Decreased expression of pseudogene PTENP1 promotes malignant behaviours and is associated with the poor survival of patients with HNSCC

Abstract

PTENP1, a pseudogene of PTEN, was previously reported to be a tumour suppressor in some cancer types. However, there was no evidence for the biological function and expression of PTENP1 in head and neck squamous cell carcinoma (HNSCC). Here, we evaluated the function and clinical implications of PTENP1 in HNSCC. Using RT-PCR and quantitative real-time PCR (qRT-PCR), we found that the level of PTENP1 was reduced in HNSCC specimens compared with adjacent tissues. A decrease in the PTENP1 copy number, but not in the PTEN copy number, was frequently observed in tumour cell lines (4 of 5 cell lines) by genomic real-time PCR. Decreased PTENP1 expression was significantly associated with a history of alcohol use (P = 0.034). Univariate and multivariate Cox regression analyses revealed that low expression of PTENP1 correlated with worse overall survival (OS, P = 0.005; HR:0.170; Cl:0.049 to 0.590) and disease-free survival (DFS, P = 0.009; HR:0.195; Cl:0.057 to 0.664) rates of HNSCC patients. Furthermore, ectopic PTENP1 expression inhibited the proliferation, colony formation and migration of HNSCC cells and the growth of xenograft HNSCC tumours. These results demonstrate that PTENP1 might play an important role in the initiation and progression of HNSCC.

Figure 1. The expression pattern and clinical implications of PTENP1 in HNSCCs.

(A). RT-PCR was performed to determine the expression of PTENP1 in five HNSCC cell lines. Oral mucosal epithelial cells were used as a control. The full-length gels are presented in Supplementary Figure 1A and B, (B). The results of a real-time PCR analysis of the PTENP1 expression in five HNSCC cell lines. The expression level in oral mucosal epithelial cells was normalized to 1. The data were presented as the means ± SD. *p < 0.05. (C) The results of a real-time PCR analysis of the PTENP1 expression in normal and tumour tissue specimens. *p < 0.05. (D) The cellular location of PTENP1 in HN13 and HN30 cells. U2 was used as a positive control for nuclear RNA. GAPDH was used as a positive control for cytoplasmic RNA. The full-length gels are presented in Supplementary Figure 1C–E. (E,F) Kaplan-Meier survival curve indicated overall survival (OS, (E)) and disease-free survival (DFS, (F)) by evaluation of the expression levels of PTENP1 in the cohort.

Figure 2. Expression of PTEN and copy number of PTENP1 or PTEN were detected in HNSCC cells.

(A) The results of a real-time PCR analysis of the PTEN expression in five HNSCC cell lines. The expression of oral mucosal epithelial cells was normalized to 1. The data were presented as the means ± SD. *p < 0.05. (B) Western blot of PTEN expression in the five HNSCCs cell lines. Oral mucosal epithelial cells were used as a control. The full-length blots are presented in Supplementary Figure 1F and G. (C) Genomic real-time PCR of the PTENP1 copy number in five HNSCC cells. The data were presented as the means ± SD. *p < 0.05. (D) Genomic real-time PCR of the PTEN copy number in five HNSCC cells. Primers were designed against intron 1, intron 3 and exon 9 of PTEN. The data were presented as the means ± SD. *p < 0.05.

Figure 3. Co-expression of PTENP1 and PTEN in HNSCC cells.

(A,B) The results of a real-time PCR analysis of the PTENP1 expression after PCMV-PTENP1 transfection in HN13 and HN30 cells. The data were presented as the means ± SD. *p < 0.05. (C,D) The results of a real-time PCR analysis of the PTEN expression after PCMV-PTENP1 transfection in HN13 and HN30 cells. The data were presented as the means ± SD. *p < 0.05.

Figure 4. Over-expression of PTENP1 affects tumour growth and colony formation.

(A,B) The MTT assay for evaluation of the proliferation of HN13 and HN30 cells, respectively. The absorbance was detected at 0 h, 24 h, 48 h and 72 h. The 0 h absorbance of each group was normalized to 1. The relative values were shown as growth index. The data were presented as the means ± SD. *p < 0.05. (C) The colony formation assay for evaluation of the proliferation of HN13 and HN30 cells, respectively. (D,E) Count statistics of the colony formation assay. The colony number of control cells was normalized to 100%. The data were presented as the means ± SD. *p < 0.05.

Figure 5. PTENP1 ectopic expression influences on tumour invasion migration and migration.

(A) The transwell assay for evaluation of the invasion of HN13 and HN30 cells, respectively. (B,C) The invasion rate based on the transwell assay. The value of the control was normalized to 100%. The data were presented as the means ± SD. *p < 0.05. (D,E) The scratch wound assay for evaluation of the migration of HN13 and HN30 cells, respectively.

Figure 6. Exogenous expression of PTENP1 inhibited the growth of xenograft tumour of HN13 cell lines.

(A,B) Lumps of mice carrying Mock cells and PTENP1 expressing cells in xenograft tumour model, respectively. (C) Growth curve based on the xenograft tumour assay. The tumour size was calculated using the formula length *width *width/2. The data were presented as the means ± SD.