SOX4在下咽癌中的表达及其对癌细胞恶性生物学行为的影响

The expression of SOX4 in hypopharyngeal carcinomaand its effect on biological behavior of cancer cells

目的 :分析下咽鳞状细胞癌(hypopharyngeal squamous cell carcinomas,HSCC)组织中SRY相关HMGbox转录因子4(SRY-box transcription factor-4,SOX4)的表达及其与患者临床病理特征及预后的关系。观察siRNAs敲低FaDu人咽鳞癌细胞中SOX4表达对细胞增殖、迁移及裸鼠成瘤的影响。方法:(1)选取下咽癌石蜡切片标本76例,癌旁正常组织标本18例作为对照,采用免疫组化检测SOX4和Ki67的表达水平。(2)选取6对新鲜的下咽癌组织和相邻正常组织,采用蛋白免疫印迹法(Western blot)检测SOX4表达水平。(3)运用siRNAs敲低FaDu细胞中SOX4表达。(4)饥饿释放实验检测SOX4在增殖期FaDu细胞中的表达水平,CCK-8法检测FaDu细胞增殖活性,流式细胞术检测细胞周期的分布,Transwell试验和划痕实验检测细胞迁移能力。(5)Western blot检测上皮间充质转化(epithelial mesendyinal transition,EMT)相关分子的表达。(6)裸鼠成瘤实验观察SOX4对肿瘤生长的影响。结果:(1)与癌旁正常组织相比,HSCC组织中SOX4表达明显升高,与肿瘤分化程度、肿瘤大小、临床分期、淋巴结转移及Ki67表达相关(P<0.05)。(2)SOX4高表达患者生存期较短。(3)增殖期FaDu细胞SOX4和PCNA的表达同步明显增加。(4)SOX4-Si2处理后FaDu细胞中SOX4染色强度显著降低,细胞增殖能力明显下降,细胞停滞于G1期,S期细胞减少。(5)SOX4-Si2干扰后FaDu细胞划痕愈合能力明显下降,细胞迁移数减少,上皮表型标志物β-连环蛋白和E-钙粘蛋白表达显著增加,而间质表型标志物波形蛋白和N-钙粘蛋白表达显著降低。(6)裸鼠成瘤实验显示,SOX4-Si2干扰后裸鼠肿瘤生长受到显著抑制,Ki-67阳性率增高。结论:SOX4在HSCC中过表达,SOX4在体内外对下咽癌的进展和迁移起着重要作用,可作为下咽癌患者诊断和治疗的分子靶标。

Objective:To investigate the expression of SOX4 in hypopharyngeal squamous cell carcinoma(HSCC)tissues and the relationship between SOX4 expression level and clinicopathological features and prognosis. In vivo experiments, using siRNAs to knockdown the expression of SOX4 in FaDu human hypopharyngeal squamous cells, to explore the effect on cell proliferation, migration and tumor formation in nude mice. Methods: (1)Using immunohistochemistry to detect SOX4 and Ki-67 expression in 76 cases of paraffin-embedded HSCC and 18 cases of para-cancerous tissue samples. (2)Using western blot to examine SOX4 expression in 6 fresh pairs of HSCC cancer tissues and adjacent normal tissues. (3)SOX4-siRNAs were used to knockdown the expression of SOX4 in HSCC cell line FaDu. (4)Serum-starving experiment was used to determine expression level of SOX4 in proliferating FaDu cells. Cell viability, cell cycle and cell migration were assessed by CCK-8, flow cytometry, and wound-healing and transwell assays. (5)Using western blot to detect the expression of epithelial mesenchymal transition (EMT) related molecules. (6)Nude mice subcutaneous xenografts experiment was performed to investigate the effect of SOX4 on tumor growth. Results: (1)Compared with para-cancerous tissues, SOX4 expression significantly increased in HSCC specimens and its expression level was associated with differentiation, tumor size, clinical stage, lymph node metastasis and Ki-67 expression (P<0.05). (2)The survival time of patients with high SOX4 expression was shorter. (3)The expression of SOX4 and PCNA in FaDu cells increased significantly during proliferating phase. (4)After SOX4-Si2 treatment, the SOX4 staining intensity in FaDu cells decreased significantly, the cell proliferation ability decreased, the cells stagnated in G1 phase, and the cells in S phase decreased. (5)After SOX2-Si2 interference, the scratch healing ability of FaDu cells significantly decreased, the cell migration number decreased, and the expression of β-catenin and E-cadherin which were epithelial phenotype markers increased, while the expression of vimentin and N-cadherin which were interstitial phenotype markers significantly decreased. (6)The tumor formation experiment of nude mice showed that the tumor growth of nude mice was significantly inhibited after SOX4-Si2 interference, and the positive rate of Ki-67 increased. Conclusion: SOX4 is overexpressed in HSCC and plays an important role in the progression and migration of HSCC in vitro and vivo. It can be used as a molecular target for diagnosis and treatment of HSCC.