scholarly journals UBE2S Activates NF-κB Signaling By Binding With IκBα and Promotes Metastasis of Lung Adenocarcinoma Cell

2021 ◽  
Author(s):  
Jhih-Yun Ho ◽  
Hsing-Hsien Cheng ◽  
Yu-Chieh Kuo ◽  
Yu-Lin A. Lee ◽  
Chia-Hsiung Cheng
Keyword(s):  
Cancer Cells ◽  
Immunofluorescent Staining ◽  
Luciferase Assay ◽  
Glutathione S Transferase ◽  
Protein Levels ◽  
Vitro Binding ◽  
H460 Cells ◽  
In Vitro Binding Assay ◽  
Lung Adenocarcinomas

Abstract BackgroundNuclear factor (NF)-κB signaling in cancer cells was reported to be involved in tumorigenesis. Phosphorylation and degradation of inhibitor of NF-κBα (IκBα) is a canonical pathway of NF-κB signaling. Herein, we report non-canonical activation of NF-κB signaling without phosphorylation of IκB but by directly binding by ubiquitin-conjugating enzyme E2S (UBE2S) for degradation in adenocarcinoma cells.MethodsTCGA and the Human Atlas Protein Database were used to analyze the survival rate and expression of UBE2S. PC9, H460, H441 and A549 cells were used in this study. PC9 and H460 cells were used for further analysis because of different protein levels of UBE2S. Specific IKK inhibitors, PS1145 and SC514, were used to analyze the phosphorylation of IκBα. Western blotting experiment was used to analyze the protein levels PC9 and H460 cells. Wound-healing experiment was used to analyze the migrative ability of PC9 and H460 cells. Overexpression and knockdown of UBE2S in H460 and PC9 cells were used to analyze the downstream proteins levels. Immunoprecipitation, immunofluorescent staining, a glutathione S transferase (GST) pull-down assay, and an in vitro binding assay were used to analyze the interaction of UBE2S and IκBα. Luciferase assay was used to analyze the activation of NF-κB signaling regulated by UBE2S. Zebrafish xenograft model was used analyzed the metastasis of PC9 cells regulated by UBE2S.Results UBE2S in lung adenocarcinoma patients was negatively related to the survival rate. The protein levels of UBE2S and IκBα were shown opposite change in PC9 and H460 cells. PC9 cells showed higher UBE2S expression and migrative ability than H460 cells. Phosphorylation of IκBα was not changed by treatment with IKK specific inhibitors, PS1145 and SC514, in PC9 and H460 cells. Overexpression and knockdown of UBE2S in H460 and PC9 cells showed the protein levels of IκBα were regulated. Immunoprecipitation, immunofluorescent staining, a glutathione S transferase (GST) pull-down assay, and an in vitro binding assay showed the direct binding of UBE2S with IκBα. Protein levels of nuclear p65 and luciferase assay showed the NF-κB signaling was regulated UBE2S. EMT markers and migrative ability of cancer cells were also regulated by UBE2S. Zebrafish xenograft tumor model showed the reduction of migrative PC9 cells by knockdown of UBE2S.ConclusionHigher UBE2S expressed in lung adenocarcinomas could bind with IκBα for activation of NF-κB signaling to promote metastasis of cancer cells. UBE2S might be a potential therapeutic target for lung adenocarcinomas.

scholarly journals Effect of rs67085638 in long non-coding RNA (CCAT1) on colon cancer chemoresistance to paclitaxel through modulating the microRNA-24-3p and FSCN1 signaling

2020 ◽  
Author(s):  
Caihong Zhang ◽  
Yonglin Wang
Keyword(s):  
Colon Cancer ◽  
Cancer Cells ◽  
Luciferase Assay ◽  
Pcr Analysis ◽  
Dependent Manner ◽  
Colon Cancer Cells ◽  
Protein Levels ◽  
The Difference

