Nucleostemin Knockdown Sensitizes Hepatocellular Carcinoma Cells to Ultraviolet and Serum Starvation-Induced Apoptosis

<div><p>Nucleostemin (NS) is a GTP-binding protein that is predominantly expressed in embryonic and adult stem cells but not in terminally differentiated cells. NS plays an essential role in maintaining the continuous proliferation of stem cells and some types of cancer cells. However, the role of NS in hepatocellular carcinoma (HCC) remains unclear. Therefore, this study aimed to clarify the role of NS in HCC. First, we demonstrated high expression of NS in most HCC cell lines and liver cancer tissues. NS knockdown induced a severe decline in cell viability of MHCC97H cells as detected by MTT and cell proliferation assays. Next, we used ultraviolet (UV) and serum starvation-induced apoptosis models to investigate whether NS suppression or up-regulation affects HCC cell apoptosis. After UV treatment or serum starvation, apoptosis was strongly enhanced in MHCC97H and Bel7402 cells transfected with small interfering RNA against NS, whereas NS overexpression inhibited UV- and serum-induced apoptosis of HCC cells. Furthermore, after UV irradiation, inhibition of NS increased the expression of pro-apoptosis protein caspase 3 and decreased the expression of anti-apoptosis protein Bcl-2. A caspase 3 inhibitor could obviously prevent NS knockdown-induced apoptosis. In conclusion, our study demonstrated overexpression of NS in most HCC tissues compared with their matched surrounding tissues, and silencing NS promoted UV- and serum starvation-induced apoptosis of MHCC97H and Bel7402 cells. Therefore, the NS gene might be a potential therapeutic target of HCC.</p></div

Expression of NS in HCC cell lines and tissues.

<p>(A) Western blot analysis of NS protein expression in HCC cells and the hepatic non-tumor cell line L02. (B) Quantification of NS protein levels in various HCC cell lines and L02 cells. Image J method was performed to quantificate the western blots (n = 3), and GAPDH is a loading control. *P<0.05, **P<0.01. (C) Expression of NS mRNA in 18 matched samples of liver tumor and para-cancerous tissues. (D) Representative western blot of NS in HCC and para-cancerous tissues. N, para-cancerous tissue; T, tumor. (E) NS protein expression in 16 paired samples of and HCC and para-carcinoma tissues. **P<0.01.</p

UV-induced apoptosis is regulated by NS in MHCC97H and Bel7402 cell lines.

<p>(A) UV irradiation-induced apoptosis in MHCC97H-control and MHCC97H-siNS cells. The cells were plated on 60- or 100-mm culture dishes, and siRNA or plasmids were introduced using Lipofectamine 2000 according to the manufacturer’s protocol. For UV irradiation, the culture medium was removed after transfection for 24 h, and then the culture dishes were uncovered and placed in a UV cross-linker for the appropriate times. (B) Apoptosis distributions of MHCC97H-vector and MHCC97H-NS cells after UV irradiation. (C) UV irradiation-induced apoptosis in Bel7402-control and Bel7402-siNS cells. (D) Apoptosis distributions of MHCC97H-vector and MHCC97H-NS cells after UV irradiation. All apoptosis quantification was displayed as mean ± SD values. Each treatment was repeated in triplicate with NS knockdown or overexpression was visibly higher than that of control group. **P<0.01.</p

Knockdown of NS suppresses MHCC97H cell proliferation.

<p>(A) MTT assays of MHCC97H cells treated with siNC or siNS at 1, 2, and 3 days. Each value represents the average of three independent experiments, and bars indicate the standard error of the mean. *P<0.05, **P < 0.01. (B) Cell growth curves of MHCC97H cells were constructed by measuring cell proliferation every 12 h after transfection with siNS-and siNC for 24 h. (C and D) MTT assays of L02 cells treated with siNS or overexpressing NS. Each value represents the average of four independent experiments, and bars indicate the standard error of the mean. **P<0.01. (E) Knockdown of NS in MHCC97H cells by siRNA transfection at 48 and 72 h. Western blot analysis was performed using antibodies against NS and GAPDH as the loading control. (F)Western blotting assays of transient transfection of NS siRNA or overexpressed plasmids in L02 cells.</p

Caspase 3 inhibition prevents apoptosis induced by NS knockdown.

<p>(A) Flow cytometric analysis of apoptosis after transfection with Flag-NS or the control vector. (B) Apoptosis distributions of MHCC97H cells after transfection with siNS or siNC at 48h and MHCC97H-siNC cells treated with the caspase 3 inhibitor. FITC-labeled annexin V-positive cells (upper and lower right) were considered as apoptotic cells. (C) Apoptosis distribution induced by UV irradiation in MHCC97H-siNC and MHCC97H-siNS cells cultured in the presence or absence of the caspase 3 inhibitor (10 μM) for 48 h. FITC-labeled annexin V-positive cells (upper and lower right) were considered as apoptotic cells. (D) Quantitative analysis of the percentages of apoptotic MHCC97H cells cultured in the presence or absence of the caspase 3 inhibitor (10 μM) for 48 h. Apoptosis is displayed as mean ± SD values. Each treatment was repeated in triplicate with NS knockdown or overexpression was visibly higher than that of control group. **P<0.01. (E) Western blotting was carried to confirm inhibition of caspase 3 by the caspase 3 inhibitor.</p

Knockdown of NS affects the expression of cleaved caspase 3 and Bcl-2.

