PRR11 and SKA2 promote the proliferation, migration and invasion of esophageal carcinoma cells

Proline-rich protein 11 (PRR11) together with its upstream adjacent gene, spindle and kinetochore associated 2 (SKA2), represent a classic, head-to-head gene pair. The role of the PRR11 and SKA2 gene pair has been described in various types of cancer, including breast cancer, non-small cell lung cancer, hepatocellular carcinoma and ovarian carcinoma. However, its role in esophageal carcinoma (ESCC) remains unclear. The mRNA expression levels of PRR11 and SKA2 were examined in ESCC surgical specimens. In addition, the role of PRR11 and SKA2 in the proliferation and migratory and invasive capacities of EC9706 and EC109 cell lines was examined. The results from the present study demonstrated that PRR11 and SKA2 expression levels were upregulated in ESCC tissues compared with adjacent normal tissues. Furthermore, PRRl1 and SKA2 knockdown significantly inhibited the proliferation and migratory and invasive capacities of ESCC cells. Conversely, PRRl1 and SKA2 overexpression significantly promoted the proliferation and migratory and invasive capacities of ESCC cell lines via activation of the AKT signaling pathway and certain markers of epithelial-mesenchymal transition, including Snail and N-cadherin. The results from the present study suggested that the PRR11 and SKA2 gene pair may represent a potential target in the diagnosis and treatment of ESCC.


Introduction
Esophageal squamous cell carcinoma (ESCC), which is one of the most common types of cancer, was the sixth leading cause of cancer-associated mortality in China in 2016 (1). In 2012, an estimated 455,800 new esophageal cancer cases and 400,200 deaths were reported (2). Although surgery and radiation therapy are effective therapeutic strategies for certain tumors diagnosed at an early stage, a number of patients eventually progress to advanced stages of cancer (3). Based on previous epidemiologic studies, it is widely accepted that genetics serve a crucial role in the development and progression of ESCC (4,5); however, the underlying mechanisms remain unclear. It is therefore important to fully understand the development and progression of ESCC, in order to identify novel therapeutic targets.
The proline-rich protein 11 (PRR11) gene is located on chromosome 17q22 and contains a bivalent nuclear localization signal, two proline-rich regions and a zinc finger domains (6). In recent years, PRR11 has been reported to serve as a candidate oncogene in various types of cancer, including pancreatic cancer, breast cancer, non-small cell lung cancer, hepatocellular carcinoma and ovarian carcinoma (7)(8)(9). PRR11 and its upstream adjacent gene, spindle and kinetochore associated 2 (SKA2), are a classic head-to-head gene pair, driven by a prototypical and bidirectional promoter (10). SKA2, which is involved in the formation of the Ska complex, is involved in the maintenance of the mitotic mid-plateau and shut-down of the spindle checkpoint (11)(12)(13)(14). Currently, studies on PRR11 and SKA2 mainly focus on lung and breast cancer (7,8,10); however, the role of PRR11 and SKA2 in ESCC remains unclear.
The present study evaluated the importance of PRR11 and SKA2 in the progression of ESCC, and demonstrated that the expression level of these genes was highly expressed in human ESCC tissues. Furthermore, the present study revealed that PRR11 and SKA2 serve important roles in the proliferation and migratory and invasive capacities of ESCC cells in vitro. In addition, the underlying mechanism of PRR11 and SKA2 in the progression of ESCC was investigated.     Crystal violet assay. Control and transfected cells were seeded into 6-well plates at a density of 1,000 cells/well. Cells were cultured in DMEM with 10% FBS and medium was changed every three days. After two weeks of culture, the culture medium was removed and cells were stained with crystal violet (1 ml 0.5% crystal violet solution in 20% methanol) for 10 min at room temperature. Cells were washed with PBS and images were captured using a digital camera. The optical density (OD) value was measured at 600 nm with a microplate reader.  Fig. 1A). Similarly, SKA2 expression level was significantly increased in tumor tissues compared with normal tissues (P<0.05; Fig. 1B). Furthermore, the mRNA and protein levels of PRR11 and SKA2 in the two ESCC cell lines EC109 and EC9706 were assessed. The results demonstrated that the mRNA level of both PRR11 and SKA2 were significantly higher in EC9706 cells compared with EC109 cells. The protein expression of both PRR11 and SKA2 was also higher in EC9706 cells ( Fig. 1C and D).

