The Overexpression of NMHC IIA Promoted Invasion and Metastasis of Nasopharyngeal Carcinoma Cells

Abstract

Background: Nasopharyngeal carcinoma (NPC) is a kind of head and neck squamous cell carcinoma (HNSCC) with a strong tendency for metastasis and recurrence. Non-muscle myosin heavy chain IIA (NMHC IIA) plays important roles in recurrence and metastasis of cancers. However, the function and mechanism of NMHC IIA expression in NPC remain unclear. Methods: A receiver operating characteristic (ROC) curve was constructed for 141 specimens of HNSCC tissues and 44 control samples from The Cancer Genome Atlas (TCGA) database. Co-expressed genes with MYH9 were identified using LinkedOmics. Transcription factors (TFs) and miRNA regulation network were constructed using Networkanalyst. The migration and invasion ability of nasopharyngeal carcinoma cells were evaluated by in vitro migration and matrigel invasion assays, respectively. Results: The public microarray results showed that MYH9 expression levels were upregulated in HNSCC tissues compared with the matched adjacent normal tissues in this study (p<0.0001). The AUC of MYH9 reached up to 0.8303 at a cutoff value of 175.2, with a sensitivity and specificity of 70.21% and 86.36%, respectively. MYH9 expression was increased in lymph node metastasis HNSCC tumors compared with that in tumors without lymph node metastasis (p<0.05) and showed a strong positive association with expression of FLNA. High MYH9 and FLNA expression were related with poorer overall survival in HNSCC. MYH9 with positively associated genes regulated focal adhesion, cell-substrate junction assembly and cell morphogenesis were involved in differentiation using GO and KEGG analysis. MYH9 was correlated with a network of TFs including SP1, SRF, JUN and FOS in HNSCC. The suppression of endogenous NMHC IIA decreased cellular migration and invasion in HNE1 cells and reduced the expression of phosphorylation of EGFR, AKT and ERK. The over-expression of NMHC IIA increased cellular migration and invasion in COS-7 cells and increased the expression of phosphorylation of EGFR, AKT and ERK. Conclusion: Expression of NMHC IIA mRNA was higher in HNSCC than in the adjacent normal tissues. NMHC IIA expression was increased in lymph node metastasis HNSCC tumors compared with tumors without lymph node metastasis. High MYH9 was association with poorer overall survival in HNSCC. NMHC IIA expression increased the invasion and metastasis abilities of the nasopharyngeal cancer cell line in vitro by augmenting the expression of phosphorylation of EGFR, AKT and ERK. These findings will be beneficial for providing an effectively therapeutic strategy for NPC.

Keywords: NMHC IIA; head and neck squamous cell carcinoma; invasion; metastasis; nasopharyngeal cancer.

Figure 1

NMHC IIA mRNA expression levels in HNSCC cancer tissues and adjacent normal tissues. A. Enhanced expression of NMHC IIA mRNA expression levels in HNSCC cancer tissues compared with adjacent normal tissues (normal=44, cancer=141, p<0.01). B. NMHC IIA mRNA expression levels in HNSCC cancer of different pathological stages (T=141, p<0.01). C. The ROC curve for differentiating HNSCC tissues from controls. D. NMHC IIA had increased expression in lymph-node metastasis HNSCC compared with lymph-node non-metastasis tumors.

Figure 2

Genes differentially expressed in correlation with MYH9 in HNSCC. A. A Pearson test was used to analyze correlations between MYH9 and genes expressed in HNSCC. B-C. Heat maps showing genes positively and negatively correlated with MYH9 (TOP 50). Red indicates positively correlated genes and green indicates negatively correlated genes. D-E. The significantly enriched GO annotations and KEGG pathways of MYH9 of top 50 significant gene sets (D) positively and (E) negatively correlated with MYH9. F-G. Network module obtained from the (F) positively and (G) negatively genes protein-protein interaction network.

Figure 3

The network and survival analysis of FLNA and MYH9. A. Transcription factors and miRNA of FLNA and MYH9. B-C. Kaplan-Meier analysis of overall survival in (B) MYH9 and (C) FLNA.

Figure 4

Suppression of endogenous NMHC IIA reduced cellular migration and invasion in HNE1. A-C. NMHC IIA expression was confirmed by Quantitative real-time PCR and western-blot in HNE1 cells expressing scrambled shRNA or NMHC IIA shRNA. D-E. The analysis of the migration and invasive properties in HNE1 cells expressing scrambled shRNA or NMHC IIA shRNA. The migratory or invasive cells were stained by crystal violet and then photographed by fluorescence inversion microscope system. Original magnification ×200. F. Migratory cells were plotted as the average number of cells per field of view from ten random fields. G. Invasive cells were plotted as the average number of cells per field of view from ten random fields.

Figure 5

The exogenous expression of NMHC IIA enhanced the migration and invasiveness in COS7 cells. A. Quantitative real time PCR analysis of MYH9 gene in COS7 cells expressing control vector PLNCX2 or PLNCX2/NMHC IIA. The relative fold increase of transcripts was normalized to the amount of RNA harvested from cells expressing control vector PLNCX2. GAPDH served as the internal control. The data were presented as the mean ± SD (n = 3). B. Western blot analysis of NMHC IIA protein in COS7 cells expressing control vector PLNCX2 or PLNCX2/NMHC IIA. β-actin was used as a loading control. C. The migratory and invasive abilities induced by NMHC IIA were analyzed in COS7 cells expressing control vector PLNCX2 or PLNCX2/NMHC IIA. The migratory or invasive cells were stained by crystal violet and then photographed by fluorescence inversion microscope system (200×). E. Migratory cells or invasive cells were plotted as the average number of cells per field of view from ten random fields.

Figure 6

NMHC IIA have effect on the expression of phosphorylation of EGFR, AKT and ERK. A. Silencing endogenous NMHC IIA in HNE1 cells decreased expression of phosphorylation of EGFR, AKT and ERK. B. Overexpression of NMHC IIA in COS7 cells enhances the expression and phosphorylation of EGFR, AKT and ERK.