• BMB Reports
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• 제55권2호
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• pp.104-109
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• 2022

Skeletal myogenesis is essential to keep muscle mass and integrity, and impaired myogenesis is closely related to the etiology of muscle wasting. Recently, miR-141-3p has been shown to be induced under various conditions associated with muscle wasting, such as aging, oxidative stress, and mitochondrial dysfunction. However, the functional significance and mechanism of miR-141-3p in myogenic differentiation have not been explored to date. In this study, we investigated the roles of miR-141-3p on CFL2 expression, proliferation, and myogenic differentiation in C2C12 myoblasts. MiR-141-3p appeared to target the 3'UTR of CFL2 directly and suppressed the expression of CFL2, an essential factor for actin filament (F-actin) dynamics. Transfection of miR-141-3p mimic in myoblasts increased F-actin formation and augmented nuclear Yes-associated protein (YAP), a key component of mechanotransduction. Furthermore, miR-141-3p mimic increased myoblast proliferation and promoted cell cycle progression throughout the S and G2/M phases. Consequently, miR-141-3p mimic led to significant suppressions of myogenic factors expression, such as MyoD, MyoG, and MyHC, and hindered the myogenic differentiation of myoblasts. Thus, this study reveals the crucial role of miR-141-3p in myogenic differentiation via CFL2-YAP-mediated mechanotransduction and provides implications of miRNA-mediated myogenic regulation in skeletal muscle homeostasis.

• BMB Reports
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• 제57권4호
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• pp.194-199
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• 2024

Overexpression of mitofusin-2 (MFN2), a mitochondrial fusion protein, is frequently associated with poor prognosis in cervical cancer patients. Here, I aimed to investigate the involvement of MFN2 in cervical cancer progression and determine the effect of MFN2 on prognosis in cervical cancer patients. After generating MFN2-knockdown SiHa cells derived from squamous cell carcinoma, I investigated the effect of MFN2 on SiHa cell proliferation using the Cell Counting Kit-8 assay and determined the mRNA levels of proliferation markers. Colony-forming ability and tumorigenesis were evaluated using a colony-formation assay and tumor xenograft mouse models. The migratory and invasive abilities associated with MFN2 were measured using wound-healing and invasion assays. Wnt/β-catenin-mediated epithelial-mesenchymal transition (EMT) markers related to MFN2 were assessed through quantitative RT-PCR. MFN2-knockdown SiHa cells exhibited reduced proliferation, colony formation, migration, invasion, and tumor formation in vivo. The motility of SiHa cells with MFN2 knockdown was reduced through Wnt/β-catenin-mediated EMT inhibition. MFN2 promoted cancer progression and tumorigenesis in SiHa cells. Overall, MFN2 could serve as a therapeutic target and a novel biomarker for cervical cancer.

• Asian-Australasian Journal of Animal Sciences
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• 제31권9호
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• pp.1420-1430
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• 2018

Objective: Epigallocatechin-3-gallate (EGCG) is a major ingredient of catechin polyphenols and is considered one of the most promising bioactive compounds in green tea because of its strong antioxidant properties. However, the protective role of EGCG in bovine oocyte in vitro maturation (IVM) has not been investigated. Therefore, we aimed to study the effects of EGCG on IVM of bovine oocytes. Methods: Bovine oocytes were treated with different concentrations of EGCG (0, 25, 50, 100, and $200{\mu}M$), and the nuclear and cytoplasmic maturation, cumulus cell expansion, intracellular reactive oxygen species (ROS) levels, total antioxidant capacity, the early apoptosis and the developmental competence of in vitro fertilized embryos were measured. The mRNA abundances of antioxidant genes (nuclear factor erythriod-2 related factor 2 [NRF2], superoxide dismutase 1 [SOD1], catalase [CAT], and glutathione peroxidase 4 [GPX4]) in matured bovine oocytes were also quantified. Results: Nuclear maturation which is characterized by first polar body extrusion, and cytoplasmic maturation characterized by peripheral and cortical distribution of cortical granules and homogeneous mitochondrial distribution were significantly improved in the $50{\mu}M$ EGCG-treated group compared with the control group. Adding $50{\mu}M$ EGCG to the maturation medium significantly increased the cumulus cell expansion index and upregulated the mRNA levels of cumulus cell expansion-related genes (hyaluronan synthase 2, tumor necrosis factor alpha induced protein 6, pentraxin 3, and prostaglandin 2). Both the intracellular ROS level and the early apoptotic rate of matured oocytes were significantly decreased in the $50{\mu}M$ EGCG group, and the total antioxidant ability was markedly enhanced. Additionally, both the cleavage and blastocyst rates were significantly higher in the $50{\mu}M$ EGCG-treated oocytes after in vitro fertilization than in the control oocytes. The mRNA abundance of NRF2, SOD1, CAT, and GPX4 were significantly increased in the $50{\mu}M$ EGCG-treated oocytes. Conclusion: In conclusion, $50{\mu}M$ EGCG can improve the bovine oocyte maturation, and the protective role of EGCG may be correlated with its antioxidative property.

