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

Clincopathological Characteristics.

<p>Clincopathological Characteristics.</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

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