Identification of a novel regulatory mechanism involved in inhibition of transcription of suvivin mRNA in breast cancer cells via p21cip–mediated regulation

Purpose: To evaluate the effect of p21Cip1 on survivin transcription levels in breast carcinoma, and to investigate the potential mechanisms.Methods: Epirubicin, a p21Cip1 activator, was used to treat MCF7 cells. Under the action of normal biological functions of p53, pEGFP-C2-p21 was transfected into MCF7 cells by lipofectamine and positive clones were screened out with G418. The expression levels of p21cip1, p53 and survivin mRNA were quantitated by real-time fluorescent polymerase chain reaction (RQ-PCR). MTT assay was utilized to measure cellular viability and proliferation after transfection. Flow cytometry was employed to determine the cell cycle. Hoechst 33342 staining was carried out to assess cell apoptosis. Lastly, several transcription factor sites located at the promoter region of survivin gene, such as, sp1 site, E2F site and p300/CBP, were measured by p21 overexpression using RT-PCR.Results: Following epirubicin treatment, within 24 h, the expression levels of endogenous p21cip1 and p53 were up-regulated, whereas that of survivin was down-regulated. After transfection treatment, p21 inhibited the proliferation of MCF7 cells on days 3 and 4, and MCF7 cells overexpressed p21 mRNA, whereas the level of survivin mRNA in MCF7-p21 groups was markedly down-regulated relative to control group, but overexpression of p21 was not sufficient to cause changes in p53 gene expression. The overexpressed p21 resulted in G1/G0 phase arrest based on cell cycle analysis, but apoptosis was not induced. In addition, co-transcription factors E2F-1, sp1 and p300/CBP mRNA levels decreased significantly compared with normal p21 expression groups.Conclusion: P21cip1 may down-regulate the expression of survivin gene partially by inhibiting the expression level of HAT.Keywords: Cyclin-dependent kinase inhibitor 1, Phosphoprotein p53, Survivin, Breast carcinoma, G1/G0 phase arrest, Epirubicin, Lipofectamin

Decelerating Airy pulse propagation in highly non-instantaneous cubic media

The propagation of decelerating Airy pulses in non-instantaneous cubic medium is investigated both theoretically and numerically. In a Debye model, at variance with the case of accelerating Airy and Gaussian pulses, a decelerating Airy pulse evolves into a single soliton for weak and general non- instantaneous response. Airy pulses can hence be used to control soliton generation by temporal shaping. The effect is critically dependent on the response time, and could be used as a way to measure the Debye type response function. For highly non- instantaneous response, we theoretically find a decelerating Airy pulse is still transformed into Airy wave packet with deceleration. The theoretical predictions are confirmed by numerical simulations

Upregulation of Endogenous HMOX1 Expression by a Computer-Designed Artificial Transcription Factor

Heme oxygenase-1 (HO-1) is well known as a cytoprotective factor. Research has revealed that it is a promising therapeutic target for cardiovascular diseases. In the current study, an HMOX1 (HO-1 gene) enhancer-specific artificial zinc-finger protein (AZP) was designed using bioinformatical methods. Then, an artificial transcription factor (ATF) was constructed based on the AZP. In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included. We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression. The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells. With further research, the ATF could be developed as a potential drug for cardiovascular diseases