• 대한본초학회지
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• 제31권4호
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• pp.101-109
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• 2016

Objectives : HT070 is a mixture of herbal extracts from root of Scutellaria baicalensis and stem bark of Eleutherococcus senticosus , which have long been used for stroke therapy in traditional Korean Medicine. The purpose of this study was to investigate the neuroprotective effects of HT070 on global cerebral ischemia and its potential mechanisms.Methods : Transient global cerebral ischemia was produced by 10 min of four-vessel occlusion (4-VO) in male Wistar rats. HT070 was administered orally at a dosage of 200 mg/kg twice at 0 and 90 min after reperfusion. Hippocampal neuronal damage was measured 7 days after reperfusion. To explore the potential mechanisms, we used hydrogen peroxide (H₂O₂)-induced rat pheochromocytoma (PC12) cells as an in vitro model. PC12 cells were pretreated with HT070 for 1 h and then exposed to 100 μM H₂O₂ for 6 h in the presence of HT070. Cell viability was measured by MTT assay and the mRNA expression of Bax, Bcl-2, iNOS and COX-2 were measured by quantitative RT-PCR.Results : Oral administration of HT070 at a dose of 200 mg/kg significantly reduced neuronal death in the hippocampal CA1 region by 13.4% as compared to the vehicle-treated group. HT070 increased cell viability, reversed the down-regulated Bcl-2 mRNA level, and suppressed the up-regulated mRNA expressions of Bax, iNOS, and COX-2 in H₂O₂-treated PC12 cells.Conclusions : HT070 protects against delayed neuronal death after global cerebral ischemia and its neuroprotection properties might be attributed to the inhibition of mitochondrial apoptosis and ROS-generating enzymes.

• 한국동물위생학회지
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• 제35권3호
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• pp.215-222
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• 2012

Species-specific $TaqMan^{(R)}$ probe-based real-time PCR assays were developed for detection of beef, pork, chicken, duck, goose and turkey. The primer and probe sets used in this study were designed to be complementary to fibroblast growth factor (FGF) for cattle and pig, mitochondrial NADH dehydrogenase (ND) subunit 3 and ND2 for chicken and duck, 12S rRNA for goose and turkey, respectively. As internal positive control we used conserved region in the ribosomal 18S RNA gene to ensure the accuracy of the detection of target DNA by real-time PCR. We confirmed that real-time PCR assays with the primer and probe sets were positive for cattle, pig and chicken intended target animal species with no cross-reactivity with other non-target animal species. Only >50 ng DNA of beef show cross-reactivity in the determination of duck. Using species-specific primer and probe sets, it was possible to detect amounts of 0.1 ng DNA of cattle and pig, 1.0 pg DNA of chicken, duck and turkey, and 0.1 pg DNA of goose for raw samples, respectively. The detection limits were 0.1 ng DNA of cattle, 1.0 ng DNA of pig and 1.0 pg DNA of chicken for DNA mixtures (beef, pork and chicken) extracted from heat-treated ($121^{\circ}C$/5 min) meat samples. In conclusion, it can be suggested that the $TaqMan^{(R)}$ probe-based assay developed in this study might be a rapid and specific method for the identification of meat species in raw or cooked meat products.

• 한국식품위생안전성학회지
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• 제29권4호
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• pp.347-355
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• 2014