Abstract BackgroundIt has been reported that rs67085638 in lncRNA-CCAT1 was associated with the risk of tumorigenesis. Also, CCAT1 could affect chemoresistance of cancer cells to PTX via regulating miR-24-3p and FSCN1 expression. In this study, we aimed to investigate the effect of rs67085638 on the expression of CCAT1/miR-24-3p/FSCN1 and the response of colon cancer to the treatment of PTX.Method48 colon cancer patients were recruited and grouped by their genotypes of rs67085638 polymorphism as a CC group (N=28) and a CT group (N=20). Colon cancer cells were collected from the patients and cancer cell xenografts were transplanted into mice. PCR analysis, IHC assay and Western blot were performed to observe the expression of lncRNA-CCAT1, miR-24-3p and FSCN1 in vivo and in vitro, and the relationships among the expression of lncRNA-CCAT1, miR-24-3p and FSCN1 were validated by computational analysis and luciferase assay. TUNEL assay and flow cytometry were conducted to observe tumor cell apoptosis and survival.ResultLncRNA-CCAT1 and FSCN1 mRNA/protein were over-expressed while miR-24-3p was down-regulated in the CT-genotyped patients and cells compared with those in the CC-genotyped patients and cells. The survival of colon cancer cells was decreased while the apoptosis of colon cancer cells was increased by PTX treatment in a dose-dependent manner. However, the survival rate of CT-genotyped cells was higher while the apoptosis rate of CT-genotyped cells was lower than that of the CC-genotyped cells, and the difference was partly eliminated by the knockdown of lncRNA-CCAT1. MiR-24-3p was validated to target lncRNA-CCAT1 and FSCN1 mRNA, and the over-expression of CCAT1 could reduce the expression of miR-24-3p while elevating the expression of FSCN1. The growth of CT-genotyped tumors in mice was more suppressed in compared with the growth of CC-genotyped tumors, while the knockdown of lncRNA-CCAT1 partly reversed the effect of the CT genotype. Furthermore, compared with the rs67085638-CC mice, the lncRNA-CCAT1 and FSCN1 mRNA/protein levels in the rs67085638-CT+NC shRNA mice were increased while their miR-24-3p level was decreased, and the knockdown of lncRNA-CCAT1 partly reversed the dysregulation of these genes.ConclusionThe findings of this study demonstrated that the presence of the minor allele of rs67085638 increased the expression of CCAT1 and accordingly enhanced the resistance to PTX. Downregulation of CCAT1 partially, but significantly, re-stored the sensitivity to PTX of colon cancer cells.

scholarly journals BIOM-55. DGKζ-TARGETED REGULATION OF MIR-34A IN THE PROLIFERATION AND TUMORIGENICITY OF HUMAN GLIOBLASTOMA

2020 ◽  
Vol 22 (Supplement_2) ◽  
pp. ii13-ii13
Author(s):  
Wangxian Gu ◽  
Guoqing Wan ◽  
Yanjun Zheng ◽  
Xintong Yang ◽  
Peng Zhang ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Precancerous Lesions ◽  
Lipid Kinase ◽  
Human Glioblastoma ◽  
Kinase Substrate ◽  
Downstream Target ◽  
Protein Levels ◽  
Type Iv

Abstract Diacylglycerol kinase (DGK) is a lipid kinase that catalyzes the phosphorylation of diacylglycerol (DAG) to produce phosphatidic acid (PA), which uses ATP as a phosphate donor. Diacylglycerol kinases ζ(DGKζ) is characterized as specific type IV due to its myristoylated alanine-rich C-kinase substrate (MARCKS), ankyrin, and PDZ binding domain. Similar to other DGKs, DGKζ is also reported to be abnormally expressed in human colorectal cancer cells, and it is indispensable for the proliferation of cancer cells. However, its implications in human glioblastoma (GBM) is largely unknown. Both the mRNA and protein levels of DGKζ were significantly higher in GBM tissues than in precancerous lesions. Knockdown of DGKζ inhibited GBM cell proliferation, cell cycle and promoted apoptosis of GBM cells. Moreover, down-regulation of DGKζ markedly reduced in vitro colony formation and in vivo tumorigenic capability. Furthermore, we confirmed that DGKζ was the downstream target of miR-34a. The expression level of DGKζ was negatively correlated with miR-34a in GBM tissues. Overexpression of DGKζ reversed the tumor suppressive roles of miR-34a in GBM cells. Taken together, DGKζ can act as a potential prognostic biomarker for GBM patients and promote the growth of GBM cells was regulated by miR-34a, and it may represent a promising therapeutic target for patients with GBM.

