Downregulation of GBAS regulates oral squamous cell carcinoma proliferation and apoptosis via the p53 signaling pathway

Abstract

Purpose: Oral squamous cell carcinoma (OSCC) is the most common and severe type of head and neck malignancy. The mechanisms by which OSCC arises depend on changes in a number of different factors and genes and the clinicopathological stage of the tumors. Better understanding the possible mechanisms of OSCC would help to identify a new target for molecular targeted therapy. The current study was focused on elucidating the significance of the glioblastoma-amplified sequence (GBAS) on malignant behaviors in OSCC, including proliferation and apoptosis. Patients and methods: In this study, we measured the levels of mRNA in OSCC and normal oral tissue samples using Affymetrix microarrays. We examined GBAS expression in OSCC tissues and the effect of GBAS knockdown on cell proliferation and apoptosis in vitro and in vivo. The mechanisms underlying GBAS were investigated. Results: In the present study, GBAS expression was substantially elevated in the majority of tested OSCC tissues. Further, knockdown of GBAS using lentiviral-delivered shRNA in cells had significant effects on cell proliferation, apoptosis and the cell cycle. A xenograft model was also used to assess the tumorigenicity of the GBAS knockdown on OSCC cells in vivo. Mechanistically, GBAS activated p53 signaling by regulating the mRNA and protein expression of CHEK1, AKT1, AKT2 and Bax. Finally, we also investigated the expression of GBAS in patients with OSCC, and the data revealed that GBAS expression was correlated with the rates of relapse and tumor grade. Conclusion: Our studies provide evidence that GBAS regulates OSCC cell proliferation and apoptosis via p53 signaling, which may be a candidate biomarker for the prognosis and treatment of OSCC.

Keywords: GBAS; apoptosis; oral squamous cell carcinoma; p53 signaling pathway.

Figure 1

Affymetrix microarray and high-content screening identified GBAS as a critical gene in the progression of OSCC. Notes: (A) Heat map showing gene expression profiles. Each row represents a gene and each column represents a sample. Red indicates high expression, whereas green indicates low expression. (B) A total of 17 genes were selected for validation by high-content screening. (C) Representative fluorescence images of high-content screening for GBSA and TMEM5. (D) The mRNA expression of GBSA by the qRT-PCR, **P<0.01. Abbreviations: OSCC, oral squamous cell carcinoma; GBAS, glioblastoma amplified sequence; TMEM5, transmembrane protein 5.

Figure 2

Representative figures of immunohistochemical staining for GBAS in OSCC tissues and paired adjacent normal tissue. Abbreviation: OSCC, oral squamous cell carcinoma.

Figure 3

Knockdown of GBAS inhibits the cell viability of 2 OSCC cell lines (CAL-27, Tca-8113). Notes: (A, B) MTT assay showed that the cell viability of CAL-27 (A) and Tca-8113 (B) was significantly inhibited after decreasing the expression of GBAS by sh-GBAS. (C, D) Celigo analysis was used to determine the cell viability of CAL-27 (C) and Tca-8113 (D) by the treatment of sh-GBAS. In all above experiments, the data were performed independently in triplicate and presented as mean ± SD, **P<0.01. Abbreviations: OSCC, oral squamous cell carcinoma; GBAS, glioblastoma amplified sequence.

Figure 4

4 Knockdown of GBAS influences the cell viability of 2 OSCC cell lines (CAL-27, Tca-8113). Notes: (A, B) Apoptosis was determined by flow cytometry assays in CAL-27 (A) and Tca-8113 (B) with GBAS silence and control cells. The apoptotic rate was calculated as the percentage of Annexin FITC positive cell. (C, D) Flow cytometry was also used to analysis the cell cycle of CAL-27 (C) and Tca-8113 (D) by the treatment of sh-GBAS. In all above experiments, the data were performed independently in triplicate and presented as mean ± SD, *P<0.05 and **P<0.01. Abbreviations: OSCC, oral squamous cell carcinoma; GBAS, glioblastoma amplified sequence; FITC: fluorescein isothiocyanate.

Figure 5

Knockdown of GBAS in CAL-27 cells inhibited tumorigenicity in nude mice. sh-GBAS or sh-Ctrl transfected cells were transplanted into inoculated subcutaneously into nude mice for up to 26 days (n = 10). Note: (A) After scarification, xenograft tumors in vivo and excised tumors were recorded. (B) Tumor volume was estimated based on the following equation: volume =1/2 × (largest diameter) × (smallest diameter)2 and data were shown as mean ± SD (n = 10 in each group), **P<0.01. (C) Tumor weight was obtained at the end of experiment and was displayed as mean ± SD (n = 10 in each group), **P<0.01. (D) Tumor growth was systemically administered and monitored by FI. At the end of the experiments, tumor tissues were exposed to FI. Images represent the radiant efficiency. (E) Regions of interest calculated for the tumor and results were expressed as total radiant efficiency ([p/s]/[μW/cm2]), **P<0.01. Abbreviations: GBAS, glioblastoma amplified sequence; NC, normal control; KD, knockdown; Avg, average; FI, fluorescence imaging.

Figure 6

IPA identified proteins networks showing inter-relationships and pathways. Note: (A) Network analysis revealed that some genes may be activated by GBAS (NIPSNAP2) and its partners. Binding partner proteins were uploaded into the IPA and the top biological networks generated a global view. (B) The altered pathways were demonstrated. Orange marks indicated activated (Z-score>0) and the blue marks indicated suppressed pathways (Z-score<0). Abbreviations: GBAS, glioblastoma amplified sequence; IPA, ingenuity pathway analysis; NIPSNAP2, 4-nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 2.

Figure 7

The expression of AKT1, AKT2, Bax, CHEK1 and GBAS by the qRT-PCR and western blotting in tumor tissues in the sh-GBAS groups. Note: (A) The protein expression of AKT1, AKT2, Bax and CHEK1 by western blotting. GAPDH was used as a loading control. (B) The mRNA expression of AKT1, AKT2, Bax, CHEK1 and GBAS by the qRT-PCR. Histogram representing indicated the results of three independent experiments **P<0.01. Abbreviations: GBAS, glioblastoma amplified sequence; AKT1, AKT serine/threonine kinase 1; AKT2, AKT serine/threonine kinase 2; Bax, BCL2 associated X; CHEK1, checkpoint kinase 1; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT-PCR, reverse transcription-polymerase chain reaction; NC, normal control; KD, knockdown.