본 연구에서는 식품 중 동물성 사용원료의 진위 판별을 위하여 분자생물학적 기법을 이용한 시험법을 개발하였다. 동물성 식품원료의 종 판별을 위한 유전자로는 미토콘드리아 DNA에 존재하는 COI, Cytb, 및 16S rRNA 유전자를 대상으로 하였으며, 가공식품에 적용하기 위하여 PCR 산물의 크기는 200 bp 내외가 되도록 종 특이 프라이머를 설계하였다. 대상종으로는 가축류 2종, 가금류 6종, 민물어류 2종, 해양어류 13종 및 갑각류 1종, 총 24종을 선정하였으며 종 특이 프라이머를 이용하여 예상되는 PCR 산물의 생성 유무를 확인하였다. PCR을 수행한 결과 토끼, 여우, 꿩, 집비둘기, 멧비둘기, 메추리, 참새, 제비, 메기, 쏘가리, 날치, 열빙어, 청어, 까나리, 멸치, 참조기, 넙치, 조피볼락, 홍어, 가오리, 말쥐치, 농어, 성게 및 바닷가재에 대하여 각각 156, 204, 152, 160, 113, 163, 167, 152, 165, 121, 136, 151, 178, 178, 146, 188, 177, 166, 179, 218, 188, 185, 127 및 172 bp에서 PCR 증폭 산물을 확인하였다. 그리고 프라이머 별로 비교종에서는 비특이적 PCR 산물(non-specific PCR product)은 생성되지 않았다. 본 연구에서 개발된 유전자 분석법을 이용하여 동물성 식품원료가 사용된 식품 원료 및 가공식품의 진위 판별에 활용이 가능할 것이며, 불량식품 근절에 크게 기여할 것으로 기대된다.

• 한국식품영양과학회지
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• 제31권5호
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• pp.788-795
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• 2002

본 연구는 프락토즈 중합체인 레반의 지질 감소 효과와 비만 유전자인 UCP를 통한 에너지 대사에 미치는 효과를 살펴보기 위하여 성장기 6주간 AIN-76A diet로 사육한 흰쥐에게 레반을 식이 섭취량의 1%, 2%로 5주간 경구투여하여, 혈중 지질 수준과 체지방 형성 및 혈중 인슬린, 렙틴 농도와 갈색 지방 조직, 백색지방 조직, 골격 근육, 시상하부에서 의 UCP mRNA 발현량 변화를 관찰하였다. 연구 결과를 종합해 보면 다음과 같다. 1) 레반군에서 혈청 중성지방과 총 콜레스테롤은 감소하고 HDL 콜레스테롤 수준은 영향을 미치지 않아 1%와 2% 레반 공급이 지질 대사를 개선시킬 수 있음을 보여주었다. 2) 1%와 2% 레반군에서 내장 지방 무게가 감소하여 적은 양의 레반 공급으로 체지방 축적이 억제될 수 있음을 보여주었다. 3) 갈색 지방 조직과 부고환 지방, 복막 지방 무게는 그룹간 차이가 나타나지 않았다. 4) 혈청 인슬린 농도는 1% 레반군에서, 혈청 렙틴 농도는 1% 레반군, 2% 레반군에서 대조군에 비해 감소하는 경향을 나타내었으나 통계적 유의성은 나타나지 않았다. 5) 1%와 2% 레반 공급에 의하여 갈색 지방 조직과 골격근육, 시상하부에서의 UCP mRNA발현량에는 변화가 없었다. 6) 백색 지방 조직의 UCP 2 mRNA 발현량이 레반 공급에 의해 증가하여 1%와 2%레반 공급에 의해 에너지 소비율이 증가할 수 있음이 나타났다 결론적으로 저농도의 1%와 1% 액상 레반 공급은 총 식이의 ,3%～5% 레반 공급과 비교하여 혈중 총 콜레스테롤과 중성 지방 감소 효과에는 차이가 있으나 UCP 발현 증가에 미치는 영향은 적은 것으로 보인다. 그러나 저농도의 1～2% 액상 레반 공급으로 지질 대사를 개선하고 체지방 축적을 어느 정도 억제하는 효과를 나타냄을 알 수 있으며 이에 관한 임상 연구로 레반의 고지혈증과 비만을 조절하는 기능성 식품 소재로서의 가능성 검색이 요망된다.

• 대한본초학회지
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• 제30권6호
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• pp.7-15
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• 2015

Objectives : Rosa laevigata Michx. has been used for the treatment of renal disease in traditional Korean medicine. In this study, we investigated cytoprotective effect of R. laevigata water extract (RLE) against oxidative stress induced by arachidonic acid (AA) + iron.Methods : To evaluate the protective effects of RLE against AA + iron-induced oxidative stress in HepG2 cell, cell viability and changes on apoptosis-related proteins were assessed by MTT and immunoblot analyses. The effects of RLE on reduced glutathione level, production of reactive oxygen species and mitochondrial membrane potential were also monitored. Furthermore, to verify underlying molecular mechanism, NF-E2-related factor 2 (Nrf2) was examined by immunoblot analysis. Additionally, Nrf2 transactivation and its downstream target genes expression were also determined by reporter gene and realtime RT-PCR analyses.Results : RLE pretreatment (30-300 μg/ml) prevented cells from AA + iron-mediated cell death in a concentration dependent manner. In addition, 100 μg/ml RLE inhibited AA + iron-induced glutathione depletion, reactive oxygen species production and mitochondrial dysfunction. RLE accumulated nuclear Nrf2 and also transactivated Nrf2, which was evidenced by antioxidant response element- and glutathione S-transferase A2-driven luciferase activities and mRNA level of glutamate-cysteine ligase catalytic subunit, NAD(P)H:quinone oxidoreductase 1 and sestrin 2. Moreover, protective effect of RLE against AA + iron was abolished in Nrf2 knockout cells.Conclusions : These results indicate that RLE has the ability to protect hepatocyte against oxidative stress through Nrf2 activation.

