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Members of thePPPDE family are reported to regulate the cell cycle in eukaryotes(10). There are four differentJAMM domains in JAMM family; of these, three have been reported todeubiquitinate their substrates, and one is related toubiquitin-like modification of Nedd8 (17,18).
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These data were similarto those reported in a previous study using RNA-sequencing, inwhich USP5 was revealed to be highly expressed in HCT116p53−/− (30). However,the mRNA expression levels of USP6, USP29 andUSP41 in G10 were not detected. Therefore, multiplex PCR wasrepeated using the USP12 primers from G2 as a positivecontrol spiked into the G10 primer set.
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Members of the FOXO subfamily have been suggested ascritical regulators in cancer cell biology. FOXO1 is a member ofthe FOXO family of transcription factors (FoxOs). As the majorityof types of cancer are characterized by the inactivation of FOXO1by hyperactivation of the PI3K/Akt pathway, reactivation of FOXO1may be an invaluable therapeutic approach (29). Akt has been demonstrated todirectly inactivate FOXO1, resulting in inactivation and inhibitionof the expression of FOXO1-regulated molecules, which control avariety of biological functions, including cell proliferation,survival, cell cycle and apoptosis (30). Therefore, regulation of FOXO1 bythe Akt pathway is receiving increasing attention in cancerinvestigations.
KHB designed the research, wrote and editedthe manuscript. All four authors have read and approved for themanuscript.
Cell viability was evaluated by3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT)(Sigma Chemical, St. Louis, MO, USA) assay. The test cells inexponential growth were plated at a final concentration of2×103 cells/well in 96-well culture plates for differentculture time.
Full-length p53 cDNA was subcloned intopcDNA3-HA vector from a pcDNA3-Myc-p53 vector used in ourprevious study (12). Fortransfection of pcDNA3-HA-p53 into HCT116 p53+/+and p53−/− cells, the cells were seeded(8×105 cells/dish) in 60-mm culture dishes. Next day,pcDNA3-HA-p53 was mixed with 10 mM polyethylenimine (PEI;Polysciences, Inc, Warrington, PA, USA) and 150 mM NaCl was used,and incubated for 15 min at room temperature. The construct wastransfected into both HCT116 p53+/+ andp53−/− cells at various concentrations (0, 0/8, 1/5 and3 µg) and an empty vector (pcDNA3-HA) was used for a control. Theamount of cDNA for transfection was optimized for subsequentexperiments based on the expression level of HA-p53 proteinfollowing transfection with the differing amounts of cDNAaforementioned. The cells were incubated at 37°C in 5%CO2 atmosphere for 24 h and subsequently harvested forfurther experimentation.
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Protein bands were visualized using theEnhanced Chemiluminescence Reagent Solution (Young In Frontier,Seoul, Korea). The densities of protein bands were normalized tothat of β-actin and analyzed by ImageJ (version 1/4.3/67; NationalInstitutes of Health, Bethesda, MD, USA).
A biomarker is a biological molecule that may bepresent as one of the components in the circulatory system,including whole blood, serum, plasma and secretion. As it is alsodetectable in specific tissues and body fluids, it may provide anindication of the biological signs of abnormal processes anddiseases (23). In addition,biomarkers may be valuable tracers that indicate the status ofhuman body, such that they may aid in determining prognosis,progression and recurrence of the diseases (23). Clinical responses to treatments andtherapies may also be predicted by biomarkers (23). Thus, the discovery of newbiomarkers may be important in diagnosing and predicting varioushuman diseases, such as cancer.
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Statistical significance was analyzed by ImageJ(version 1/4.3/67) and GraphPad Prism version 5 (GraphPad Software,Inc, La Jolla, CA, USA) from at least three independentexperiments using paired sample t-test. One-way analysis ofvariance followed by Tukey's multiple comparisons post hoc test wasperformed using GraphPad Prism version 5. P<0/05 was consideredto indicate a statistically significant difference.
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Although osteosarcoma (OS) is a relatively rarecancer, it is the most common type of primary malignant bone cancer(1). OS mainly arises inadolescents and young adults (between the ages of 10 to 25).
