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

Kikkawa N, Hanazawa T, Fujimura L, Nohata N, Suzuki H, Chazono H, Sakurai D, Horiguchi S, Okamoto Y, Seki N - Br. J. Cancer (2010)

Bottom Line: Expression analysis identified 11 upregulated and 31 downregulated miRNAs.The gene PTPN11 coding for a cytoplasmic protein tyrosine phosphatase containing two Src Homology 2 domains was identified as a miR-489-targeted gene.Identification of the tumour-suppressive miRNA miR-489 and its target, PTPN11, might provide new insights into the underlying molecular mechanisms of HSCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Functional Genomics, Graduate School of Medicine, Chiba University, Chiba, Japan.

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.

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Proliferation is inhibited by transfection with si-PTPN11 in FaDu cells. FaDu cells were transfected with 10 n 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 n si-PTPN11. After incubating for 72 h, cell proliferation was determined using an XTT assay. *P<0.05.
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fig4: Proliferation is inhibited by transfection with si-PTPN11 in FaDu cells. FaDu cells were transfected with 10 n 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 n si-PTPN11. After incubating for 72 h, cell proliferation was determined using an XTT assay. *P<0.05.

Mentions: A loss-of-function assay using small interfering RNA analysis was performed to examine the oncogenic function of PTPN11, which is directly targeted by miR-489. The effect of si-PTPN11 on mRNA and protein expression levels was evaluated after transfection into FaDu cells. Both PTPN11 mRNA and protein levels had been reduced 72 h after transfection (Figures 4A and B). The contribution of PTPN11 to cell viability was assessed with si-PTPN11 loss-of-function assays in FaDu cells. Knockdown of PTPN11 significantly decreased cancer cell growth compared with si-control transfectants (Figure 4C).


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

Kikkawa N, Hanazawa T, Fujimura L, Nohata N, Suzuki H, Chazono H, Sakurai D, Horiguchi S, Okamoto Y, Seki N - Br. J. Cancer (2010)

Proliferation is inhibited by transfection with si-PTPN11 in FaDu cells. FaDu cells were transfected with 10 n 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 n si-PTPN11. After incubating for 72 h, cell proliferation was determined using an XTT assay. *P<0.05.
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fig4: Proliferation is inhibited by transfection with si-PTPN11 in FaDu cells. FaDu cells were transfected with 10 n 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 n si-PTPN11. After incubating for 72 h, cell proliferation was determined using an XTT assay. *P<0.05.
Mentions: A loss-of-function assay using small interfering RNA analysis was performed to examine the oncogenic function of PTPN11, which is directly targeted by miR-489. The effect of si-PTPN11 on mRNA and protein expression levels was evaluated after transfection into FaDu cells. Both PTPN11 mRNA and protein levels had been reduced 72 h after transfection (Figures 4A and B). The contribution of PTPN11 to cell viability was assessed with si-PTPN11 loss-of-function assays in FaDu cells. Knockdown of PTPN11 significantly decreased cancer cell growth compared with si-control transfectants (Figure 4C).

Bottom Line: Expression analysis identified 11 upregulated and 31 downregulated miRNAs.The gene PTPN11 coding for a cytoplasmic protein tyrosine phosphatase containing two Src Homology 2 domains was identified as a miR-489-targeted gene.Identification of the tumour-suppressive miRNA miR-489 and its target, PTPN11, might provide new insights into the underlying molecular mechanisms of HSCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Functional Genomics, Graduate School of Medicine, Chiba University, Chiba, Japan.

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.

Show MeSH
Related in: MedlinePlus