抑制酰基-辅酶A去饱和酶1表达对食管鳞癌细胞增殖和凋亡的影响及其分子机制探讨

Down-regulation of stearoyl-CoA desaturase 1 expression inhibits cell proliferation and induces apoptosis of esophageal squamous cell carcinoma and their related molecular mechanism

目的:检测酰基-辅酶A去饱和酶1(stearoyl-CoA desaturase-1,SCD-1)在食管鳞癌组织和细胞中的表达,分析其表达下调对食管鳞癌EC1细胞增殖和凋亡的影响,并探讨其相关的分子机制。方法:采用免疫组织化学和原位杂交法检测60例食管鳞癌组织及其相应癌旁正常食管黏膜组织中SCD-1mRNA和蛋白的表达,实时荧光定量PCR和蛋白质印迹法检测正常食管组织(作为阴性对照)和食管鳞癌细胞株中SCD-1mRNA和蛋白的表达。不同浓度SCD-1小干扰RNA(small interfering RNA,siRNA)转染食管鳞癌EC1细胞后,采用实时荧光定量PCR和蛋白质印迹法检测SCD-1mRNA和蛋白的表达,应用CCK-8法检测转染前后EC1细胞增殖的变化,FCM检测转染前后EC1细胞凋亡的改变,蛋白质印迹法检测总Akt、p-Akt、bcl-2和bax蛋白的表达。结果:食管鳞癌组织中SCD-1mRNA和蛋白表达的阳性率显著高于正常食管黏膜组织(P<0.05),其表达上调与肿瘤组织分级、TNM分期和淋巴结转移密切相关(P<0.05)。此外,与正常食管组织相比,食管鳞癌EC9706、Eca109和EC1细胞中SCD-1mRNA和蛋白表达均显著上调(P<0.05),其中EC1细胞中SCD-1的表达水平最高。50nmol/LSCD-1siRNA能显著下调EC1细胞中SCD-1mRNA和蛋白的表达。SCD-1表达下调可显著抑制EC1细胞的增殖,诱导细胞凋亡;同时,显著上调bax蛋白的表达,并下调p-Akt和bcl-2蛋白的表达,但不改变总Akt的表达水平。结论:SCD-1可能在食管鳞癌的发生和发展中具有重要作用,抑制SCD-1的表达有望成为食管鳞癌重要的分子治疗策略之一。

Objective： To investigate the expression of stearoyl-CoA desaturase-1 （SCD-1） in esophageal squamous cell carcinoma （ESCC） cells and tissues and the effect of downregulation of SCD-1 expression on cell proliferation and apoptosis of ESCC EC1 cells, and to explore their related molecular mechanisms. Methods： The expressions of SCD-1 mRNA and protein were detected in 60 cases of ESCC tissues and the corresponding paracancerous normal esophageal epithelial tissues by immunohistochemistry and in situ hybridization method, respectively. The expressions of SCD-1 mRNA and protein in normal esophageal epithelium and different esophageal squamous cell carcinoma cell lines were detected by real-time fluorescence quantitative PCR （RFQ-PCR） and Western blotting, respectively. After the ESCC EC1 cells were transfected with different concentrations of SCD-1 siRNAs, the expression levels of SCD-1 mRNA and protein were detected by RFQ-PCR and Western blotting, respectively. Subsequently, CCK-8 kit was utilized to analyze the change of cell proliferation of EC1 cells, and the flow cytometry （FCM） was used to detect the change of cell apoptosis of EC1 cells. Finally, the expressions of total Akt, p-Akt, bcl-2 and bax proteins were examined by Western blotting. Results： The percentages of positive cells expressing SCD-1 mRNA and protein were significantly higher in ESCC tissues than those in normal esophageal epithelial tissues （P 〈 0.05）, and the up-regulation of SCD-1 mRNA and protein expression levels were closely associated with histological grade, TNM stage and lymph node metastasis （P 〈 0.05）. Furthermore, as compared with the normal esophageal epithelium, the expressions of SCD-1 mRNA and protein were both markedly up-regulated in ESCC cell lines EC9706, Eca109 and EC1 （P 〈 0.05）, in which the EC1 cells displayed the highest SCD-1 expression level. The expression levels of SCD-1 mRNA and protein in EC1 cells were obviously down-regulated after tranfection with 50 nmol/L SCD-1 siRNA. The down-regulation of SCD-1 expression evidently inhibited the cell proliferation, induced the apoptosis, elevated the expression of bax, and reduced the expressions of p-Akt and bcl-2, but not changed the expression level of total Akt in EC1 cells. Conclusion： SCD-1 may play a pivotal role in the occurrence and development of ESCC. Inhibition of SCD-1 expression may be an important way for molecular therapy of ESCC.