| Oral squamous cell carcinoma (OSCC) is the most common malignant tumor of oral cancer in the world. Radical surgery is now the standard treatment of patients with OSCC. However, most of these patients are diagnosed in advanced stages with poor prognosis due to OSCC prone to recurrence and metastasis. OSCC metastasis is closely related to their rapid proliferation ability. Thus, the key therapeutic strategy of OSCC is to effectively inhibit malignant cell proliferation.Abnormality of chromosome segregation is important reasons for genetic instability in turn leading to tumorigenesis and progression. Chromosome alignment and segregation require that all kinetochores (KTs) establish stable bioriented attachments to spindle microtubules (MTs). Central to this process is the spindle and kinetochore-associated protein (SKA) complex (SKA1,2and3). SKA2and SKA3have been identified to be associated with tumorigenesis and progression. But the role of SKA1in oncogenesis is largely unexploited. The present study was to investigate whether SKA1is a key molecule to regulate oral squamous cell carcinoma malignant proliferation.Firstly, we collected and analyzed the SKA1gene expression in36cases oral squamous cell carcinoma and31cases of non-tumor tissue or adjacent tissues by immunohistochemistry. Secondly, lentivirus-mediated short hairpin RNA (shRNA) was employed to science SKA1gene expression to study the molecular mechanism of SKA1in OSCC. RT-PCR and western-blot were used to detect the silence efficiency of SKA1mRNA and protein levels. The effect of LV-shSKAl on cell growth, colony formation and cell cycle regulation were investigated by MTT, colony formation assay and flow cytometry respectively. Finally, to further study the molecular mechanisms of SAK1in oral squamous cell malignant proliferation, microarray analysis was performed using Agilent Gene Chip to analyze the differentially expressed genes in CAL-27cells before and after SKA1gene silence. We selected the part of the differentially expressed gene (fold change≧2) to detect its expression by real-time fluorescence quantitative polymerase chain reaction (Real-time PCR) for validation.The results show that SKA1expression was frequently observed in tumor tissues, most in cytoplasm and membrane, and little in nucleus. The positive rate of SKA1was88.9%(32/36), and the high-positive rate was25%(9/36) in tumors tissue. While the positive rate of SKA1protein was0%(0/31), weakly positive rate was9.6%(3/31) in31cases of non-tumor tissue or adjacent tissues, the results indicated that SKAlwas highly expressed in oral squamous cell. LV-shSKAl can efficiently infect CAL-27cells, and effectively silence SKA1mRNA and protein expression. MTT, colony formation assay and flow cytometry showed that silencing the target gene SKA1, oral squamous cell proliferation, clonal growth capacity were significantly reduced, S phase cells were significantly reduced, G0/G1and G2/M phase cell cycle were arrested. The present study suggest that LV-shSKAl can effectively silence SKA1gene expression in oral squamous cell, significantly inhibited cell proliferation and affect cell cycle distribution. Microarray results showed different expression genes (fold change≧2) include NDC80, CDKN1B and GPR110, etc. Real-time PCR confirmed these three genes expression were consistent with microaaray results.In summary, SKA1protein expression was increased in oral squamous cell carcinoma. SKA1silence resulted in a significant reduction in OSCC cell proliferation and colony formation ability along with cell cycle arresting. Microarray analysis suggested that SKA1might inhibit cell growth via CDK inhibitor, CDKN1B. SKA1plays an essential role in the development of OSCC, and may serve as a promising molecular target in OSCC therapy. |
PDF Full Text Request