Expression Of Aurora-A In Human Esophageal Squamous Cell Carcinoma And Effect On The Cell Lines

Background and objectiveEsophageal cancer is one of the most common cancers and the major causes of cancer death in the world, especially in China. Its five years survival rate is less than 10%. At present, esophageal cancer has been on the 4th leading cause of cancer death in urbanite, and 3th in rural area of China. There are 2 main histopathological types; one is squamous cell carcinoma (SCC), the other is adenocarcinoma (ADC). In China, SCC occurred far more frequently than ADC. Therefore, it is important to investigate some mechanisms of esophageal squamous cell carcinoma (ESCC). With the molecular biotechnology development, people learn more about tumor. It was regarded as a kind of genic disease. So it is important to do some research on ESCC-related genes for the mechanisms of ESCC tumorigenesis, invasive and metastasis. It is thought that some novel findings will be shown up for the molecular pathological diagnosis, evaluation of prognosis and selection of therapy methods.Aurora-A protein consists of 403 amino acids and it is a kind of centrosome related serine/threonine kinase. Aurora-A has been first identified from breast cancer. It locates within the 20ql.3 chromosome region that is frequently amplified in various epithelial malignant tumors. Aurora-A plays important roles in the completion of essential mitotic events such as centrosome separation, spindle assembly and spindle maintenance. Aurora-A interactes with multiple important cellular proteins such as p53, BRCA1 and plays an essential role in the control of cell proliferation.resulting in errors in cell division. Aurora-A over-expression disrupts the G2 checkpoint and the spindle checkpoint, as well as the Gl checkpoint, and produced the chromosome instability and aneuploidy. Rodent fibroblast cells transfected with Aurora-A form tumors in nude mice, indicating that Aurora-A is an oncogene. Elevated levels of Aurora-A mRNA and protein expression have been observed in many tumors, including breast, colon, bladder, ovarian and pancreatic cancers. Recently, several inhibitors of Aurora kinases have been developed. Among them, VX-680 has shown promising in animal studies. However, the function of this enzyme during tumorigenesis is still unclear. How did it express in the ESCC, one of the most common cancers in our country? What's its role in the ESCC development? To find some clues to these questions, we carried out this project. In this study, we focused on the expression of Aurora-A protein in ESCC samples and the normal adjacent tissues firstly. Then transfected this gene into the ESCC cell showed low level of Aurora-A expression and investigated the phenotypic alterations of these cells. Furthermore, in the ESCC cells which showed high level of Aurora-A, this gene was knocked-out by small interfering RNA transfection and investigated the mobility alteration. Materials and Methods1. Twenty ESCC samples and the normal adjacent tissues were collected and Aurora-A expression was examined by Western Blot analysis. Sixty-four ESCC specimens and sixty-one normal adjacent tissue samples were collected, Aurora-A protein expression and location was analyzed with immunohistochemical approach. The histopathological classification of these sixty-four ESCC tissues was well differentiation in fourteen samples, moderate differentiation in forty-one sample and poor differentiation in nine samples. Among these sixty-four ESCC tissues, there are forty-one samples of carcinoma in situ and twenty-three samples of invasive carcinoma.2. Twelve ESCC cell lines were cultured and made the protein for Western Blot analysis of Aurora expression. The KYSE30 cell that had low level of Aurora-A expression was selected for transfecting into the plasmid containing Aurora-A cDNA,and developed the Aurora-A over-expressing cell lines. Then investigated the phenotype alterations, such as cell proliferation, cell adhesion, mobility.3. Mobility of the twelve ESCC cell lines were examined by transwell assay and analyzed their relationship with Aurora-A expression. Moreover, in the EC9706 and Colo-680 cells that had high level of Aurora-A, this gene was knocked-out by small interfering RNA transfection, and mobility alterations of these cells were investigated by transwell assay. Results1. Protein of twenty tumor samples and their normal adjacent tissues were collected and assayed for Aurora-A expression by Western Blot analysis. Aurora-A over-expression was detected in twelve tumor samples. Sixty-four ESCC tissues and sixty-one normal adjacent tissues were analyzed for Aurora-A expression and location by immunohistochemical approach. Among sixty-four human ESCC samples, strong cytoplasmic staining of Aurora-A protein was detected in forty-five tumors. In contrast, only one sample exhibited strong staining for Aurora-A protein among normal adjacent tissues, P<0.05.2. There are twenty-eight samples exhibited strong staining in forty-one moderate differentiation tumors (28/41), nine exhibited strong staining in nine poor differentiation tumors (9/9), and nine in the fourteen well differentiation tumors (9/14) among the sixty-four ESCC samples. Tumors with moderate or poor differentiation exhibited higher expression levels of Aurora-A protein compared with tumors with well differentiation, P<0.05, P<0.01 respectively. According to infiltration pattern, there are forty-one carcinoma in situ tissues in which twenty-two exhibited strong staining (22/41), twenty-three invasive tissues in which twenty samples exhibited strong staining (20/23).3. The plasmid, which contains Aurora-A cDNA, was transfected into KYSE30 cell for developing Aurora-A overexpressing cells: KYSE30-16 and KYSE30-35. It was fund that the Aurora-A overexpressing cells showed faster proliferation; somewhat easily attached to the ECM. The adhesion rate of KYSE30 and KYSE30-16was 20.62±9.12E-2%, 39.09+12.5% after cultured 30min, p<0.05, and 33.23 + 11.31%, 56.84+15.47% after cultured 2h, P<0.05.4. Mobility of 12 ESCC cells were correlated with their Aurora-A levels, p< 0.05; Aurora-A over-expressing cells KYSE30-16 and KYSE30-35 exhibited stronger motility than KYSE30 cell. Cells of passing through the 8um polycarbouate membrane of KYSE30-16 and KYSE30-35 in the transwell assay was more than six times than KYSE30; EC9706 and Colo-680 cells were treated with Aurora-A siRNA for 48h or 72h, more than 60% of endogenous Aurora-A was suppressed, at the same time the mobility reduced about 50%. Conclusion1. Over-expression of Aurora-A is detected in ESCC tissues and correlates with the differentiation and invasion of ESCC. It may be considered as a candidate molecular parameter indicating the tumor invasion and prognosis of ESCC.2. Aurora-A may contribute to invasive growth and metastasis of human ESCC through an unknown molecular pathway that promoting cell proliferating, adhesion to ECM and as well as mobility.