S期激酶相关蛋白2在喉癌组织中的表达及调控增殖和凋亡的机制

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

目的检测S期激酶相关蛋白2（S-phase kinase associated protein 2，Skp2）在不同分化程度喉癌组织标本中的表达，并探讨应用RNA干扰技术抑制skp2基因表达对喉癌Hep2细胞生长、凋亡、p27表达以及细胞周期的影响。方法应用免疫组织化学方法检测Skp2蛋白在不同分化程度喉癌组织标本中的表达，根据设计siRNA的原则，针对人Skp2的mRNA序列，设计并合成编码siRNA的两条寡核苷酸序列，构建重组质粒pGPU6Skp2，转染重组质粒至Hep-2细胞中，应用流式细胞术检测skp2蛋白的表达。采用四甲基偶氮唑蓝法（MrIT）检测细胞增殖变化。采用流式细胞术检测细胞凋亡、细胞周期和p27蛋白表达的变化。结果52例喉癌组织标本中skp2蛋白均呈阳性表达，高分化喉癌组织标本Skp2蛋白表达低于中、低分化组（X2=7．33，P〈0．05）。经酶切和DNA测序鉴定证实重组质粒上连接序列确实为所设计序列。pGPU6-Skp2转染组skp2蛋白表达明显低于空质粒对照组（f=19．42，P〈0．01）；pGPU6-Skp2转染组细胞增殖抑制率为26．93％，明显高于空质粒对照组2．47％（t=15．23，P〈0．01）；pGPU6-Skp2转染组细胞凋亡率为11．71％，明显高于空质粒对照组1．93％（t=17．92，P〈0．01）；pGPU6-Skp2转染组s期细胞所占比例明显高于空质粒对照组（t=7．73，P〈0．05）；pGPU6-Skp2转染组p27蛋白表达明显高于空质粒对照组（t=2．86，P〈0．05）。结论skp2蛋白在喉癌中的表达与癌组织的分化程度有关。抑制喉癌细胞系Hep-2细胞skp2的表达可以抑制细胞增殖，并促进细胞凋亡p27蛋白表达提高以及细胞s期阻滞可能是其作用机制之一。

Objective 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 poliferation and apoptosis and the expressing of p27 protein as well as change of cell cycle in laryngeal carcinoma cell line Hep-2 cell. Methods The expression of Skp2 in laryngeal carcinoma tissues of different diffrentiaton 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 MTY assay. Flow cytometry was performed to analyze apoptosis and cell cycle. Results Positive expression of Skp2 was detected in all 52 cases of laryngeal carcinoma tissues. The positive rate of expression of Skp2 in well-diffentiated larygeal carcinoma group was lower than that in middle-poorly diffrentiated group （ χ^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 ）. Conclusions The high level expressln of Skp2 in laryngeal carcinoma is significantly related to the diffrentiation 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 werease of p27 protein level and Sphase cell cycle arrest of Hep-2 cell.