Wu Zilong, Yu Boyao, Jiang Lei
Department of Thoracic Surgery, The First Affiliated Hospital of Nanchang University, Nanchang, China.
Xiangya School of Medicine, Central South University, Changsha, China.
J Thorac Dis. 2020 Aug;12(8):4357-4367. doi: 10.21037/jtd-20-2558.
Esophageal squamous cell carcinoma (ESCC) is the predominant subtype of esophageal cancer in East Asia, with approximately half of ESCC cases occurring in China. ESCC poses a serious threat to the quality of life of patients. MicroRNAs (miRNAs) are extremely important in the occurrence and development of ESCC. Current studies have shown that miR-212-3p is expressed at low levels in esophageal adenocarcinoma tumor tissues; however, its function and mechanism in ESCC have not been studied.
The expression levels of miR-212-3p and SOX4 were detected by quantitative polymerase chain reaction (qPCR) in ESCC tissues and adjacent tissues, and ESCC cell lines from 60 patients. The interaction of miR-212-3p and SOX4 was determined using a dual-luciferase reporter gene system. EC9706 Cells were transfected with miR-212-3p mimic, NC mimic, si-NC and si-SOX4 and miR-212-3p mimic + overexpressing-SOX4, and the effects on miR-212-3p and SOX4 expression were observed, respectively. An MTT assay was carried out to detect the proliferation ability of ESCC. The invasion ability and apoptosis level of the cells were determined by Transwell assay and flow cytometry, respectively. qPCR was used to detect expression of miR-212-3p and SOX4. Western blot was performed to observe the expression of SOX4, Wnt1, β-catenin, c-Myc, and Cyclin D1.
miR-212-3P was observed to be down-regulated in ESCC tissues and cells, while SOX4 expression was up-regulated; the two were negatively correlated. The dual-luciferase reporter gene further confirmed that miR-212-3p targeted SOX4. miR-212-3p overexpression and interference with SOX4 significantly inhibited the proliferation and invasion of ESCC EC970 cells, and promoted apoptosis. Furthermore, the results of Western blot confirmed that miR-212-3p overexpression and interference with SOX4 down-regulated the expression of Wnt1, β-catenin, c-Myc, and Cyclin D1. Meanwhile, SOX4 overexpression reversed the effect of up-regulation of miR-212-3p on EC970 function.
miR-212-3p mediates the apoptosis and invasion of ESCC cells through inhibiting the Wnt/β-catenin signal pathway by targeting SOX4.
食管鳞状细胞癌（ESCC）是东亚地区食管癌的主要亚型，约一半的ESCC病例发生在中国。ESCC对患者的生活质量构成严重威胁。微小RNA（miRNA）在ESCC的发生和发展中极为重要。目前的研究表明，miR-212-3p在食管腺癌肿瘤组织中表达水平较低；然而，其在ESCC中的功能和机制尚未得到研究。
采用定量聚合酶链反应（qPCR）检测60例患者的ESCC组织、癌旁组织及ESCC细胞系中miR-212-3p和SOX4的表达水平。使用双荧光素酶报告基因系统确定miR-212-3p与SOX4的相互作用。将EC9706细胞分别转染miR-212-3p模拟物、NC模拟物、si-NC和si-SOX4以及miR-212-3p模拟物+过表达-SOX4，分别观察对miR-212-3p和SOX4表达的影响。进行MTT试验检测ESCC的增殖能力。分别通过Transwell试验和流式细胞术测定细胞的侵袭能力和凋亡水平。使用qPCR检测miR-212-3p和SOX4的表达。进行蛋白质免疫印迹法观察SOX4、Wnt1、β-连环蛋白、c-Myc和细胞周期蛋白D1的表达。
观察到miR-212-3p在ESCC组织和细胞中表达下调，而SOX4表达上调；二者呈负相关。双荧光素酶报告基因进一步证实miR-212-3p靶向SOX4。miR-212-3p过表达和干扰SOX4可显著抑制ESCC EC970细胞的增殖和侵袭，并促进凋亡。此外，蛋白质免疫印迹法结果证实miR-212-3p过表达和干扰SOX4可下调Wnt1、β-连环蛋白、c-Myc和细胞周期蛋白D1的表达。同时，SOX4过表达逆转了miR-212-3p上调对EC970细胞功能的影响。
miR-212-3p通过靶向SOX4抑制Wnt/β-连环蛋白信号通路，介导ESCC细胞的凋亡和侵袭。
