Cancerous Inhibitor Of Protein Phosphatase 2a Expression On Proliferation Of Laryngeal Squamous Cell Carcinomas Cells HEP-2 And Its Mechanism

Objective:Laryngeal squamous cell carcinoma (LSCC) derived from mucosa epithelium tissue is one of the most common malignancies of human head and neck cancers and has an obviously growing trend in recent years. The major cause of low 5-year survival rate and poor prognosis in LSCC patients is relapse and metastasis of tumor cells. Surgical resection treatment seriously affects voice function and quality of life in the postoperative patients and tumor drug resistance limtes the treatment of laryngeal cancer, highlighting the urgent need for identifation of effective drug targets for LSCC therapy. CIP2A is a human oncoprotein that prevents c-myc protein degradation by inhibiting PP2A-mediated dephosphorylation of c-myc at serine 62, promotes anchorage-independent cell growth and malignant transformation. CIP2A overexpression has been found in a variety of human malignancies, including those of lung cancer, ovarian, osteosarcoma, pancreatic ductal adenocarcinomas, colon, and so on. In our previous study, we found that CIP2A highly expressed higher in the LSCC tissues than in the nomal laryngeal tissues, and was significantly associated with clinical tumor staging, lymph nodes metastases, histological differentiation and prognosis. Furthermore, currently the biology effect of CIP2A and its mechanism in LSCC is unclear. In this work, the expression of CIP2A in LSCC was inhibited by RNA interference. This article is just to investigate the effect of CIP2A depletion on cell proliferation, apoptosis and the sensitivity to cisplatin and its mechanism in laryngeal squamous cell carcinomas cells Hep-2, in order to provide the experimental and theoretical basis for the early molecule diagnosis and clinical therapy of LSCC.Methods:siRNA-mediated CIP2A and negative control sequences were performed in Hep-2 cells. The experiment was designed into CIP2A siRNA group and Control siRNA group. Colony formation assay was used to examine the role of CIP2A on cell proliferation. The impact of CIP2A on cell growth and cisplatin sensitivity was evaluated using MTT assay. Flow cytometry was used to examine cell cycle distribution and apoptotic rate. Quantitative real-time PCR and Western blot were applied to assess the expression of CIP2A, c-myc, AKT, p-AKT, and cyclinDl.Results:CIP2A knockdown in Hep-2 cells markedly decreased both CIP2A mRNA and protein expression, reduced the proliferation speed and colony formation(Control group was 339±34,CIP2A siRNA group was 126±33, P=0.010) and increased the drug sensitivity of the cells to cisplatin (Control group was 6.67±0.54ug/ml,CIP2A siRNA group was 3.53 ±0.32μg/ml,P=0.001). In addition, CIP2A depletion blocked cell cycle at Gl phase (Control group was 66.860±1.972,CIP2A siRNA group was 74.613±3.357,P=0.026) and decreased the percentage of S stage cells (Control group was 23.713±1.564,CIP2A siRNA group was 17.747±1.255,P=0.007), and did not significantly alter cell apoptosis rate(P=0.216). Western blot showed that CIP2A depletion downregulated CyclinD1, c-myc, p-AKT expression (P=0.011,P=0.011,P=0.010), but had no effects on the expression of total AKT protein (P=0.962).Conclusion:CIP2A depletion inhibited cell proliferation and increased the sensitivity to cisplatin, mechanism of which may be related with decreased c-myc, cyclinDl, and p-AKT.CIP2A may be a potential maker in the diagnosis and gene treatment of laryngeal squamous cell carcinoma.