PTPN12 down-regulated by miR-146b-3p gene affects the malignant progression of laryngeal squamous cell carcinoma

Abstract

Laryngeal squamous cell carcinoma (LSCC) is a common malignancy among men in the anatomical position of head and neck. Hoarseness, pharyngalgia, and dyspnea are common symptoms. LSCC is a complex polygenic carcinoma that is caused by many factors involving polygenic alteration, environmental pollution, tobacco, and human papillomavirus. Classical protein tyrosine phosphatase nonreceptor type 12 (PTPN12) has been extensively studied to decipher its mechanism as a tumor suppressor gene in various human carcinomas; however, there is no comprehensive elucidation of the PTPN12 expression and its regulatory mechanisms in LSCC. As such, we expect to provide new insights for finding new biomarkers and effective therapeutic targets in LSCC. Immunohistochemical staining, western blot (WB), and quantitative real-time RT-PCR (qRT-PCR) were used for the messenger RNA (mRNA) and protein expression analyses of PTPN12, respectively. 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, clone formation, transwell migration, and transwell invasion assays were used to assess the proliferation, migration, and invasion ability of LSCC cells. Online prediction and design software tools (http://www.targetscan.org/ and http://www.microRNA.org) were used to predict associated miRNA. Studying the targeted regulatory relationship between miR-146b-3p and PTPN12 was based on dual luciferase reporter gene analysis. qRT-PCR was used to assess miR-146b-3p expression in LSCC. miR-146b-3p inhibitor and mimic were transfected, followed by qRT-PCR and WB assays to detect the expression of PTPN12. The gain and loss functional experiments were used to investigate the effects of miR-146b-3p transfection on the proliferation, migration, and invasion of tumor cells. Online bioinformatics prediction software (https://cn.string-db.org/ and https://www.genecards.org/) was used to determine potential downstream target genes of PTPN12. qRT-PCR and WB analyses were used to assess the mRNA and protein expression levels of target genes. Our study showed significantly decreased mRNA and protein expression levels of PTPN12 in LSCC compared with the adjacent normal tissues. The lower PTPN12 mRNA expression was correlated with pathological differentiation, and lower PTPN12 protein expression was correlated with the TNM stage in LSCC tissues. The subsequent in vitro functional analyses showed the inhibitory effect of PTPN12 over-expression on the proliferation, migration, and invasiveness abilities of LSCC cell line. Using online prediction and design software, miR-146b-3p was searched to target PTPN12. The miR-146b-3p was expressed at a high level in LSCC tissues and cell lines. Luciferase reporter assay exhibited that miR-146b-3p inhibited the luciferase activity of PTPN12 markedly. The functional analyses showed the tumor-promoting role of miR-146b-3p on the proliferation, migration, and invasiveness abilities of LSCC cell. Furthermore, co-transfection of cells with miR-146b-3p and PTPN12 significantly restored the inhibitory effect of PTPN12 on LSCC cell growth, migration, and invasiveness. This phenomenon unveiled that miR-146b-3p regulated the proliferation, migration, and invasion of LSCC cells by targeting PTPN12. EGFR and ERBB2 were selected as the downstream-regulation target genes. Up-regulation of PTPN12 significantly suppressed EGFR expression. Accordingly, the miR-146b-3p mimic significantly up-regulated the EGFR expression. However, up-regulation of PTPN12 and miR-146b-3p mimic suppressed ERBB2 protein expression but induced its gene expression. Down-regulation of PTPN12 is associated with up-regulation of miR-146b-3p in LSCC. Moreover, PTPN12 serves as a tumor suppressor gene through regulating the proliferation, migration, and invasion of LSCC cells. miR-146b-3p/PTPN12 axis is expected to be a novel therapeutic target in LSCC.

Keywords: Laryngeal squamous cell carcinoma; PTPN12; invasion; miR-146b-3p; migration.

Figure 1

PTPN12 expression in LSCC tissues and cell lines. (a). Relative expression of PTPN12 mRNA in LSCC tiusses and paired paracancerous tissues. (b). Comparison of the relative expression of PTPN12 mRNA between tumor tissues and paired adjacent normal tissues. (c). PTPN12 protein expression in LSCC tissues and paired paracancerous tissues from two patients. (d). Relative expression of PTPN12 mRNA in two laryngeal cancer cell lines. The Pool represents mean expression level of PTPN12 mRNA in 20 para-cancer normal tissues. (e) The protein expression levels of PTPN12 in LSCC cells and five normal tissue samples. Data are presented as the mean ± SD. Student*s t-test was used for P value assessment. *P < 0.05, **P < 0.01.