scholarly journals Maternal obesity during pregnancy leads to adipose tissue ER stress in mice via miR-126-mediated reduction in Lunapark

Diabetologia ◽  
2021 ◽  
Author(s):  
Juliana de Almeida-Faria ◽  
Daniella E. Duque-Guimarães ◽  
Thomas P. Ong ◽  
Lucas C. Pantaleão ◽  
Asha A. Carpenter ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Glucose Tolerance ◽  
Er Stress ◽  
Maternal Obesity ◽  
Luciferase Assay ◽  
Whole Body ◽  
Mrna Levels ◽  
Protein Levels ◽  
Novel Targets

Abstract Aims/hypothesis Levels of the microRNA (miRNA) miR-126-3p are programmed cell-autonomously in visceral adipose tissue of adult offspring born to obese female C57BL/6J mice. The spectrum of miR-126-3p targets and thus the consequences of its dysregulation for adipocyte metabolism are unknown. Therefore, the aim of the current study was to identify novel targets of miR-126-3p in vitro and then establish the outcomes of their dysregulation on adipocyte metabolism in vivo using a well-established maternal obesity mouse model. Methods miR-126-3p overexpression in 3T3-L1 pre-adipocytes followed by pulsed stable isotope labelling by amino acids in culture (pSILAC) was performed to identify novel targets of the miRNA. Well-established bioinformatics algorithms and luciferase assays were then employed to confirm those that were direct targets of miR-126-3p. Selected knockdown experiments were performed in vitro to define the consequences of target dysregulation. Quantitative real-time PCR, immunoblotting, histology, euglycaemic–hyperinsulinaemic clamps and glucose tolerance tests were performed to determine the phenotypic and functional outcomes of maternal programmed miR-126-3p levels in offspring adipose tissue. Results The proteomic approach confirmed the identity of known targets of miR-126-3p (including IRS-1) and identified Lunapark, an endoplasmic reticulum (ER) protein, as a novel one. We confirmed by luciferase assay that Lunapark was a direct target of miR-126-3p. Overexpression of miR-126-3p in vitro led to a reduction in Lunapark protein levels and increased Perk (also known as Eif2ak3) mRNA levels and small interference-RNA mediated knockdown of Lunapark led to increased Xbp1, spliced Xbp1, Chop (also known as Ddit3) and Perk mRNA levels and an ER stress transcriptional response in 3T3-L1 pre-adipocytes. Consistent with the results found in vitro, increased miR-126-3p expression in adipose tissue from adult mouse offspring born to obese dams was accompanied by decreased Lunapark and IRS-1 protein levels and increased markers of ER stress. At the whole-body level the animals displayed glucose intolerance. Conclusions/interpretation Concurrently targeting IRS-1 and Lunapark, a nutritionally programmed increase in miR-126-3p causes adipose tissue insulin resistance and an ER stress response, both of which may contribute to impaired glucose tolerance. These findings provide a novel mechanism by which obesity during pregnancy leads to increased risk of type 2 diabetes in the offspring and therefore identify miR-126-3p as a potential therapeutic target. Graphical abstract

scholarly journals Identification and purification of a protein that binds DNA cooperatively with the yeast SWI5 protein.

