Expression of S-phase kinase associated protein 2 in laryngeal carcinoma and its molecular mechanisms involved in regulation of proliferation and apoptosis of Hep-2 cells / 中华耳鼻咽喉头颈外科杂志

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the expression of S-phase kinase associated protein 2 (Skp2) in human laryngeal squamous cell carcinomas and to explore the effect of silencing of Skp2 by RNAi on the proliferation and apoptosis and the expressing of p27 protein as well as change of cell cycle in laryngeal carcinoma cell line Hep-2 cell.</p><p><b>METHODS</b>The expression of Skp2 in laryngeal carcinoma tissues of different differentiation grade was detected by immunohistochemistry. According to the encoding sequence of mRNA of Skp2, two pieces of oligonucleotide sequences were designed and synthesized. The annealed oligonucleotide fragments were subcloned into pGPU6/Neo siRNA expression vector. The recombinant plasmids pGPU6Skp2 were transfected into Hep-2 cells induced by lipofectamine(TM) 2000. The expression level of Skp2 and p27 were examined by flow cytometry. The cell proliferation was examined by MTT assay. Flow cytometry was performed to analyze apoptosis and cell cycle.</p><p><b>RESULTS</b>Positive expression of Skp2 was detected in all 52 cases of laryngeal carcinoma tissues. The positive rate of expression of Skp2 in well-differentiated laryngeal carcinoma group was lower than that in middle-poorly differentiated group (chi(2) = 7.33, P < 0.05). DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pGPU6 vector, and Skp2 protein expression in the transfected cells were inhibited significantly. The fluorescence index of Skp2 protein expression in pGPU6-Skp2 group was significantly inhibited compared with that in blank pGPU6 group (t = 19.42, P < 0.01). The inhibition ratio of cell proliferation in pGPU6-Skp2 group was 26.93% which was strikingly higher than that of blank pGPU6 group 2.47% (t = 15.23, P < 0.01). The apoptosis ratio in pGPU6-Skp2 group was 11.71% which was increased significantly compared with that of their blank pGPU6 group 1.93% (t = 17.92, P < 0.01). In cell cycle study the percentage of S phase cells in pGPU6-Skp2 group was significantly higher than that in blank pGPU6 group (t = 7.73, P < 0.05). The fluorescence index of p27 protein level was significantly higher than that in blank pGPU6 group (t = 2.86, P < 0.05).</p><p><b>CONCLUSIONS</b>The high level expression of Skp2 in laryngeal carcinoma is significantly related to the differentiation of laryngeal carcinoma. Silencing of Skp2 expression could inhibit cell proliferation and increase cell apoptosis in Hep-2 cells which might be related to the were of p27 protein level and S phase cell cycle arrest of Hep-2 cell.</p>