[关键词]
[摘要]
目的:分析整合素连接激酶(ILK)基因在食管鳞状细胞癌(ESCC)组织中的表达水平及其与患者临床病理特征之间的关系,探讨其对KYSE-150细胞增殖、凋亡和裸鼠皮下移植瘤生长的影响。方法:选取2012年1月至2014年12月手术切除并经病理证实的75例ESCC患者的癌组织和其配对的癌旁组织标本,用组织芯片技术及免疫组织化学染色法检测ESCC组织和癌旁组织中ILK 的表达情况;qPCR法检测ESCC细胞ECA109、TE-1、EC9706、KYSE-150中ILK mRNA的表达,选用ILK表达最高的KYSE-150细胞进行后续细胞功能学研究。使用ILK干扰慢病毒感染KYSE-150细胞下调ILK的表达,qPCR和WB法检测ILK基因敲降效率;MTT实验、克隆形成实验和FACS检测干扰ILK表达对KYSE-150细胞增殖能力和凋亡水平的影响;裸鼠皮下成瘤实验检测干扰ILK对KYSE-150细胞移植瘤生长的影响。结果:ESCC 组织中 ILK 蛋白阳性表达率高于癌旁组织(P<0.05),且ILK高表达与淋巴结转移有关联(P<0.05)。ILK干扰慢病毒感染的KYSE-150细胞中ILK mRNA表达明显受到抑制(P<0.05),ILK蛋白水平表达下调,以上结果提示ILK敲降成功。与感染阴性对照病毒的KYSE-150细胞相比,ILK干扰慢病毒感染的 KYSE-150细胞的增殖能力、克隆形成数均显著降低(均 P<0.05),但细胞凋亡率升高(P<0.05)。与对照组相比,干预组裸鼠移植瘤生长缓慢,移植瘤的质量及体积均较小(均 P<0.05)。结论:ESCC组织中ILK的表达高于癌旁组织,且ILK高表达与患者发生淋巴结转移有关联;抑制ILK基因可导致KYSE-150细胞增殖能力降低,促进细胞凋亡而抑制裸鼠移植瘤生长。
[Key word]
[Abstract]
Objective: To analyze the expression level of integrin-linked kinase (ILK) gene in esophageal squamous cell carcinoma (ESCC) tissues and its relationship with the clinicopathological characteristics of patients, and to explore its effect on KYSE-150 cell proliferation, apoptosis and the growth of subcutaneous xenograft tumor in nude mice. Methods: The cancer tissues and paired paracancerous tissues of 75 patients with ESCC who had surgical resection and confirmed by pathological examination from January 2012 to December 2014 were selected. Tissue chip technology and immunohistochemical staining were used to detect the expression of ILK in ESCC tissues and para-cancerous tissues; qPCR was used to detect the ILK expression in ESCC cell ECA109, TE-1, EC9706 and KYSE-150, and the KYSE-150 cells with the highest ILK expression was selected for subsequent cell functional studies. The KYSE-150 cells were transfected with ILK interference lentivirus to down-regulate the expression of ILK, and the knockdown efficiency was verified by qPCR and WB methods; the effects of interfering ILK expression on the proliferation and apoptosis of KYSE-150 cells were detected by MTT assay, clone formation assay and FACS; subcutaneous tumorigenesis assay in nude mice was used to detect the effect of interfering ILK on the growth of KYSE-150 cells transplanted tumors. Results: The positive rate of ILK protein in ESCC tissues was higher than that in para-cancerous tissues (P<0.05), and the high expression of ILK was associated with lymph node metastasis (P<0.05). The mRNA expression of ILK in shILK-infected KYSE-150 cells was significantly inhibited (P<0.05)and the expression of ILK protein was significantly down-regulated in the shILK group, which indicated the successful knockdown of ILK. Compared with the cells transfected with shCtrl, the proliferation ability and colony formation number of KYSE-150 cells in the shILK group were significantly reduced (both P<0.05), but the apoptosis rate was significantly increased (P<0.05). Compared with the NC group, the growth of transplanted tumors in the nude mice of the KD group was slower, and the weight and volume of the tumor were smaller (both P<0.05). Conclusion: The expression of ILK in ESCC tissues is higher than that in para-cancerous tissues, and the high ILK expression is associated with lymph node metastasis. Silencing ILK gene can inhibit the proliferation but promote the apoptosis of KYSE-150 cells and inhibit tumorigenesis in nude mice.
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[基金项目]
新疆维吾尔自治区自然科学基金资助项目(No.2020D01C258)