Design of a zinc finger protein binding a sequence upstream of the A20 gene

<p>Abstract</p> <p>Background</p> <p>Artificial transcription factors (ATFs) are composed of DNA-binding and functional domains. These domains can be fused together to create proteins that can bind a chosen DNA sequence. To construct a valid ATF, it is necessary to design suitable DNA-binding and functional domains. The Cys₂-His₂zinc finger motif is the ideal structural scaffold on which to construct a sequence-specific protein. A20 is a cytoplasmic zinc finger protein that inhibits nuclear factor kappa-B activity and tumor necrosis factor (TNF)-mediated programmed cell death. A20 has been shown to prevent TNF-induced cytotoxicity in a variety of cell types including fibroblasts, B lymphocytes, WEHI 164 cells, NIH 3T3 cells and endothelial cells.</p> <p>Results</p> <p>In order to design a zinc finger protein (ZFP) structural domain that binds specific target sequences in the A20 gene promoter region, the structure and sequence composition of this promoter were analyzed by bioinformatics methods. The target sequences in the A20 promoter were submitted to the on-line ZF Tools server of the Barbas Laboratory, Scripps Research Institute (TSRI), to obtain a specific 18 bp target sequence and also the amino acid sequence of a ZFP that would bind to it. Sequence characterization and structural modeling of the predicted ZFP were performed by bioinformatics methods. The optimized DNA sequence of this artificial ZFP was recombined into the eukaryotic expression vector pIRES2-EGFP to construct pIRES2-EGFP/ZFP-flag recombinants, and the expression and biological activity of the ZFP were analyzed by RT-PCR, western blotting and EMSA, respectively. The ZFP was designed successfully and exhibited biological activity.</p> <p>Conclusion</p> <p>It is feasible to design specific zinc finger proteins by bioinformatics methods.</p

Upregulation of Endogenous HMOX1 Expression by a Computer-Designed Artificial Transcription Factor

Heme oxygenase-1 (HO-1) is well known as a cytoprotective factor. Research has revealed that it is a promising therapeutic target for cardiovascular diseases. In the current study, an HMOX1 (HO-1 gene) enhancer-specific artificial zinc-finger protein (AZP) was designed using bioinformatical methods. Then, an artificial transcription factor (ATF) was constructed based on the AZP. In the ATF, the p65 functional domain was used as the effector domain (ED), and a nuclear localization sequence (NLS) was also included. We next analyzed the affinity of the ATF to the HMOX1 enhancer and the effect of the ATF on endogenous HMOX1 expression. The results suggest that the ATF could effectively upregulate endogenous HMOX1 expression in ECV304 cells. With further research, the ATF could be developed as a potential drug for cardiovascular diseases

Research on lightning transient distribution characteristics of cable-stayed bridges

Bridges are usually located at the junction of land and water, where the surrounding area is open terrain, making them to be easily damaged by lightning strikes. The quantitative analysis of lightning transient characteristics and the impulse effect on bridges can provide scientific data to support the lightning protection design for bridges. In this study, a single-tower cable-stayed bridge is taken as a representative case. The CDEGS (Current Distribution Electromagnetic Interference Grounding and Soil Structure Analysis) software is used to establish a three-dimensional simulation model of the bridge. With this model, the magnetic field, step voltage, and lightning current distribution on the tower top, stay cables, and bridge deck under the most severe direct lightning strike scenario typical for cable-stayed structures are simulated. The results are as follows: (1) when the stay cables are struck by lightning, the peak of the magnetic field intensity is highest at the location of electronic information equipment, followed by that at the top of the tower, and the lowest at the bridge deck. The peak step voltage at the ground below the bridge is the largest when lightning strikes the cable, and that is the smallest when lightning strikes the bridge deck. (2) The magnitude distribution of the lightning current on the stay cables is related to the strike location, the distance between the strike point and the grounding system, and the length of the stay cables. When the stay cables are closer to the grounding system and the length is shorter, the lightning current that flows through them is larger. (3) The magnitude distribution of the lightning current on the grounding system is associated with the location of the strike and the position of the grounding system. The closer the grounding system is to the strike point, the larger the amplitude of the lightning current on the grounding system. The grounding system positioned in the middle significantly reduces the lightning current due to the shielding effect. The grounding system located at the edge shows minimal variation in the time domain characteristics of the lightning current, resulting in less reduction of the initial steepness of the lightning wave. However, the grounding system at the middle position experiences a significant increase in the temporal characteristics of lightning current, reducing the hazards caused by the steepness of the original lightning wave