1993 ◽  
Vol 13 (9) ◽  
pp. 5524-5537 ◽  
Author(s):  
R M Brazas ◽  
D J Stillman
Keyword(s):  
Zinc Finger Protein ◽  
Cooperative Interaction ◽  
Protein Fusion ◽  
Glutathione S Transferase ◽  
Binding Studies ◽  
Eukaryotic Transcription ◽  
Low Concentrations ◽  
Vitro Binding

The Saccharomyces cerevisiae SWI5 gene encodes a zinc finger protein required for the expression of the HO gene. A protein fusion between glutathione S-transferase and SWI5 was expressed in Escherichia coli and purified. The GST-SWI5 fusion protein formed only a low-affinity complex in vitro with the HO promoter, which was inhibited by low concentrations of nonspecific DNA. This result was surprising, since genetic evidence demonstrated that SWI5 functions at the HO promoter via this site in vivo. A yeast factor, GRF10 (also known as PHO2 and BAS2), that promoted high-affinity binding of SWI5 in the presence of a large excess of nonspecific carrier DNA was purified. Final purification of the 83-kDa GRF10 protein was achieved by cooperative interaction-based DNA affinity chromatography. In vitro binding studies demonstrated that SWI5 and GRF10 bind DNA cooperatively. Methylation interference and missing-nucleoside studies demonstrated that the two proteins bind at adjacent sites, with each protein making unique DNA contacts. SWI5 and GRF10 interactions were not detected in the absence of DNA. The role of cooperative DNA binding in determining promoter specificity of eukaryotic transcription factors is discussed.

scholarly journals Exosomal miR-25-3p from mesenchymal stem cells alleviates myocardial infarction by targeting pro-apoptotic proteins and EZH2

2020 ◽  
Vol 11 (5) ◽  
Author(s):  
Yi Peng ◽  
Ji-Ling Zhao ◽  
Zhi-Yong Peng ◽  
Wei-Fang Xu ◽  
Guo-Long Yu
Keyword(s):  
Myocardial Infarction ◽  
Inflammatory Responses ◽  
Luciferase Assay ◽  
Protective Effects ◽  
Functional Studies ◽  
Protein Levels ◽  
Cardioprotective Effects ◽  
Apoptotic Proteins

Abstract Mesenchymal stem cell (MSC) therapy is a promising approach against myocardial infarction (MI). Studies have demonstrated that MSCs can communicate with other cells by secreting exosomes. In the present study, we aimed to identify exosomal microRNAs that might contribute to MSC-mediated cardioprotective effects. Primary cardiomyocytes were deprived of oxygen and glucose to mimic MI in vitro. For the animal model of MI, the left anterior descending artery was ligated for 1 h, followed by reperfusion for 12 h. MSC-derived exosomes were used to treat primary cardiomyocytes or mice. Cardioprotection-related microRNAs were determined, followed by target gene identification and functional studies with quantitative PCR, western blotting, MTT assay, flow cytometry assay, chromatin immunoprecipitation and dual-luciferase assay. We found that MSC co-culture reduced OGD-induced cardiomyocyte apoptosis and inflammatory responses. Cardioprotection was also observed upon treatment with MSC-derived exosomes in vitro and in vivo. In line with this, exosome uptake led to a significant increase in miR-25-3p in cardiomyocytes. Depletion of miR-25-3p in MSCs abolished the protective effects of exosomes. Mechanistically, miR-25-3p directly targeted the pro-apoptotic genes FASL and PTEN and reduced their protein levels. Moreover, miR-25-3p decreased the levels of EZH2 and H3K27me3, leading to derepression of the cardioprotective gene eNOS as well as the anti-inflammatory gene SOCS3. Inhibition of EZH2 or overexpression of miR-25-3p in cardiomyocytes was sufficient to confer cardioprotective effects in vitro and in vivo. We concluded that exosomal miR-25-3p from MSCs alleviated MI by targeting pro-apoptotic proteins and EZH2.

scholarly journals Knockdown of Ezrin inhibited migration and invasion of cervical cancer cells in vitro

2020 ◽  
Vol 34 ◽  
pp. 205873842093089
Author(s):  
Meili Xi ◽  
Wenbin Tang
Keyword(s):  
Cervical Cancer ◽  
Cancer Cells ◽  
Messenger Rna ◽  
Migration And Invasion ◽  
Protein Levels ◽  
Sulforhodamine B ◽  
Ezrin Expression ◽  
Cervical Cancer Cells ◽  
Polymerase Chain