<p>(A) Changes in the expression pattern of certain apoptosis regulatory genes in siNS-treated MHCC79H cells. For UV irradiation, the culture medium was removed after transfection for 24 h. The culture dishes were then uncovered and placed in a UV cross-linker, and the cells were cultured for the appropriate times. For serum starvation, the culture medium was replaced with DMEM without FBS, followed by 24 h of culture. Total proteins were extracted for western blot analysis. Expression of pro-apoptotic caspase 3 was higher than in the control after irradiation with 50 J/m2 UV-C or serum starvation for 24 h, while expression the anti-apoptotic protein Bcl-2 was lower. (B) Quantification of NS, caspase 3, and Bcl-2 protein levels in MHCC97H cell lines. Image J analysis was performed to quantificate the western blots (n = 3), and GAPDH is a loading control. *P<0.05, **P<0.01. (C) After UV-induction or serum starvation, changes in the expression pattern of certain apoptosis regulatory genes in siNS-treated Bel7402 cells. (D) Quantification of NS, caspase 3, and Bcl-2 protein levels in Bel7402 cell lines. Image J analysis was performed to quantificate the western blots (n = 3), and GAPDH is a loading control. *P<0.05, **P<0.01.</p

Clincopathological Characteristics.

<p>Clincopathological Characteristics.</p

Exfoliation of LiNi_1/3Mn_1/3Co_1/3O₂ into Nanosheets Using Electrochemical Oxidation and Reassembly with Dialysis or Flocculation

Two-dimensional (2D) materials such as nanosheets are increasingly attracting attention for applications in energy storage and conversion. Many conventional battery compounds have layered structures, which can facilitate the exfoliation of these materials into nanosheet morphologies. In this work, LiNi_1/3Mn_1/3Co_1/3O₂ (NMC) particles were exfoliated into nanosheets using an electrochemical oxidation method enabled by the intercalation of tetraethylammonium cations into the interlayer space. The exfoliated materials were monolayer or double-layer nanosheets with hexagonal shapes and sizes of <50 nm. Two different methods were used to reassemble the nanosheets into NMC particles: (1) a slow, dialysis-based approach and (2) direct flocculation. Characterization of the NMC materials at different stages in the exfoliation and reassembly processes was performed using compositional analysis, X-ray diffraction, electron microscopy, and electrochemical methods. The dialysis reassembly method allowed for the restacking of the nanosheets into faceted, hexagonally shaped nanoplatelets, and the flocculation approach yielded only ill-defined particles. The differences in the observed potential-dependent redox behavior and electrochemical cycling characteristics are attributed to the role of the reassembly method in the formation of phase-segregated domains, with the particles reassembled using the dialysis approach displaying the best performance

Engineering the Microstructure of Electrospun Fibrous Scaffolds by Microtopography

Controlling the structure and organization of electrospun fibers is desirable for fabricating scaffolds and materials with defined microstructures. However, the effects of microtopography on the deposition and, in turn, the organization of the electrospun fibers are not well understood. In this study, conductive polydimethylsiloxane (PDMS) templates with different micropatterns were fabricated by combining photolithography, silicon wet etching, and PDMS molding techniques. The fiber organization was varied by fine-tuning the microtopography of the electrospinning collector. Fiber conformity and alignment were influenced by the depth and the slope of microtopography features, resulting in scaffolds comprising either an array of microdomains with different porosity and fiber alignment or an array of microwells. Microtopography affected the fiber organization for hundreds of micrometers below the scaffold surface, resulting in scaffolds with distinct surface properties on each side. In addition, the fiber diameter was also affected by the fiber conformity. The effects of the fiber arrangement in the scaffolds on the morphology, migration, and infiltration of cells were examined by in vitro and in vivo experiments. Cell morphology and organization were guided by the fibers in the microdomains, and cell migration was enhanced by the aligned fibers and the three-dimensional scaffold structure. Cell infiltration was correlated with the microdomain porosity. Microscale control of the fiber organization and the porosity at the surface and through the thickness of the fibrous scaffolds, as demonstrated by the results of this study, provides a powerful means of engineering the three-dimensional structure of electrospun fibrous scaffolds for cell and tissue engineering

Facile Green Synthesis of Magnetic Porous Organic Polymers for Rapid Removal and Separation of Methylene Blue

Herein, magnetic porous organic polymers composites (MOPs) with abundant free phenolic hydroxyl group were synthesized via a simple and green azo coupling reaction under mild conditions in water. Remarkably, Fe₃O₄@SiO₂ nanoparticles were encapsulated in the disordering porous organic polymers during the coupling reaction. The prepared MOPs possessed excellent stability, high special surface areas, controllable magnetism, and high adsorption capacity (1153 mg g^–1) toward cationic dye methylene blue. Moreover, the MOPs could be easily and rapidly recovered from the solution by using a magnet due to their superparamagnetic property and could be reused at least 5 times without decreasing the adsorption capacity, demonstrating a potential application in pollutants removal