• BMB Reports
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• 제52권2호
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• pp.157-162
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• 2019

Our previous study found that two novel cancer-related genes, PRR11 and SKA2, constituted a classic gene pair that was regulated by p53 and NF-Y in lung cancer. However, their role and regulatory mechanism in breast cancer remain elusive. In this study, we found that the expression levels of PRR11 and SKA2 were upregulated and have a negative prognotic value in breast cancer. Loss-of-function experiments showed that RNAi-mediated knockdown of PRR11 and/or SKA2 inhibited proliferation, migration, and invasion of breast cancer cells. Mechanistic experiments revealed that knockdown of PRR11 and/or SKA2 caused dysregulation of several downstream genes, including CDK6, TPM3, and USP12, etc. Luciferase reporter assays demonstrated that wild type p53 significantly repressed the PRR11-SKA2 bidirectional promoter activity, but not NF-Y. Interestingly, NF-Y was only essential for and correlated with the expression of PRR11, but not SKA2. Consistently, adriamycin-induced (ADR) activation of endogenous p53 also caused significant repression of the PRR11 and SKA2 gene pair expression. Notably, breast cancer patients with lower expression levels of either PRR11 or SKA2, along with wild type p53, exhibited better disease-free survival compared to others with p53 mutations and/or higher expression levels of either PRR11 or SKA2. Collectively, our study indicates that the PRR11 and SKA2 transcription unit might be an oncogenic contributor and might serve as a novel diagnostic and therapeutic target in breast cancer.

• 생명과학회지
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• 제18권10호
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• pp.1348-1354
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• 2008

mi transcription factor (MITF)는 비만세포의 분화를 조절하는 중요한 전사인자이다. 특히 MITF는 결합조직형 비만세포에서 일반적으로 발현하는 비만세포 특이적 세린 단백분해효소의 일종인 mMCP-6 유전자의 전사를 조절한다. 본 연구는 마우스 골수유래 배양비만세포에서 mMCP-6 유전자의 전사를 조절하는 MITF이형체를 규명하였다. MITF 이형체들의 발현은 RT-PCR로 확인하였다. IL-3존재 하에서 배양한 점막형 비만세포들은 MITF-A,-E, -H, -Mc 등이 발현하였다. 반면에 SCF존재 하에서 배양한 결합조직형 비만세포들은 MITF-A가 발현하였다. MITF이형체를 과발현시키면 NIH-3T3 세포에서 mMCP-6 promoter를 통한 luciferase 활성을 증가시키고, MC/9 비만세포주에서는 증가된 mMCP-6발현을 유도하였다. 더불어 비만세포에서의 mMCP-6 발현은 MITF-A 고갈로 인하여 유의적으로 억제되었다. MITF-A의 전사활성과 DNA결합은 MITF-E, -H, -Mc 등의 타 이형체들의 결과와 유사하였다. 따라서 본 연구의 결과들은 MITF-A가 마우스 결합조직형 비만세포에서 발현하여 mMCP-6 전사를 조절하는 중요한 이형체임을 제시한다.

• Molecules and Cells
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• 제37권12호
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• pp.873-880
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• 2014

In our previous study, miRNA-183, a miRNA in the miR-96-182-183 cluster, was significantly over-expressed in esophageal squamous cell carcinoma (ESCC). In the present study, we explored the oncogenic roles of miR-183 in ESCC by gain and loss of function analysis in an esophageal cancer cell line (EC9706). Genome-wide mRNA micro-array was applied to determine the genes that were regulated directly or indirectly by miR-183. 3'UTR luciferase reporter assay, RT-PCR, and Western blot were conducted to verify the target gene of miR-183. Cell culture results showed that miR-183 inhibited apoptosis (p < 0.05), enhanced cell proliferation (p < 0.05), and accelerated G1/S transition (p < 0.05). Moreover, the inhibitory effect of miR-183 on apoptosis was rescued when miR-183 was suppressed via miR-183 inhibitor (p < 0.05). Western blot analysis showed that the expression of programmed cell death 4 (PDCD4), which was predicted as the target gene of miR-183 by microarray profiling and bioinformatics predictions, decreased when miR-183 was over-expressed. The 3'UTR luciferase reporter assay confirmed that miR-183 directly regulated PDCD4 by binding to sequences in the 3'UTR of PDCD4. Pearson correlation analysis further confirmed the significant negative correlation between miR-183 and PDCD4 in both cell lines and in ESCC patients. Our data suggest that miR-183 might play an oncogenic role in ESCC by regulating PDCD4 expression.

