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, 10 (8), 8324-8333
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CCNA2 Facilitates Epithelial-To-Mesenchymal Transition via the Integrin αvβ3 Signaling in NSCLC

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CCNA2 Facilitates Epithelial-To-Mesenchymal Transition via the Integrin αvβ3 Signaling in NSCLC

Jun Shan Ruan et al. Int J Clin Exp Pathol.

Abstract

Non-small cell lung carcinoma (NSCLC) is the most common malignancy with the highest morbidity and mortality. Studies have demonstrated that the abnormal expression of cyclin-A2 (CCNA2) is associated with multiple malignancies, yet its functional role in NSCLC metastasis remains to be elucidated. In the present study, we investigated the role of CCNA2 in regulating migration and invasion of NSCLC cells by establishing NSCLC cell strains with constitutively silenced or elevated CCNA2 expression. We demonstrated that ectopic expression of CCNA2 accelerates NSCLC cells migration and invasion in vitro through cell wound scratching and Transwell invasion assays. Conversely, further analysis indicated that suppression of CCNA2 expression via siRNA inhibits metastasis of NSCLC cells. In addition, we studied the correlation between CCNA2 expression and overall survival using the Kaplan-Meier Plotter database in NSCLC cancers. There was correlation between CCNA2 expression levels and patient survival. Finally, our findings demonstrate that CCNA2 promotes invasion and migration of NSCLC cells through integrin αVβ3 signaling pathway. Collectively, this study provides novel insights into that CCNA2 represents a crucial regulator of NSCLC cells metastasis and suggests targeted treatment of CCNA2-expressing cancer serves as a new therapeutic target for NSCLC.

Keywords: CCNA2; EMT; NSCLC; integrin.

Conflict of interest statement

None.

Figures

Figure 1
Figure 1
CCNA2 is upregulated in human NSCLC specimens. A. NSCLC expression in normal lung tissue and NSCLC specimens. Images were taken from the Human Protein Atlas (http://www.proteinatlas.org) online database. B. Oncomine data showing CCNA2 expression in normal vs. tumor of NSCLC. CCNA2 mRNA expression in the Hou lung and Su lung data set. C. Kaplan-Meier survival curves for the NSCLC patients. The overall survival times in the low (green, n=88) and high CCNA2 (red, n=87) groups, with a hazard ratio of 1.88 (95% confidence interval (CI) 1.2-2.94) and P=0.005. D. qPCR analysis of CCNA2 mRNA levels in various NSCLC cell lines relative to the normal bronchial epithelial cells BEAS-2B. PCR values were normalized to the levels of β-actin. Data were presented as the mean ± SD from three independent measurements. E. Western blot analysis of CCNA2 expression in different NSCLC cell lines. β-actin was used as loading controls.
Figure 2
Figure 2
CCNA2 promotes H1975 cells migration and invasion in vitro. A. Wound healing assay. Confluent cell monolayers were wounded, and wound closure was monitored at 0 hour and 24 hour. Quantification of wound closure was calculated. B. Invasion assay. H1975 control or cells transfected with CCNA2 plasmid were subjected to a Transwell invasion assay. The invasive cells were stained with 1% crystal violet and counted. Data were collected from five fields in three independent experiments. Quantification of invasive cells per field was analyzed. For indicated comparisons, **P<0.01. C. In vitro wound healing assay with human H1975 cells after knock out with CCNA2 expression. Image was acquired at 0, 24 h time points after scratching (left panel). Quantification of wound closure was calculated (right panel). D. Representative staining of invasive potentials of human H1975 cells from in vitro Transwell assay (left panel). Quantification of invasive cells per field was analyzed (right panel). Statistical analyses were performed by the Student’s t test. The following symbols were used to indicate significant differences: **P<0.01.
Figure 3
Figure 3
CCNA2 regulates EMT in NSCLC cells. A. Morphological changes by CCNA2 in H1975 and H1975 cells. B. Western blot (left panel) and qRT-PCR (right panel) shown down-regulated expression of E-cadherin and upregulated expression of N-cadherin in H1975-CCNA2 cells. C. In contrast, silencing of CCNA2 resulted in increased expression of E-cadherin and decreased expression of N-cadherin in H1975-CCNA2si cells. For indicated comparisons, **P<0.01.
Figure 4
Figure 4
CCNA2 expression regulates integrin αvβ3/MMPs signaling in NSCLC cells. A. CCNA2 over-expression enhances integrin αvβ3 expression, whereas knockdown of CCNA2 inhibits integrin αvβ3 expression. B. Indicated H1975 cells were immunostained with rabbit anti-CCNA2 antibody (green) and DAPI (blue) for observation by laser confocal microscopy. C. The expression of MMP-2 and MMP-9 in H1975 NSCLC cells transfected with the vector expressing CCNA2 plasmid or CCNA2-siRNA was evaluated by immunoblotting.
Figure 5
Figure 5
Inhibitor of integrin αvβ3 reduces CCNA2-induced EMT, cell migration and invasion. A. H1975-CCNA2 cells were treated or not with 20 μM cilengitide during 24 h after which proteins were analyzed by western blot with specific antibodies against E-cadherin and N-cadherin. B. Analysis of migration potential from cilengitide and vehicle treated H1975-CCNA2 cells by a wound healing assay (left) and the quantification of wound closure (right). Bars show means ± SD of three independent experiments. Statistical analyses were performed by using the Student’s t test. C. Analysis of invasion potential from cilengitide and vehicle treated H1975-CCNA2 cells by a wound healing assay (left) and the quantification of wound closure (right). Bars show means ± SD of three independent experiments. Statistical analyses were performed by using the Student’s t test. For indicated comparisons, **P<0.01.

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