• 한국식품영양과학회지
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• 제40권12호
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• pp.1654-1661
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• 2011

AKE의 농도별 처리가 인체 유방암 세포 MDA-MB-231의 세포사멸에 미치는 영향을 확인하기 위하여 세포 화학적인 방법인 MTT 분석, 이중 핵 염색법(Hoechst 33342/EtBr staining), FACS를 통하여 세포사멸을 관찰하였다. MTT 분석 결과, 150 ${\mu}g$/mL 처리 군에서 대조군에 대비하여 약 50%의 세포사멸을 나타내었으며 세포사멸이 농도 의존적으로 증가되었고(p<0.05), 이중 핵 염색법을 이용하여 세포사의 구분 결과 능동적 세포예정사인 apoptosis가 농도 의존적으로 급격히 증가하였으며(p<0.05), 특히 150 ${\mu}g$/mL 처리군에서 현저한 증가율을 나타내었다. 보다 더 명확한 세포사멸을 확인하기 위하여 FACS를 이용한 apoptosis 측정 결과, 처리군 간 크게 차이를 보이며 농도 의존적으로 증가되었다. 세포사멸관련 mRNA 유전자 발현을 관찰한 결과, 세포사멸 억제 유전자 Bcl-2는 처리농도가 증가할수록 유의적 증가를 보였으며(p<0.05), 세포사멸 유도 유전자 Bax는 유의적 감소를 나타내었다(p<0.05). 세포사멸의 지표인 Bcl-2/Bax의 비율은 농도 의존적인 감소를 나타내었으며(p<0.05), 세포사멸유도의 마지막 단계의 실행자인 caspase-3의 활성도 첨가 농도 의존적으로 증가하여 세포사멸을 유도하는 것으로 확인되었다(p<0.05). 결론적으로, AKE는 유방암 세포 MDA-MB-231의 세포사멸을 유도하는 것으로 나타나 신선초의 항암효과의 가능성을 제시해주었다. 향후 in vivo 실험에서도 신선초의 항암효과에 대한 심층적 연구가 이뤄져야 할 것으로 사료된다.

• 한국식품영양과학회지
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• 제42권1호
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• pp.8-16
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• 2013

본 연구는 체내 세포배양을 통한 적양배추 추출물의 항유방암 효과를 검정하기 위해 세포활성, 세포사멸, ROS 축적 및 세포사멸 관련 유전자 발현을 분석하였다. 적양배추 추출물을 0.5, 0.75, 1.0, 1.25, 1.5, 1.75 및 2.0 mg/mL의 농도로 24시간 처리하여 세포 생존율(MTT assay)을 분석한 결과 농도 의존적으로 감소되었다(p<0.05). 또한 세포사멸/괴사분석(Hoechst33342/ethidium bromide 염색법), flow cytometry assay, ROS 측정(DCF-DA 염색법) 등의 세포 화학적 방법을 통해 분석한 결과 처리 농도가 증가할수록 세포사멸이 유의적으로 증가하였고, ROS 생성 또한 증가하였다. 특히 2.0 mg/mL의 농도에서는 다른 농도에 비해 유의적으로 높은 사멸율을 나타내었으며, ROS 또한 다량으로 생성되는 것을 관찰할 수 있었다(p<0.05). RT-PCR을 통해 세포사멸관련 유전자인 Bcl-2, Bax, caspase-3의 mRNA 발현 정도를 관찰한 결과 농도 의존적으로 Bcl-2는 유의적으로 감소하였고, Bax와 caspase-3는 유의적으로 증가하였으며, Bcl-2/Bax의 비율은 유의적으로 감소하였다(p<0.05). 이상의 결과로 보아 적양배추 추출물이 인체 유방암세포 MDAMB-231의 세포사멸을 증가시키는 효과가 있음을 알 수 있었다.