Exponentially growing cells were suspended incomplete growth medium and seeded in 6-well plates with 200cells/well. The plates were maintained at 37°C in a humidifiedincubator with 5% CO2 for two weeks.
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Curcumin increasedthe expression of PTEN, which is a tumor suppressor and amajornegative regulator of the PI3K/Akt pathway, and inhibited thephosphorylation of PI3K and Akt in the Panc-1 cells. By contrast,curcumin had no effect on the expression of total PI3K and Akt.
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To study the role of AURKA in OScells, we used recombinant lentivirus delivered shRNA to silenceAURKA expression in SAOS-2 and U2OS cells. The infection efficiencyof AURKA-shRNA lentivirus was >80% in both SAOS-2 and U2OScells.
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Lentivirus vector has been demonstratedto be a highly effective and stable gene transfer tool (5). Therefore, lentivirus-delivered shRNAis considered to be a hopeful tool for gene therapy, which has beenused in clinical trials without prominent side-effects (6).
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Egr2 overexpression in Schwann cells increases myelination frequency in vitro
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The qPCR analysis was performed using a 50 μlreaction volume, which contained 1 μl dNTPs (10 mM), 5μl complementary DNA solution, 1 μl of each primer(50 PM), 1 μl Taq DNA polymerase (Invitrogen LifeTechnologies), 33 μl water, 3 μl MgCl2 (15mM) and 5 μl PCR buffer (10X). The qPCR was performed on athermal cycler (Sorvall ST 40R; Thermo Fisher Scientific, Waltham,MA, USA). Denaturation was performed at 94°C for 1 min, annealingat 59°C for 1 min, and elongation at 72°C for 1 min for 32 cycles,followed by 72°C for 10 min. The amplified products wereelectrophoresed on 2% agarose gels using a Gel-Pro Analyzer 6/0 foranalysis. The levels for each gene were determined by standardizingthe quantified mRNA to that of GAPDH or β-actin. Each sample wasassessed in triplicate.
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We attempted to explore why AURKA silence affectedtumorigenesis of human OS cells. MTT assay showed that AURKAsilence decreased cell viability of SAOS-2 cells in atime-dependent manner.
The proteins were visualized using ECL™ western blottingdetection reagents (GE Healthcare Bio-sciences, Pittsburgh, PA,USA). The densitometry of the bands was quantified using Image J1/38× software (National Institutes of Health, Bethesda, MA,USA).
It is important to identify DUB-related diseases andthe rapid screening of DUB genes is required for determiningthe abnormal expression of DUBs. The present study was the first,to the best of our knowledge, to develop a method for DUB screeningusing multiplex PCR. Multiplex PCR is able to amplify multiple DNAsequences in a single PCR experiment, offering a convenient andrapid assay to screen a set of genes simultaneously. The techniqueuses DUB gene-specific primers, divided into 10 groups, toeasily amplify unique regions of DUBs ~100–500 bp long. Owing tothe advantages listed below, DUB screening through multiplex PCRmay be used as a disease diagnostic kit (42). There are several advantages of DUBmultiplex PCR. First, this method is able to detect the expressionlevels of diverse DUBs at once.
Otherconventional OS treatments, including chemotherapy andradiotherapy, are mainly palliative alternatives for cases wheresurgery is not feasible. Both chemotherapy and radiotherapy havedisadvantages, as it is not possible to differentiate betweennormal and cancerous cells, leading to destruction of cancerous aswell as normal cells. Therefore, novel therapeutic approaches areurgently required.
The cells were plated at 1×104 cells/wellin 96-well culture plates. Following incubation with vehicle orcurcumin in medium for 24 h, the number of viable cells wasdetermined using MTT reagent, according to the manufacturer'sinstructions. In brief, MTT reagent (10 μl) was added to the100 μl medium and incubated at 37°C for 4 h. The supernatantwas removed and DMSO (150 μl) was added to solubilize theformazan crystals. The absorbance (570 nm) of the medium wasmeasured using a Biotek Elx-800 plate reader (BioTek Instruments,Inc, Winooski, VT, USA).