• Advances in nano research
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• 제12권5호
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• pp.501-514
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• 2022

This study aimed to investigate the effects and potential mechanisms of Chikusetsusaponin V (CsV) on endothelial nitric oxide synthase (eNOS) and vascular endothelial cell functions. Different concentrations of CsV were added to animal models, bovine aorta endothelial cells (BAECs) and human umbilical vein endothelial cells (HUVECs) cultured in vitro. qPCR, Western blotting (WB), and B ultrasound were performed to explore the effects of CsV on mouse endothelial cell functions, vascular stiffness and cellular eNOS mRNA, protein expression and NO release. Bioinformatics analysis, network pharmacology, molecular docking and protein mass spectrometry analysis were conducted to jointly predict the upstream transcription factors of eNOS. Furthermore, pulldown and ChIP and dual luciferase assays were employed for subsequent verification. At the presence or absence of CsV stimulation, either overexpression or knockdown of purine rich element binding protein A (PURA) was conducted, and PCR assay was employed to detect PURA and eNOS mRNA expressions, Western blot was used to detect PURA and eNOS protein expressions, cell NO release and serum NO levels. Tube formation experiment was conducted to detect the tube forming capability of HUVECs cells. The animal vasodilation function test detected the vasodilation functions. Ultrasonic detection was performed to determine the mouse aortic arch pulse wave velocity to identify aortic stiffness. CsV stimulus on bovine aortic cells revealed that CsV could upregulate eNOS protein levels in vascular endothelial cells in a concentration and time dependent manner. The expression levels of eNOS mRNA and phosphorylation sites Ser1177, Ser633 and Thr495 increased significantly after CsV stimulation. Meanwhile, CsV could also enhance the tube forming capability of HUVECs cells. Following the mice were gavaged using CsV, the eNOS protein level of mouse aortic endothelial cells was upregulated in a concentration- and time-dependent manner, and serum NO release and vasodilation ability were simultaneously elevated whereas arterial stiffness was alleviated. The pulldown, ChIP and dual luciferase assays demonstrated that PURA could bind to the eNOS promoter and facilitate the transcription of eNOS. Under the conditions of presence or absence of CsV stimulation, overexpression or knockdown of PURA indicated that the effect of CsV on vascular endothelial function and eNOS was weakened following PURA gene silence, whereas overexpression of PURA gene could enhance the effect of CsV upregulating eNOS expression. CsV could promote NO release from endothelial cells by upregulating the expression of PURA/eNOS pathway, improve endothelial cell functions, enhance vasodilation capability, and alleviate vessel stiffness. The present study plays a role in offering a theoretical basis for the development and application of CsV in vascular function improvement, and it also provides a more comprehensive understanding of the pharmacodynamics of CsV.

• Animal Bioscience
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• 제35권9호
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• pp.1327-1339
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• 2022

Objective: Fat deposition in poultry is an important factor in production performance and meat quality research. miRNAs also play important roles in regulating adipocyte differentiation process. This study was to investigate the expression patterns of miRNAs in duck adipocytes after differentiation and explore the role of miR-214 in regulating carnitine palmitoyltransferases 2 (CPT2) gene expression during duck adipocyte differentiation. Methods: Successful systems for the isolation, culture, and induction of duck primary fat cells was developed in the experiment. Using Illumina next-generation sequencing, the miRNAs libraries of duck adipocytes were established. miRanda was used to predict differentially expressed (DE) miRNAs and their target genes. The expression patterns of miR-214 and CPT2 during the differentiation were verified by quantitative real-time polymerase chain reaction and western blot. Luciferase reporter assays were used to explore the specific regions of CPT2 targeted by miR-214. We used a miR-214 over-expression strategy in vitro to further investigate its effect on differentiation process and CPT2 gene transcription. Results: There were 481 miRNAs identified in duck adipocytes, included 57 DE miRNA candidates. And the 1,046 targets genes of DE miRNAs were mainly involved in p53 signaling, FoxO signaling, and fatty acid metabolism pathways. miR-214 and CPT2 showed contrasting expression patterns before and after differentiation, and they were selected for further research. The expression of miR-214 was decreased during the first 3 days of duck adipocytes differentiation, and then increased, while the expression of CPT2 increased both in the transcriptional and protein level. The luciferase assay suggested that miR-214 targets the 3'untranslated region of CPT2. Overexpression of miR-214 not only promoted the formation of lipid droplets but also decreased the protein abundance of CPT2. Conclusion: Current study reports the expression profile of miRNAs in duck adipocytes differentiated for 4 days. And miR-214 has been proved to have the regulator potential for fat deposition in duck.

