miRNA-221 promotes cutaneous squamous cell carcinoma progression by targeting PTEN

Abstract

Background: Cutaneous squamous cell carcinoma (CSCC) is a common type of skin malignancy. MicroRNA-221 (miRNA-221) is a critical non-coding RNA in tumor initiation and progression. However, the molecular mechanisms of miRNA-221 in the development of CSCC remain unknown. This study investigated the expression of miRNA-221 in CSCC and its potential tumor biological functions.

Methods: MTT assay, colony assay, PCR, and Western blot were adopted.

Results: In this study, miRNA-221 expression was significantly higher in CSCC tissues and cell lines than in normal tissues and cells (P < 0.05). Further functional experiments indicated that miRNA-221 knockdown inhibited the proliferation and cell cycle, while upregulation of miRNA-221 presented the opposite role. The dual reporter gene assays indicated that PTEN is a direct target gene of miRNA-221. PTEN protein or mRNA levels were decreased after the cells were transfected with miR-221 mimics.

Conclusions: Taken together, the obtained results indicated that miR-221 plays an oncogenic function in CSCC by targeting PTEN and further suggest that miR-221 may be a potential target for CSCC diagnosis and treatment.

Keywords: Cutaneous squamous cell carcinoma; PTEN; Proliferation; Treatment; miRNA-221.

Fig. 1

Expression of miR-221 in CSCC tissues and cell lines. a qPCR analysis of miR-221 in tumor and adjacent non-tumor tissues. b Average relative miR-221 level in CSCC cell lines (A431, SCC13, HSC-5 and SCL-1) and the human normal skin cell line HaCaT. Data are means ± SD of three independent experiments. * P < 0.05, compared with control

Fig. 2

miR-221 regulates cell proliferation in CSCC. Expression level of miR-221 in cells after transfection with miR-221 mimics (a) and inhibitors (b), as detected by quantitative RT-PCR. miR-221 overexpression (c) or silencing (d) regulated cell viability in cells at 24, 48, and 72 h by MTT assay. Quantitative results of colony formation assay in A431 (e) and SCC13 (f) cells transfected with miR-221 inhibitor or mimic, respectively. * P < 0.05, compared with control

Fig. 3

miR-221 regulates cell cycle in CSCC. Quantitative results of cell-cycle assay in A431 (a) and SCC13 (b) cells transfected with miR-221 inhibitor or mimic, respectively. * P < 0.05, compared with control

Fig. 4

PTEN is a direct target of miR-221. a Binding sequences for miR-221 in the 3′-UTR of PTEN, and the mutations in the 3′-UTR of PTEN are presented. b Luciferase activity of the wild type PTEN 3′-UTR (Wt) and mutant T PTEN 3′-UTR (Mut) co-transfected with miR-221 mimics or a negative control (miR-NC) was measured. c RT-qPCR analysis of PTEN mRNA in A431 and SCC13 cells following transfection with miR-221 inhibitor or mimics. d Western blotting was used to detect PTEN protein expression in A431 and SCC13 cells following transfection with miR-221 inhibitor or mimics. e Relative PTEN mRNA expression levels were determined using RT-qPCR in CSCC tissues and adjacent non-tumorous gastric mucosae tissues. f Analysis of correlation between miR-221 and PTEN mRNA expression in CSCC tissues

Fig. 5

Impact of miR-221 in Akt pathway. a, b Western blot analysis of pAkt, cyclin D, Bcl-2, and MMP2/9 of cells following transfection with miR-221 inhibitor or mimics. β-actin was used as a negative control