PRR11 and/or SKA2 knockdown inhibits the proliferation, migration, and invasion of ESCC.
To identify the function of the PRR11 and SKA2 gene pair in ESCC cells, specific shRNA targeting PRR11 and/or SKA2 were used in EC9706 cells. The efficiency of these knockdowns was confirmed by western blotting (Fig. 2A). Furthermore, the results from MTT and crystal assays demonstrated that transfection with shPRR11 or SKA2 significantly reduced EC9706 cell proliferation compared with untransfected cells. In addition, double transfection induced a more significant decrease in cell proliferation compared with single transfections (P<0.01  (15), Tumor-Node-Metastasis. and P<0.001; Fig. 2B and C). In addition, as presented in Fig. 2D, the results from Boyden chamber assay demonstrated that the migratory ability of EC9706 cells was significantly decreased following PRR11 or SKA2 knockdown compared with untransfected cells (P<0.01 and P<0.001, respectively; Fig. 2D). Cell migratory capacity was also significantly more decreased following double knockdown of PRR11 and SKA2 compared with single transfections (P<0.001; Fig. 2C). Furthermore, the results from transwell assay indicated that the invasive capacity of EC9706 cells was significantly reduced following PRR11 and/or SKA2 knockdown (P<0.01 and P<0.001; Fig. 2E).
PRR11 and/or SKA2 overexpression promotes the proliferation, migration, and invasion of ESCC. PRR11 and/or SKA2 were overexpressed in EC109 cells by transfection. The successful overexpression of PRR11 and/or SKA2 was confirmed by western blotting (Fig. 3A). The results from MTT and crystal violet assays demonstrated a significantly increased cell proliferation following PRR11 or SKA2 overexpression compared with untransfected cells. In addition, the double transfection induced a more significant increase in cell proliferation compared with single transfections (P<0.01 and P<0.001; Fig. 3B and C). Furthermore, as presented in Fig. 3D, the results from Boyden chamber assay demonstrated that the migratory ability of EC109 cells was significantly increased following PRR11 or SKA2 overexpression compared with untransfected cells (P<0.01; Fig. 3D). Cell migratory capacity was more significantly increased following overexpression of PRR11 and SKA2 compared with single transfections (P<0.001; Fig. 3D). In addition, the results from transwell assay reported that overexpression of the gene pair significantly promoted the invasive ability of EC109 cells (P<0.01 and P<0.001; Fig. 3E).

PRR11 and SKA2 gene pair activates AKT and epithelialmesenchymal transition (EMT) signaling pathways.
In order to explore the underlying mechanism of the gene pair PRR11 and SKA2 on ESCC progression, the protein expression of p-AKT, total AKT, Snial and N-cadherin of EMT, and Cyclin D1 of cell cycle signal in EC9706 and EC109 cells by western blotting. The results demonstrated that expression levels of p-AKT, Snail and N-cadherin were reduced following PRR11 and SKA2 knockdown in EC9706 cells, and double transfection induced a more notable decrease in the expressions of these proteins compared with single transfections (Fig. 4A). Opposite effects were observed in EC109 cells overexpressing PRR11 and SKA2 (Fig. 4B). In addition, PRR11 and SKA2 overexpression or knockdown had no effect on Cyclin D1 expression. Furthermore, PRR11 and  SKA2 overexpression increased the expression of the proliferation marker PCNA, whereas their knockdown decreased PCNA expression (Fig. 4A and B).

Discussion
Over the past few decades, the prognosis of ESCC has been relatively poor in China, with the 5-year overall survival rate <20% and most patients died within 1 year of diagnosis (2). In addition, this disease lacks sensitive and specific early diagnosis method. Surgical and radiation therapies are the main treatments for ESCC; however, numerous patients eventually develop advanced stages of ESCC (4). The present study aimed therefore to identify potential intervention targets for ESCC. Recent studies reported that the gene pair of PRR11 and SKA2 is involved in tumorigenesis and cancer progression. Zhu et al (9) reported that PRR11 overexpression facilitates ovarian carcinoma cell proliferation, migration, and invasion through activation of the PI3K/AKT/β-Catenin pathway. Furthermore, it was reported that the gene pair PRR11 and SKA2 is negatively regulated by p53 through nuclear factor Y in lung cancer cells (10). Also, PRR11 silencing leads to cell cycle arrest, suppresses colony formation, decreases cell proliferation and inhibits tumorigenic potential of lung cancer cells (19). In addition, the PRR11 and SKA2 gene pair has been hypothesized as a potential new target for the diagnosis and treatment of lung cancer (20). It has reported that overexpression of PRR11 could promote breast cancer progression by activating EMT (7). Qiao et al (6) demonstrated that proline-rich protein 11 silencing inhibits hepatocellular carcinoma growth and epithelial-mesenchymal transition through β-catenin signaling.
The present study demonstrated that PRR11 and SKA2 mRNA levels were significantly increased in ESCC tissues compared with adjacent normal tissues. Furthermore, cell proliferation, migratory and invasive capacities were significantly increased following PRR11 and SKA2 overexpression. In addition, PRR11 and SKA2 knockdown inhibited the proliferation, invasive and migratory capacities of ESCC cells. Subsequently, in order to investigate the underlying mechanism, this study demonstrated that PRR11 and SKA2 overexpression significantly activated the AKT signaling pathway and certain markers, including Snail and N-cadherin of EMT. AKT signaling pathway activation is implicated in the development of a numerous human cancers, including ESCC (21)(22)(23). Furthermore, EMT represents a critical event in the transition from early to invasive carcinomas, and it was demonstrated that N-cadherin upregulation is associated with poor prognosis and lower survival in patients with cancer (24)(25)(26). These findings are consistent with the results from the present study. To the best of our knowledge, this study was the first to demonstrate the involvement of PRRl1 and SKA2 in the progression of ESCC.
In summary, this study demonstrated that the gene pair PRRl1 and SKA2 may serve a crucial role in the proliferation, migratory and invasive abilities of ESCC cells. These results provided a better understanding of the underlying mechanisms of PRRl1 and SKA2 in ESCC tumor development, and PRRl1 and SKA2 may be considered as potential targets for the diagnosis and/or treatment of ESCC.