Knockdown of NOB1 expression by RNAi inhibits cellular proliferation and migration in human gliomas

Abstract

NOB1 (NIN1/RPN12 binding protein 1 homolog), a ribosome assembly factor, is thought to be essential for the processing of the 20S pre-rRNA into the mature 18S rRNA. It is also reported to participate in proteasome biogenesis. However, the contribution of NOB1 gene dysfunction to the pathology of human diseases, such as gliomas, has not been addressed. Here, we detected expression levels of NOB1 mRNA in U251, U87, U373, and A172 cells by quantitative real-time PCR. To analyze the expression levels of NOB1 protein in glioma tissues, we performed immunohistochemistry on 56 pathologically confirmed glioma samples (7 Grade I cases, 19 Grade II cases, 16 Grade III cases, and 14 Grade IV cases). A recombinant lentivirus expressing NOB1 short hairpin RNA (shNOB1) was constructed and infected into U251 and U87-MG human glioma cells. We found that NOB1 mRNA was expressed in all four cell lines. The expression level of the NOB1 protein was significantly higher in high-grade gliomas than in low-grade gliomas. Knockdown of the NOB1 gene resulted in suppression of the proliferation and the colony-forming abilities of U251 and U87-MG cells, cell cycle arrest during the G0/G1 phase, and a significant enhancement of cell apoptosis. In addition, cell migration was significantly suppressed in U251 and U87-MG cells that were infected with the shNOB1-expressing lentivirus. These results suggest that NOB1 promotes glioma cell growth and migration and could be a candidate for molecular targeting during gene therapy treatments of glioma.

Keywords: 26S proteasome non-ATPase regulatory subunit 8 binding protein 1; 3,3′-diaminobenzidine; ATCC; American Type Culture Collection; BCA; BSA; Bicinchoninic acid; Bovine serum albumin; DAB; DMEM; Dulbecco's modified Eagle's medium; FACS; FBS; Fetal bovine serum; Fluorescence-activated cell sorting; GAPDH; GFP; Glioma; Glyceraldehyde3-phosphate dehydrogenase; Green Fluorescent Protein; HEK; Homologues of the pilT N-terminal domain; Human embryonic kidney; IHC; Immunohistochemistry; M-MLV Reverse Transcriptase; M-MLV-RT; MOI; MTT; Methylthiazoletetrazolium; Migration; Multiplicity of infection; NIN1/RPN12 binding protein 1 homolog; NOB1; PI; PIN domain; PSMD8BP1; Proliferation; Propidium iodide; RNA interference; RNAi; Short-hairpin RNA; Small interfering RNA; TRITC; Tetraethyl rhodamine isothiocyanate; WHO; World Health Organization; shRNA; siRNA.