Cervical cancer is the fourth most common malignancy in women. The aim of this study was to investigate the functions of Ezrin in cervical cancer cells. Two cervical cancer cell lines, SiHa and CaSki, were cultured in vitro. Following the knockdown of Ezrin using siRNA, real-time reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analysis were applied to analyze Ezrin expression at the messenger RNA (mRNA) and protein levels. Subsequently, wound healing assay, transwell assay, and sulforhodamine B (SRB) assay were used to detect the migration, invasion, and viability of cervical cancer cells, respectively. Results revealed that Ezrin siRNA can notably inhibit the migration and invasion of SiHa and CaSki cells ( P  < 0.05). However, knockdown of Ezrin shows no effects on the viability of SiHa and CaSki cells ( P  < 0.05). It is indicated that Ezrin plays a possible role in promoting the migration and invasion of cervical cancer cells and may be a therapeutic target to prevent metastasis of cervical cancer.

scholarly journals Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions.

1994 ◽  
Vol 127 (6) ◽  
pp. 1617-1626 ◽  
Author(s):  
M Furuse ◽  
M Itoh ◽  
T Hirase ◽  
A Nagafuchi ◽  
S Yonemura ◽  
...  
Keyword(s):  
Amino Acids ◽  
Fusion Protein ◽  
Tight Junctions ◽  
Integral Membrane Protein ◽  
Deletion Mutants ◽  
Glutathione S Transferase ◽  
E Coli ◽  
Vitro Binding ◽  
Peripheral Proteins

Occludin is an integral membrane protein localizing at tight junctions (TJ) with four transmembrane domains and a long COOH-terminal cytoplasmic domain (domain E) consisting of 255 amino acids. Immunofluorescence and laser scan microscopy revealed that chick full-length occludin introduced into human and bovine epithelial cells was correctly delivered to and incorporated into preexisting TJ. Further transfection studies with various deletion mutants showed that the domain E, especially its COOH-terminal approximately 150 amino acids (domain E358/504), was necessary for the localization of occludin at TJ. Secondly, domain E was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and this fusion protein was shown to be specifically bound to a complex of ZO-1 (220 kD) and ZO-2 (160 kD) among various membrane peripheral proteins. In vitro binding analyses using glutathione-S-transferase fusion proteins of various deletion mutants of domain E narrowed down the sequence necessary for the ZO-1/ZO-2 association into the domain E358/504. Furthermore, this region directly associated with the recombinant ZO-1 produced in E. coli. We concluded that occludin itself can localize at TJ and directly associate with ZO-1. The coincidence of the sequence necessary for the ZO-1 association with that for the TJ localization suggests that the association with underlying cytoskeletons through ZO-1 is required for occludin to be localized at TJ.

scholarly journals The Ras-related protein R-ras interacts directly with Raf-1 in a GTP-dependent manner

1994 ◽  
Vol 300 (2) ◽  
pp. 303-307 ◽  
Author(s):  
M Spaargaren ◽  
G A Martin ◽  
F McCormick ◽  
M J Fernandez-Sarabia ◽  
J R Bischoff
Keyword(s):  
Amino Acids ◽  
Small Gtpases ◽  
Dependent Manner ◽  
Downstream Effector ◽  
Yeast Two Hybrid System ◽  
Vitro Binding ◽  
Ras Family ◽  
In Vitro Binding Assay ◽  
Two Hybrid

R-ras is a member of the ras family of small GTPases that associates with the apoptosis-suppressing proto-oncogene product Bcl-2. Using the yeast two-hybrid system we provide evidence for an interaction between R-ras and the Raf-1 kinase. This interaction requires only the N-terminal regulatory domain (amino acids 1-256) of Raf-1, and is observed with both the wild type and a constitutively active R-ras mutant, but not with a deletion mutant that lacks the potential effector domain or a mutant of R-ras impaired for GTP binding. Moreover, using an in vitro binding assay we show a direct GTP-dependent interaction of purified R-ras with a purified Raf-1 fragment corresponding to the proposed 81-amino-acid H-Ras-binding domain of Raf-1 (amino acids 51-131). Taken together, these data indicate that R-ras may exert its biological effect by means of modulating the activity of the Raf-1 kinase as its direct downstream effector.

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