Gene silencing of NOB1 by lentivirus suppresses growth and migration of human osteosarcoma cells

Abstract

NIN1/RPN12 binding protein 1 homolog (Saccharomyces cerevisiae) (NOB1) encodes a chaperone protein that joins the 20S proteasome with the 19S regulatory particle in the nucleus and facilitates the biogenesis of the 26S proteasome, which plays a role in maintaining cellular homeostasis by controlling protein degradation. In order to investigate the role of NOB1 in osteosarcoma, NOB1 protein expression in human osteosarcoma cell lines was assessed using western blot analysis. Lentivirus-mediated short hairpin RNA was employed to knock down NOB1, and the effects of NOB1 silencing on cell growth were assessed using MTT, colony formation and cell cycle assays. Cell migration was observed using the Transwell assay. In addition, the expression levels of E-cadherin and β-catenin were examined by western blot analysis. Functional analysis indicated that NOB1-knockdown markedly inhibited cell growth and caused G2/M-phase arrest in human osteosarcoma cells. Furthermore, NOB1 inhibition decreased cell migration and increased E-cadherin and β-catenin expression in U2OS cells. In conclusion, the present study suggested that NOB1 depletion may inhibit osteosarcoma development by increasing E-cadherin and β-catenin expression and, for the first time, indicated the potential of NOB1 as a target in osteosarcoma treatment.

Figure 1

NOB1-knockdown by a lentivirus-mediated RNA interference system. (A) Expression levels of Nin one binding protein in five osteosarcoma cell lines (Saos-2, U2OS, MG-63, SW1353 and SF-86) using western blot analysis. (B and C) Fluorescence photomicrographs of (B) Saos-2 and (C) U2OS cells infected by the lentivirus. Pictures were captured 96 h after infection (magnification, ×100). (D and E) Identification of NOB1-knockdown efficiency via quantitative polymerase chain reaction in (D) Saos-2 and (E) U2OS cells. ^*P<0.05, ^**P<0.01 compared with Lv-shCon. Con, no lentivirus treatment; Lv-shCon, control lentivirus; Lv-shNOB1, lentivirus containing short hairpin RNA targeting NOB1; NOB1, NIN1/RPN12 binding protein 1 homolog (Saccharomyces cerevisiae); GFP, green fluorescent protein.

Figure 2

Effect of NOB1 inhibition on the growth of Saos-2 cells. (A) Cell proliferation assay was performed using methylthiazoletetrazolium staining. The absorbance of the plate was recorded at 595 nm. (B) Saos-2 cells were allowed to grow into natural colonies in a six-well plate. Following staining with Giemsa, the number of colonies was counted in the three groups. (C) Fluorescence and light photomicrographs of Saos-2 cell monoclones in the three groups (magnification, ×40). ^***P<0.001 compared with Lv-shCon. Con, no lentivirus treatment; Lv-shCon, control lentivirus; Lv-shNOB1, lentivirus containing short hairpin RNA targeting NOB1; NOB1, NIN1/RPN12 binding protein 1 homolog (Saccharomyces cerevisiae); OD, optical density.

Figure 3

Effect of NOB1 inhibition on the growth of U2OS cells. (A) Cell proliferation assay was performed using methylthiazoletetrazolium staining. The absorbance of the plate was recorded at 595 nm. (B) U2OS cells were allowed to grow into natural colonies in a six-well plate. Following staining with Giemsa, the number of colonies was counted in the three groups. (C) Fluorescence and light photomicrographs of U2OS cell monoclones in the three groups (magnification, ×40). ^***P<0.001 compared with Lv-shCon. Con, no lentivirus treatment; Lv-shCon, control lentivirus; Lv-shNOB1, lentivirus containing short hairpin RNA targeting NOB1; NOB1, NIN1/RPN12 binding protein 1 homolog (Saccharomyces cerevisiae); OD, optical density.

Figure 4

NOB1 inhibition induces G2/M arrest in Saos-2 cells. (A) Flow cytometry histograms of Saos-2 cells following lentivirus infection in the three groups (Con, Lv-shCon and Lv-shNOB1). (B) Quantification of cell cycle distribution in Saos-2 cells by fluorescence-activated cell sorting. ^**P<0.01 compared with Lv-shCon. Con, no lentivirus treatment; Lv-shCon, control lentivirus; Lv-shNOB1, lentivirus containing short hairpin RNA targeting NOB1; NOB1, NIN1/RPN12 binding protein 1 homolog (Saccharomyces cerevisiae).

Figure 5

NOB1 inhibition suppresses U2OS cell migration. U2OS cells were subjected to three treatments (Con, Lv-shCon or Lv-shNOB1) for 72 h. Cells were then seeded onto the upper chamber of the Transwell plate. Migrated cells were (A) stained with crystal violet (magnification, ×100) and (B) counted. (C) Stained cells were dissolved and color intensity was assessed using a spectrophotometer. ^**P<0.01, ^***P<0.001 compared with Lv-shCon. Con, no lentivirus treatment; Lv-shCon, control lentivirus; Lv-shNOB1, lentivirus containing short hairpin RNA targeting NOB1; NOB1, NIN1/RPN12 binding protein 1 homolog (Saccharomyces cerevisiae); OD, optical density.

Figure 6

Effect of NOB1 inhibition on cancer cell regulators. The protein levels of E-cadherin and β-catenin were examined using western blot analysis. ^**P<0.01, ^***P<0.001 compared with Lv-shCon. Con, no lentivirus treatment; Lv-shCon, control lentivirus; Lv-shNOB1, lentivirus containing short hairpin RNA targeting NOB1; NOB1, NIN1/RPN12 binding protein 1 homolog (Saccharomyces cerevisiae).