miR-489 is a tumour-suppressive miRNA target PTPN11 in hypopharyngeal squamous cell carcinoma (HSCC)

Abstract

Background: Hypopharyngeal squamous cell carcinoma (HSCC) is an aggressive malignancy with one of the worst prognoses among all head and neck cancers. Greater understanding of the pertinent molecular oncogenic pathways could help improve diagnosis, therapy, and prevention of this disease. The aim of this study was to identify tumour-suppressive microRNAs (miRNAs), based on miRNA expression signatures from clinical HSCC specimens, and to predict their biological target genes.

Methods: Expression levels of 365 human mature miRNAs from 10 HSCC clinical samples were screened using stem-loop real-time quantitative PCR. Downregulated miRNAs were used in cell proliferation assays to identify a tumour-suppressive miRNA. Genome-wide gene expression analyses were then performed to identify the target genes of the tumour-suppressive miRNA.

Results: Expression analysis identified 11 upregulated and 31 downregulated miRNAs. Gain-of-function analysis of the downregulated miRNAs revealed that miR-489 inhibited cell growth in all head and neck cancer cell lines examined. The gene PTPN11 coding for a cytoplasmic protein tyrosine phosphatase containing two Src Homology 2 domains was identified as a miR-489-targeted gene. Knockdown of PTPN11 resulted in the inhibition of cell proliferation in head and neck SCC cells.

Conclusion: Identification of the tumour-suppressive miRNA miR-489 and its target, PTPN11, might provide new insights into the underlying molecular mechanisms of HSCC.

Figure 1

Effect of transfection with 31 downregulated miRNAs on cancer cell proliferation. Cancer cells were transfected with 10 nM of the indicated mature miRNA. After incubation for 72 h, cell proliferation was determined using XTT assays. (A) FaDu cells; (B) HSC2 cells; (C) HSC3 cells; (D) D562 cells. The darkly shaded bars represent a decrease in cell proliferation of more than 30% compared with control transfections.

Figure 2

miR-489 negatively regulates PTPN11 expression. FaDu cells were transfected with miR-489. After incubation for 72 h, total RNA and proteins were isolated. (A) FaDu cells were treated with a miR-negative control (10 nM) or miR-489 (10 nM). After 72 h, miR-489 expression was measured by TaqMan quantitative real-time PCR. The results are normalised to RNU44 expression. (B) PTPN11 mRNA expression was analysed by TaqMan quantitative real-time PCR. The results are normalised to GAPDH expression and are presented relative to control expression. ^* P<0.05. (C) Cell lysates were analysed by immunoblotting. Membranes were incubated with anti-PTPN11 IgG and anti-β-actin IgG. The autoradiographic density of each protein band was quantified using NIH ImageJ software. The results are standardised against β-actin levels and are presented as the relative density.

Figure 3

miR-489 binds to the 3′ UTR of PTPN11 mRNA. A luciferase reporter assay used a vector encoding the partial PTPN11 3′ UTR (position 3300–3850). Renilla luciferase values are normalised against firefly luciferase values. Luciferase reporter assays were repeated using mutated vectors in which the candidate sites targeted by the miR-489 were deleted. ^*P<0.05

Figure 4

Proliferation is inhibited by transfection with si-PTPN11 in FaDu cells. FaDu cells were transfected with 10 nM si-PTPN11. Total RNA and proteins were isolated after 72-h incubation. (A) PTPN11 mRNA expression was analysed by TaqMan quantitative real-time PCR. The results are normalised to GAPDH expression and are presented as relative to control expression. ^*P<0.05. (B) Cell lysates were analysed by immunoblotting. Membranes were incubated with anti-PTPN11 IgG and anti-β-actin IgG. The autoradiographic density of each protein band was quantified using NIH ImageJ software. The results are standardised against β-actin levels and are presented the relative density. (C) FaDu cells were transfected with 1 or 10 nM si-PTPN11. After incubating for 72 h, cell proliferation was determined using an XTT assay. ^*P<0.05.

Figure 5

PTPN11 overexpression in clinical HSCC specimens. (A) PTPN11 mRNA expression levels were analysed by TaqMan quantitative real-time PCR and normalised to 18S rRNA expression. PTPN11 mRNA expression was compared between matched HSCC and non-cancerous tissues in 16 patients. Data were analysed using the paired t-test. N, non-cancerous tissues; C, cancer tissues. (B) Correlation between PTPN11 and miR-489 expression in HSCC clinical specimens.