Figure 2

The biological function of AMC-HN-8 overexpressing PTPN12 in vitro. (a). PTPN12 overexpression efficiency was verified by qRT-PCR analysis in AMC-HN-8 cells after transfection with pcDNA3.1-PTPN12 or pcDNA3.1 empty vector. (b). PTPN12 overexpression efficiency was verified by WB analysis in AMC-HN-8 cells after transfection with pcDNA3.1-PTPN12 or pcDNA3.1 empty vector. (c). The MTS assay was used to determine the cell proliferation ability. PTPN12 overexpression led to marked inhibition of tumor cell proliferation capacity than cell transfected with the pcDNA3.1 empty vector at the indicated time points. (d). A colony formation assay was carried out to analyse the cell proliferation ability. Cells transfected with pcDNA3.1-PTPN12 had decreased colony formation. (e–f). Transwell migration and invasion assays demonstrated that overexpressing PTPN12 notably decreased cell migration ability and invasion ability compared with control cells. Data are presented as the mean ± SD of three independent experiments. **P < 0.01.

Figure 3

The correlation of PTPN12 expression and miR-146b-3p expression. (a). Relative expression of miR-146b-3p in LSCC tiusses and paired paracancerous tissues. (b). Pearsons correlation analysis was performed to explore the correlation between the expression of PTPN12 and miR-146b-3p. (c). Relative expression of miR-146b-3p in two laryngeal cancer cell lines. The Pool represents mean expression level of miR-146b-3p in 20 para-cancer normal tissues. (d–e). qRT-PCR was carried out to analyse the relative expression of PTPN12 in LSCC cells transfected with miR-146b-3p mimic and inhibitor. (f). WB was carried out to analyse the PTPN12 protein expression in AMC-HN-8 cell transfected with miR-146b-3p mimic and inhibitor. **P < 0.01.

Figure 4

MiR-146b-3p directly targets PTPN12 in LSCC cell. (a). pmirGLO-PTPN12-3′-UTR-WT and pmirGLO-PTPN12-3′-UTR-Mut luciferase reporter plasmids with the miR-146b-3p binding site. (b). Luciferase activity was significantly decreased in AMC-HN-8 and 293 T cells co-transfected with PTPN12-3′-UTR-WT and miR-146b-3p mimic in vitro. **P < 0.01.

Figure 5

The biological function of miR-146b-3p in vitro. (a and b). Transfection efficiency was verified by qRT-PCR analysis in AMC-HN-8 and TU177 cells transfected with miR-146b-3p mimic and inhibitor. (c). The MTS assay was used to determine the cell proliferation ability. The miR-146b-3p inhibitor led to marked inhibition of tumor cells proliferation capacity than cells transfected with the miR-146b-3p mimic at the indicated time points. (d and e). Down-regulated miR-146b-3p expression dramatically decreased the ability of LSCC cell migration ability and invasion ability in vitro. Data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01.

Figure 6

Synergistic effect of miR-146b-3p and PTPN12 on biological behavior of laryngeal cancer cell. (a). Growth curve of AMC-HN-8 after cotransfected with miR-146b-3p mimic and pcDNA3.1-PTPN12 was determined by MTS assay. (b). A colony formation assay of AMC-HN-8 cell after cotransfected with miR-146b-3p mimic and pcDNA3.1-PTPN12. (c). Migration assay and (d). Invasion assay of AMC-HN-8 cell after cotransfected with miR-146b-3p mimic and pcDNA3.1-PTPN12. Data are presented as the mean ± SD of three independent experiments. *P < 0.05, **P < 0.01.

Figure 7

Prediction of downstream target genes of PTPN12. (a). Prediction potential downstream targets of PTPN12 was performed by the online software STRING. (b). Prediction potential downstream targets of PTPN12 was performed by the online software GeneCards database. (c). Relative expression of EGFR and ERBB2 in AMC-HN-8 after transfected with miR-146b-3p mimic and pcDNA3.1-PTPN12 was determined by qRT-PCR assay. (d). Relative expression of EGFR and ERBB2 in AMC-HN-8 after transfected with miR-146b-3p mimic and pcDNA3.1-PTPN12 was determined by WB assay. Data are presented as the mean ± SD of three independent experiments. **P < 0.01.

Figure A1

Relative expression of miR-124-3p in LSCC tiusses and paired paracancerous tissues. (b). Relative expression of miR-194-5p in LSCC tiusses and paired paracancerous tissues. (c). Relative expression of miR-338-3p in LSCC tiusses and paired paracancerous tissues. (d). Relative expression of miR-506-3p in LSCC tiusses and paired paracancerous tissues. *P < 0.05, **P < 0.01.