• 생명과학회지
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• 제26권1호
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• pp.42-49
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• 2016

레티노산(RA)는 많은 유형의 세포에서 성장 및 발달에 중요한 역할을 수행하며 생체 활성화에 적합한 RA의 농도는 CYP26A1 등 여러 가지 효소에 의해 조절된다. CYP26A1의 발현은 RA에 의해서 조절되며 CYP26A1는 RA에 반응하는 유전자 중 하나이다. CYP26A1 유전자 클로닝은 여러 동물에서 보고되어 있지만, 소에서 CYP26A1 유전자의 클로닝은 아직 보고되지 않았다. 소로부터 CYP26A1의 프로모터 부위를 중합효소 연쇄반응을 이용하여 클로닝 한 후 다른 동물과 염기 서열 비교분석 결과 RARE DR-5 (ttggg)의 존재를 확인하였고, DR-5의 염기서열은 분석한 종 에서 완전히 일치하였다. DR-5 motif를 함유한 소의 CYP26A1 프로모터 부위를luciferase리포터 유전자에 결합한 후 transient transfection에 의해 promoter 발현을 분석하였다. 폐 유래 세포주인 MTCC 세포에서 CYP26A1 promoter의 발현은 ATRA의 처리에 의하여 촉진되었다. CYP26A1 유전자의 발현은 ATRA 의존적으로 RAR-α 및 RAR-β에 의하여 현저하게 촉진되었다. 그러나 RAR-γ나 RXR-γ는 CYP26A1 발현에 별다른 영향을 미치지는 않았다. 또한 MTCC 세포주가 생산하는 내인성 CYP26A1 유전자 발현을 Q-RT-PCR로 분석한 결과 1-2일간의 ATRA 처리에 의해서는 현저한 영향을 받지 않으나, 3일 동안 ATRA를 처리한 샘플에서는 CYP26A1의 발현이 현저하게 감소하였다. 결론적으로, 소의 CYP26A1유전자의 프로모터 부위에 존재하는 DR-5 RARE는 RAR-α 및 RAR-β의 결합부위로 작용하여 MTCC 세포에서 CYP26A1 유전자 발현 조절과 RA signal의 조절에 관여하는 것을 확인하였다.

• 대한본초학회지
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• 제28권2호
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• pp.39-44
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• 2013

Objectives : Gambigyeongsinhwan 16 (GGEx16), gambigyeongsinhwan 18 (GGEx18) and gambitongseong capsule are shown to be involved in the regulation of obesity. Therefore, the aim of this study was to compare the reporter activity of anti-obesity genes such as peroxisome proliferator-activated receptor ${\alpha}$ ($PPAR{\alpha}$) and $PPAR{\delta}$ by GGEx16, GGEx18 and gambitongseong capsule. Methods : After NMu2Li liver cells, C2C12 skeletal muscle cells and 3T3-L1 preadipocytes were treated with GGEx16 (1 ${\mu}g/ml$), GGEx18 (1 ${\mu}g/ml$) and different concentrations of gambitongseong capsule, the transactivation of $PPAR{\alpha}$ and $PPAR{\delta}$ was measured by a luciferase reporter gene assay. Results : $PPAR{\alpha}$ reporter gene activity in NMu2Li liver cells and 3T3-L1 preadipocytes was significantly increased by GGEx16, GGEx18 and gambitongseong capsule compared with control, whereas $PPAR{\alpha}$ reporter gene activity in C2C12 skeletal muscle cells was significantly increased by GGEx18 only compared with control. Similarly, $PPAR{\delta}$ reporter gene activity in 3T3-L1 preadipocytes was also significantly increased by GGEx18 compared with control. $PPAR{\delta}$ reporter gene activity in C2C12 skeletal muscle cells was significantly increased by GGEx16 and GGEx18 compared with control although $PPAR{\delta}$ reporter gene activity in NMu2Li liver cells was not changed by these three formulas. Conclusions : These results suggest that all three formulas have the ability to stimulate $PPAR{\alpha}$ and $PPAR{\delta}$ transactivation in animal cell lines with high metabolic rates. In particular, this effects were most prominent in GGEx18-treated cells. In addition, it is likely that GGEx18 may be used as an effective anti-obesity composition.

