SOX9活化Wnt/β-catenin信号通路对食管鳞状细胞癌细胞增殖的影响

Effects of SOX9-activated Wnt/β-catenin signaling pathway on cellular proliferation of esophageal squamous cell carcinoma

目的探讨SOX9对食管鳞状细胞癌(esophageal squamous cell carcinoma,ESCC)细胞增殖的影响和分子机制。方法采用免疫组化EnVision法检测ESCC组织和癌旁正常组织中SOX9的表达,并分析其与临床病理特征及预后的关系。对过表达SOX9的Eca109-SOX9细胞和Eca109-Vector进行全转录芯片检测。qRT-PCR技术检测TE-1和干扰SOX9表达的TE-1细胞中差异基因的表达。Western blot法检测干扰和过表达SOX9后活化/非磷酸化β-catenin蛋白表达。使用Wnt抑制剂LGK974处理不同ESCC细胞后,通过CCK-8法检测细胞增殖能力的变化。结果SOX9在ESCC组织(4.58±3.04)中的表达显著高于癌旁正常组织(1.56±2.08,P<0.001)。SOX9表达与ESCC分化程度和浸润深度有关(P<0.05)。生存分析显示,SOX9高表达患者的总生存率明显低于低表达者(P<0.05);生物信息学分析发现AXIN2、FZD7和WNT5A在Eca109-SOX9细胞中的表达明显高于对照组;使用siRNA干扰SOX9后,AXIN2、FZD7和WNT5A的mRNA表达水平及活化/非磷酸化β-catenin的蛋白表达水平均降低,过表达SOX9则活化/非磷酸化β-catenin表达增加;经不同浓度LGK974处理的TE-1和Eca109-SOX9细胞增殖能力均显著降低,而低表达SOX9的Eca109-Vector细胞增殖能力未见明显改变,且SOX9蛋白表达不受LGK974影响。结论SOX9在ESCC中高表达,并可通过活化Wnt/β-catenin信号通路促进ESCC增殖,提示SOX9有望成为ESCC的预后标志物和药物治疗靶点。

Purpose To investigate the effect and molecular mechanism underlying SOX9 during esophageal squamous cell carcinoma(ESCC)cells.Methods Immunohistochemistry(IHC)was used to detect the expression of SOX9 in ESCC tissues and adjacent normal tissues.The correlation of SOX9 expression with clinicopathological features and prognosis of ESCC was analyzed.The differentially expressed genes in Eca109-Vector and Eca109-SOX9 cells were detected by Affymetrix miRNA array.qRT-PCR was used to determine the differential gene in TE-1 and TE-1-siSOX9 cells.The relationship between SOX9 and active/unphosphorylatedβ-catenin levels was detected by Western blot.The effects of Wnt inhibitor LGK974 on the proliferation of ESCC cells were detected by CCK-8.Results SOX9 was highly expressed in ESCC(4.58±3.04)as compared with that in adjacent normal tissues(1.56±2.08,P<0.001).SOX9 was related to histopathological grade and invasion depth(P<0.05).Kaplan-Meier analysis indicated high SOX9 expression in ESCC was significantly correlated with shorter overall survival(P<0.05).Transcriptome profiling and qRT-PCR analysis suggested that SOX9 contributed to the regulation of AXIN2,FZD7 and WNT5A.Overexpression of SOX9 in Eca109 cells increased active/unphosphorylatedβ-catenin levels,whereas silencing SOX9 caused a decrease.Significant attenuation of cell proliferation was observed at various concentrations of LGK974 without affecting SOX9 expression on SOX9-expressing cell lines.As expected,this inhibitory effect was not obvious in Eca109-Vector cells when compared with Eca109-SOX9 cells treated with the same concentration of LGK974.Conclusion SOX9 is highly expressed in ESCC and SOX9-mediated Wnt/β-catenin signal pathway activation at least partially contributes to the SOX9-induced ESCC growth.These findings suggest that SOX9 is a promising prognostic marker and therapeutic target for ESCC.