT assay was correlated with the measures obtained from the clonogenic assay. This result highlights the possibility that the MTT assay could be used in clinical specimens for individual radiosensitivity assays.

• Journal of Life Science
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• v.19 no.8
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• pp.1073-1080
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• 2009

A number of studies have demonstrated that the regular use of nonsteroidal anti-inflammatory drugs (NSAIDs) can reduce the risks of colorectal, oesophageal and lung cancers. NSAIDs have been shown to exert their anti-cancer effects through inducing apoptosis in cancer cells. The susceptibility of tumor cells to anti-tumor drug-induced apoptosis appears to depend on the balance between pro-apoptotic and anti-apoptotic programs such as nuclear factor kB (NF-kB), phosphatidylinositol 3-kinase (PI3K)-Akt/protein kinase B (PKB) and MEK1/2-ERK1/2 pathways. We examined the effects of pro-survival PI3K and ERK1/2 signal pathways on cell cycle arrest and apoptosis in response to NSAIDs including sulindac sulfide and NS398. We show that simultaneous inhibition of the Akt/PKB and ERK1/2 signal cascades could synergistically enhance the potential pro-apoptotic activities of sulindac sulfide and NS398. Similar enhancement was observed in cells treated with sulindac sulfide or NS398 and 100 ${\mu}$M genistein, an inhibitor of receptor tyrosine kinases (RTKs) that are upstream of PI3K and MEK1/2 signaling. We further demonstrate that NAG-1 is induced and plays a critical role(s) in apoptosis by NSAIDs-based combined treatment. In sum, our results show that combinatorialtreatment of sulindac sulfide or NS398 and genistein results in a highlysynergistic induction of apoptotic cell death to increase the chemopreventive effects of the NSAIDs, sulindac sulfide and NS398.

• Journal of Nutrition and Health
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• v.55 no.2
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• pp.211-226
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• 2022

Choline, an essential nutrient for humans, is required for the structural integrity of the cell membranes, methyl-group metabolism, synthesis of the neurotransmitter acetylcholine, synthesis of the membrane phospholipid components of the cell membranes, and the transport of lipids and cholesterol. Choline can be synthesized in the body, but it is insufficient to meet the daily requirements and hence it must be obtained through the diet. In the United States/Canada, Australia/New Zealand, Europe, China, and Taiwan, the adequate intake (AI) and tolerable upper intake level (UL) of choline have been established, while the establishment of the 2020 Dietary Reference Intakes for Koreans (KDRI) for choline was postponed due to the lack of a choline database for Korean foods and studies on the choline intake of Koreans. However, as part of the preparation work for the 2020 DRI revision and finalization, choline intake and the possibility of disease occurrence were verified through analysis of published data. The groundwork for the subsequent establishment of a choline DRI was laid through a literature search, evaluation, and review of the literature reported from 1949 up to 2019. This can be regarded as the culmination of this project. According to the results of randomized controlled trials (RCTs), cohort studies, case-control studies, and cross-sectional observational studies in humans, approximately 400-500 mg/day of choline intake was effective in preventing liver function damage (fatty liver), neural tube damage, cardiovascular disease, breast cancer, and cognitive function improvement. The same amount of choline intake, however, also correlated with the risk of prostate and colorectal cancer. At present, there is limited information available on choline intake and health outcomes, particularly for the Korean population. More human studies, including clinical trials on the requirements and the physiological benefits associated with dietary intake, are needed to establish the KDRI for choline.