MicroRNA-145 Regulates Chondrogenic Differentiation of Mesenchymal Stem Cells by Targeting Sox9

Chondrogenic differentiation of mesenchymal stem cells (MSCs) is accurately regulated by essential transcription factors and signaling cascades. However, the precise mechanisms involved in this process still remain to be defined. MicroRNAs (miRNAs) regulate various biological processes by binding target mRNA to attenuate protein synthesis. To investigate the mechanisms for miRNAs-mediated regulation of chondrogenic differentiation, we identified that miR-145 was decreased during transforming growth factor beta 3 (TGF-β3)-induced chondrogenic differentiation of murine MSCs. Subsequently, dual-luciferase reporter gene assay data demonstrated that miR-145 targets a putative binding site in the 3′-UTR of SRY-related high mobility group-Box gene 9 (Sox9) gene, the key transcription factor for chondrogenesis. In addition, over-expression of miR-145 decreased expression of Sox9 only at protein levels and miR-145 inhibition significantly elevated Sox9 protein levels. Furthermore, over-expression of miR-145 decreased mRNA levels for three chondrogenic marker genes, type II collagen (Col2a1), aggrecan (Agc1), cartilage oligomeric matrix protein (COMP), type IX collagen (Col9a2) and type XI collagen (Col11a1) in C3H10T1/2 cells induced by TGF-β3, whereas anti-miR-145 inhibitor increased the expression of these chondrogenic marker genes. Thus, our studies demonstrated that miR-145 is a key negative regulator of chondrogenic differentiation by directly targeting Sox9 at early stage of chondrogenic differentiation

Stanniocalcin-1 Protects a Mouse Model from Renal Ischemia-Reperfusion Injury by Affecting ROS-Mediated Multiple Signaling Pathways

Stanniocalcin-1 (STC-1) protects against renal ischemia-reperfusion injury (RIRI). However, the molecular mechanisms remain widely unknown. STC-1 inhibits reactive oxygen species (ROS), whereas most ROS-mediated pathways are associated with ischemic injury. Therefore, to explore the mechanism, the effects of STC-1 on ROS-medicated pathways were studied. Non-traumatic vascular clamps were used to establish RIRI mouse models. The serum levels of STC-1, interleukin-6 (IL-6), interferon (IFN) γ, P53, and capase-3 were measured by ELISA kits. Superoxide dismutase (SOD) and malondialdehyde (MDA) were measured by fluorescence spectrofluorometer. All these molecules changed significantly in a RIRI model mouse when compared with those in a sham control. Kidney cells were isolated from sham and model mice. STC-1 was overexpressed or knockout in these kidney cells. The molecules in ROS-medicated pathways were measured by real-time quantitative PCR and Western blot. The results showed that STC-1 is an effective ROS scavenger. The serum levels of STC-1, MDA and SOD activity were increased while the serum levels of IL-6, iIFN-γ, P53, and capase-3 were decreased in a model group when compared with a sham control (p &lt; 0.05). Furthermore, the levels of STC-1,p53, phosphorylated mitogen-activated protein kinase kinase (p-MEKK-1), c-Jun N-terminal kinase (p-JNK), extracellular signal-regulated kinase (p-ERK), IkB kinase (p-IKK), nuclear factor (NF) κB, apoptosis signal-regulating kinase 1 (ASK-1) and caspase-3 changed significantly in kidney cells isolated from a RIRI model when compared to those isolated from a sham control (p &lt; 0.05). Meanwhile, STC-1 overexpression or silence caused significant changes of the levels of these ROS-mediated molecules. Therefore, STC-1 maybe improve anti-inflammation, anti-oxidant and anti-apoptosis activities by affecting ROS-mediated pathways, especially the phospho-modifications of the respective proteins, resulting in the increase of SOD and reduce of capase-3, p53, IL-6 and IFN-γ