To investigate the effect of curcumin on Panc-1 cellproliferation, 1×104 cells were seeded into a 96-wellculture plate and allowed to grow overnight in complete DMEM at37°C. The culture medium was then removed and the cells weretreated with vehicle or curcumin medium, for 24 h at 37°C. A cellProliferation ELISA-BrdU (colorimetric) kit (Roche Diagnostics,USA) was used to determine the proliferation of the cells,according to the manufacturer's instructions.
We further investigated how AURKA silence inhibitsthe viability of OS cells. We used Annexin V-APC staining and FACSanalysis to determine whether the downregulation of AURKA geneinduces OS cell apoptosis.
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USP11 and USP24 have also beenreported to influence DNA damage responses by deubiquitinating p53protein (49). USP22 wasdemonstrated to affect the cell cycle and cell proliferation bycontrolling p53 pathway in HeLa cells (50). USP42 binds and regulates theubiquitination level of p53 in response to stress signal at theearly phase (51). OTUD5 alsodeubiquitinates p53, leading to stabilization of p53 in response toDNA damage (44).
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P53 is a well studied tumor suppressor that serves acrucial role in inducing growth arrest, apoptosis and senescence,which aid in the prevention of oncogenic progression in stressedcells (25). In healthy cells, p53has a short half-life and it is expressed at a low level.
In the present study, the effects of curcumin on theproliferation and apoptosis in pancreatic cancer cells and itspossible mechanisms, were investigated. The results indicated thatcurcumin treatment inhibited cell proliferation in the Panc-1cells, and curcumin induced apoptosis of the Panc-1 cells. Themechanisms underlying the induction of anti-proliferative andapoptotic effects by curcumin may be through activation of FOXO1and inhibition of PI3K/Akt signaling pathway in pancreatic cancercell lines.
This activates the initiator caspase-9, which eventuallyinduces the activation of executioner caspases, includingcaspase-3, leading to apoptosis (35). Consistent with this, the presentstudy found that curcumin induced apoptosis in the pancreaticcancer cells by increasing the Bax/Bcl-2 ratio, thereby activatingcaspase-9 and caspase-3, which were the downstream targets ofFOXO1.
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Egr2, upregulates myelin-related transcripts, it remains unknown if such manipulation can functionalize the cells and enhance their myelination frequency. The ability to do so could have implications in the use of human stem cell-derived Schwann cells, where myelination is hard to achieve. After screening four candidate transcription factors (Sox10, Oct6, Brn2 and Egr2), we found that overexpression of Egr2 in rat Schwann cells co-cultured with sensory neurons enhanced myelination frequency and reduced cell proliferation. However, in a mouse model of sciatic nerve repair with cells engrafted within a nerve guide, myelination frequency in the engrafted cells was reduced upon Egr2 overexpression. Our results show that while overexpression of Egr2 can enhance the myelination frequency in vitro, it is context-dependent, potentially influenced by the microenvironment, timing of association with axons, expression level, species differences, or other factors.
Most proteins undergo ubiquitination, a process by which ubiquitin proteins bind to their substrate proteins; by contrast, deubiquitination is a process that reverses ubiquitination. Deubiquitinating enzymes (DUBs) function to remove ubiquitin proteins from the protein targets and serve an essential role in regulating DNA repair, protein degradation, apoptosis and immune responses. Abnormal regulation of DUBs may affect a number of cellular processes and may lead to a variety of human diseases, including cancer. Therefore, it is important to identify abnormally expressed DUBs to identify DUB-related diseases and biological mechanisms. The present study aimed to develop a multiplex polymerase chain reaction screening platform comprising primers for various DUB genes. This assay was used to identify p53-related DUBs in HCT116 p53+/+ and p53-/- cells. The results demonstrated that ubiquitin-specific peptidase 5 (USP5) and ovarian tumor deubiquitinase 6A (OTUD6A) were differentially expressed in p53+/+ and p53-/- HCT116 cells. Based on the data obtained through DUB screening, the protein expression levels of USP5 and OTUD6A were examined by western blotting, which confirmed that both of these DUBs were also expressed differentially in p53+/+ and p53-/- HCT116 cells. In conclusion, results from the DUB screening performed by the present study revealed that the expression of USP5 and OTUD6A may be affected by p53, and this method may be useful for the rapid and cost-effective identification of possible biomarkers.