• 생명과학회지
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• 제24권1호
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• pp.1-7
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• 2014

진핵세포의 유전자 발현은 genomic DNA 부위의 프로모터라고 불리우는 지역에 전사인자와 RNA 중합효소가 자리하면서 시작되는 기작이다. 유전자 내의 프로모터를 동정하는 여러종류의 실험 방법들이 있지만, 많은 시간과 노동력이 요구되어진다. 본 연구에서는 Ensembl, NCBI, CpG plot 등과 같은 생물정보학 관련 프로그램들을 활용하여 SETDB1 유전자의 프로모터를 동정하여 클로닝하고자 하였다. PCR 증폭을 수행한 후 얻은 약 2 kb DNA 조각을 SETDB1-P1이라 명명하였으며, PCR 산물은 TA 벡터로 클로닝 후 확인하였으며, 이를 다시 제한 효소 절단을 통하여 pGL3-luc 벡터로 클로닝하였다. 클로닝된 pGL3-SETDB1-P1-luc 플라스미드를 H1299 폐암세포주에 transfection 시킨 후 여러 가지 항암제를 처리하였을 때, taxol, 5-FU, doxorubicin 처리군에서 SETDB1 프로모터 활성이 감소하는 것을 확인하였다. 이러한 결과는 웨스턴 블롯 및 RT-PCR 실험을 통해 항암제 처리 후 SETDB1 유전자 발현이 조절됨을 확인하였다. 그러므로 bioinformatics 프로그램을 통한 프로모터 동정 및 클로닝 방법을 다른 유전자들에도 적용시킨다면, 유전자 발현 연구에 매우 유용할 것으로 사료된다.

• Animal Bioscience
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• 제34권2호
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• pp.172-184
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• 2021

Objective: Vanin1 (VNN1) is a pantetheinase that can catalyze the hydrolysis of pantetheine to produce pantothenic acid and cysteamine. Our previous studies showed that VNN1 is specifically expressed in chicken liver. In this study, we aimed to investigate the roles of peroxisome proliferators activated receptor α (PPARα) and miRNA-181a-5p in regulating VNN1 gene expression in chicken liver. Methods: 5'-RACE was performed to identify the transcription start site of chicken VNN1. JASPAR and TFSEARCH were used to analyze the potential transcription factor binding sites in the promoter region of chicken VNN1 and miRanda was used to search miRNA binding sites in 3' untranslated region (3'UTR) of chicken VNN1. We used a knock-down strategy to manipulate PPARα (or miRNA-181a-5p) expression levels in vitro to further investigate its effect on VNN1 gene transcription. Luciferase reporter assays were used to explore the specific regions of VNN1 targeted by PPARα and miRNA-181a-5p. Results: Sequence analysis of the VNN1 promoter region revealed several transcription factor-binding sites, including hepatocyte nuclear factor 1α (HNF1α), PPARα, and CCAAT/enhancer binding protein α. GW7647 (a specific agonist of PPARα) increased the expression level of VNN1 mRNA in chicken primary hepatocytes, whereas knockdown of PPARα with siRNA increased VNN1 mRNA expression. Moreover, the predicted PPARα-binding site was confirmed to be necessary for PPARα regulation of VNN1 gene expression. In addition, the VNN1 3'UTR contains a sequence that is completely complementary to nucleotides 1 to 7 of miRNA-181a-5p. Overexpression of miR-181a-5p significantly decreased the expression level of VNN1 mRNA. Conclusion: This study demonstrates that PPARα is an important transcriptional activator of VNN1 gene expression and that miRNA-181a-5p acts as a negative regulator of VNN1 expression in chicken hepatocytes.

• 생명과학회지
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• 제17권2호통권82호
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• pp.167-173
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• 2007

생쥐 신경교세포 유래 신경영양인자 유도성 전사인자(mGIF)의 발현조절에 필요한 전사기작을 연구하기 위하여 mGIF cDNA를 탐침자로 이용하여 genomic clone을 분리하였다. 전체 유전자 13-kb 영역 중 전사개시점에서 4-kb 상류영역의 유전자 서열을 파악한 결과, 프로모터 영역에서 TATA box와 CAAT box는 발견할 수 없었으며 G+C content는 높은 것으로 나타났고 여러 개의 Sp1 전사인자 결합영역이 있었다. 또한 mGIF 유전자는 AP2 결합에 필요한 보존적 영역이 있었다. mGIF 유전자의 프로모터 영역의 단편들을 프로모터가 없는 pGL2-Basic 플라스미드의 luciferase 유전자의 상류에 연결하여 서로 다른 5종류의 결손 돌연변이체를 제조하고 NB41A3 세포주를 이용하여 전사활성을 측정하였다. Transient expression assays 결과, 모든 결손 돌연변이체에서 전사활성이 나타났으며 -213과 -129사이에 전사촉진 영역이 존재하며 -806과 -214사이에 전사억제 영역이 있는 것으로 나타났다. 신경세포주인 NB41A3과 신경교세포주인 C6 그리고 간세포주인 HepG2에서 mGIF 유전자 프로모터의 높은 활성이 관찰되었으며, 근육세포주인 C2C12에서는 낮은 활성이 관찰되었다. 따라서 mGIF 유전자는 조직특이적으로 발현하며 도파민 수용체 유전자와 구조적, 기능적 유사성이 있는 것을 알 수 있었다.