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Dysregulation of microRNAs (miRNAs) is acknowledged in human cutaneous squamous cell carcinoma (cSCC). We hereby evaluated the ability of miRNA-216b (miR-216b) to impact human cSCC. cSCC tissues with corresponding adjacent normal tissues were collected from 40 patients diagnosed with cSCC where the expression pattern of miR-216b and targeting protein for Xenopus kinesin-like protein 2 (TPX2) was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot analysis. A431 cells were transfected with miR-216b mimic, miR-216b inhibitor, or short interfering RNA against TPX2 to evaluate cell proliferation, invasion, migration, and apoptosis using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, scratch test, Transwell assay, and flow cytometry. TPX2 was highly expressed in cSCC tissues while miR-216b was poorly expressed in association with tumor differentiation, lymph node metastasis, and tumor node metastasis staging in patients with cSCC. In response to overexpressed miR-216b or silenced TPX2, cSCC cell proliferation, invasion, and migration were suppressed and apoptosis was stimulated, along with activated p53 signaling. Thus, upregulated miR-216b was capable of promoting apoptosis and inhibiting proliferation, invasion, and migration of cSCC cells by downregulating TPX2 through activation of the p53 signaling, highlighting a novel biomarker for novel treatment modalities against cSCC.
Figure 1. TPX2 Is Overexpressed in cSCC Tissues(A) Immunohistochemical staining of TPX2 in adjacent normal tissues and cSCC tissues (400×). (B) Quantitative analysis of positive expression level of TPX2 in adjacent normal tissues and cSCC tissues. The immunohistochemical results were expressed by measurement data and analyzed by the chi-square test. N = 40. *p < 0.05 versus the adjacent normal tissues.
Figure 2. miR-216b Is Poorly Expressed in cSCC Tissues(A) miR-216b expression and mRNA levels of TPX2, MDM2, p21, p53, PCNA, and bcl-2/bax in cSCC tissues and adjacent normal tissues determined by qRT-PCR. (B) Western blots of TPX2, MDM2, p21, p53, PCNA, and bcl-2/bax in cSCC tissues and adjacent normal tissues normalized to GAPDH. (C) Protein expressions of TPX2, MDM2, p21, p53, PCNA, and bcl-2/bax in cSCC tissues and adjacent normal tissues determined by western blot analysis. Data were expressed as mean ± standard deviation, and data among multiple groups were compared by one-way ANOVA, followed by the Tukey’s post hoc test. *p < 0.05.
Figure 3. TPX2 Is Confirmed as a Target Gene of miR-216b(A) Luciferase activity of the TPX2-WT and TPX2-Mut after transfection determined by dual luciferase reporter gene assay. (B and C) Protein bands and protein levels of TPX2 in A431 cells normalized to GAPDH determined by western blot analysis. Data were expressed as mean ± standard deviation and analyzed by the unpaired t test. *p < 0.05.
Figure 4. Overexpressed miR-216b Inhibits Proliferation and Induces Apoptosis through Activation of the p53 Signaling by Downregulating TPX2 in cSCC(A) miR-216b expression in A431 cells determined by qRT-PCR. (B) mRNA levels of p53, p21, TPX2, and MDM2 in A431 cells determined by qRT-PCR. (C) mRNA levels of proliferation marker PCNA and apoptosis-related factors bcl-2/bax in A431 cells determined by qRT-PCR. (D) Western blots of p53, p21, TPX2, MDM2, PCNA, and bcl-2/bax in A431 cells normalized to GAPDH. (E) Protein expressions of p53, p21, TPX2, MDM2 in A431 cells determined by western blot analysis. (F) Protein expressions of PCNA and bcl-2/bax in A431 cells determined by western blot analysis. Data were expressed as mean ± standard deviation, and data among multiple groups were compared by one-way ANOVA, followed by the Tukey’s post hoc test. *p < 0.05.
Figure 5. miR-216b Overexpression and TPX2 Silencing Reduce cSCC Cell Viability, Migration, and Invasion(A) Quantitative analysis for cell viability detected by MTT assay. (B) Microscopic views for cell migration detected by scratch test. (C) Quantitative analysis for cell migration detected by scratch test. (D) Microscopic views for cell invasion detected by Transwell assay (×100). (E) Quantitative analysis for cell invasion detected by Transwell assay. The data were expressed as mean ± standard deviation. Data comparisons among multiple groups were analyzed by one-way ANOVA, followed by the Tukey’s post hoc test and data at different time points were compared using repeated-measures ANOVA. *p < 0.05.
Figure 6. miR-216b Overexpression and TPX2 Silencing Block cSCC Cell-Cycle Entry and Promote Cell Apoptosis(A) Cell-cycle distribution assessed by flow cytometry. (B) Comparisons of G0/G1 and G2/M cells among each groups. (C) Apoptotic cell rate determined by flow cytometry. (D) Comparisons of apoptotic cell rate among each groups. Data were expressed as mean ± standard deviation and data among multiple groups were compared by one-way ANOVA, followed by the Tukey’s post hoc test. *p < 0.05.
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