Xu H, Meng X R, Zhou Y, Wang F
Department of General Surgery, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China.
Department of Oncology, the First Affiliated Hospital of Zhengzhou University, Zhengzhou 450000, China.
Zhonghua Zhong Liu Za Zhi. 2020 Feb 23;42(2):105-113. doi: 10.3760/cma.j.issn.0253-3766.2020.02.004.
To explore the expression of microRNA-17-5p (miR-17-5p) in esophageal squamous cell carcinoma (ESCC) and its effects on cell proliferation and invasion ability. Real-time quantitative PCR (RT-qPCR) was used to detect the miR-17-5p level in ESCC tissues and cells. MiR-17-5p inhibitor and negative control (NC) were transfected into EC9706 and TE1 cells, and miR-17-5p expression was examined by using RT-qPCR. Cell counting kit-8 (CCK-8) and EdU were conducted to detect cell proliferation and Transwell chamber was used to investigate cell invasion ability. Dual-luciferase reporter assay was used to detect the direct interaction of miR-17-5p and retinoblastoma-like protein-2 (RBL2). Western blot and RT-qPCR were used to detect the expression of RBL2 in ESCC tissues, respectively. Finally, the correlation between RBL2 and miR-17-5p was analyzed. The miR-17-5p level in ESCC tissues was 4.222±0.392, significantly higher than 1.081±0.046 in normal esophageal epithelial tissues (<0.001). The expressions of miR-17-5p in ESCC cells, including EC9706, Eca109, TE1, KYSE450, KYSE70 and KYSE520, were 13.84±1.266, 6.453±0.293, 11.41±0.520, 2.613±0.548, 5.251±0.239 and 4.251±0.195, respectively, all obviously higher than (1.007±0.079) in normal esophageal epithelial cell Het-1A (<0.05). The miR-17-5p level in patients with ESCC ⅢⅣ was 5.094±0.562, markedly higher than 2.934±0.364 in patients with ESCCⅠⅡ(<0.01). Moreover, the miR-17-5p level in ESCC patients with lymph node metastasis was 5.523±0.634, markedly higher than 3.533±0.461 in ESCC patients without lymph node metastasis (<0.05). The survival ratio of ESCC patients with higher miR-17-5p level was evidently lower than that of ESCC patients with lower miR-17-5p level (<0.05). MiR-17-5p inhibitor significantly downregulated the miR-17-5p expression in EC9706 and TE1, which suppressed cell proliferation and invasion ability. Dual-luciferase reporter assay revealed that co-transfection of 3' untranslated region-wild type (3'UTR-WT) of RBL2 and miR-17-5p mimic significantly reduced luciferase activity in EC9706 and TE1 cells (<0.01), which implicated that RBL2 was the direct target of miR-17-5p. The result of western blot revealed that RBL2 protein expressions in miR-17-5p group of EC9706 and TE1 cells were 0.936±0.055 and 0.923±0.048, obviously higher than 0.087±0.019 and 0.102±0.010 in control group (<0.001). The expression of RBL2 in ESCC tissues was 0.219±0.510, markedly lower than 0.983±0.324 in normal esophageal epithelial tissues (<0.001). The miR-17-5p level exhibited a negative correlation with RBL2 level in ESCC tissues (=-0.462, <0.001). Downregulation of RBL2 reversed the miR-17-5p inhibitor induced suppression of cell proliferation and invasion ability. MiR-17-5p participates in the carcinogenesis and development of ESCC, thus may be a potential therapeutic target for ESCC patients.
探讨微小RNA-17-5p(miR-17-5p)在食管鳞状细胞癌(ESCC)中的表达及其对细胞增殖和侵袭能力的影响。采用实时定量聚合酶链反应(RT-qPCR)检测ESCC组织和细胞中miR-17-5p水平。将miR-17-5p抑制剂和阴性对照(NC)转染至EC9706和TE1细胞中,并用RT-qPCR检测miR-17-5p表达。采用细胞计数试剂盒-8(CCK-8)和EdU检测细胞增殖,用Transwell小室检测细胞侵袭能力。采用双荧光素酶报告基因检测法检测miR-17-5p与视网膜母细胞瘤样蛋白2(RBL2)的直接相互作用。分别用蛋白质免疫印迹法和RT-qPCR检测ESCC组织中RBL2的表达。最后,分析RBL2与miR-17-5p之间的相关性。ESCC组织中miR-17-5p水平为4.222±0.392,显著高于正常食管上皮组织中的1.081±0.046(<0.001)。ESCC细胞(包括EC9706、Eca109、TE1、KYSE450、KYSE70和KYSE520)中miR-17-5p的表达分别为13.84±1.266、6.453±0.293、11.41±0.520、2.613±0.548、5.251±0.239和4.251±0.195,均明显高于正常食管上皮细胞Het-1A中的(1.007±0.079)(<0.05)。ESCCⅢⅣ期患者的miR-17-5p水平为5.094±0.562,明显高于ESCCⅠⅡ期患者的2.934±0.364(<0.01)。此外,有淋巴结转移的ESCC患者的miR-17-5p水平为5.523±0.634,明显高于无淋巴结转移的ESCC患者的3.533±0.461(<0.05)。miR-17-5p水平较高的ESCC患者的生存率明显低于miR-17-5p水平较低的ESCC患者(<0.05)。miR-17-5p抑制剂显著下调EC9706和TE1中miR-17-5p的表达,抑制细胞增殖和侵袭能力。双荧光素酶报告基因检测显示,共转染RBL2的3'非翻译区野生型(3'UTR-WT)和miR-17-5p模拟物可显著降低EC9706和TE1细胞中的荧光素酶活性(<0.01),这表明RBL2是miR-17-5p的直接靶点。蛋白质免疫印迹结果显示,EC9706和TE1细胞miR-17-5p组中RBL2蛋白表达分别为0.936±0.055和0.923±0.048,明显高于对照组中的0.087±0.019和0.102±0.010(<0.001)。ESCC组织中RBL2的表达为0.219±0.510,明显低于正常食管上皮组织中的0.983±0.324(<0.001)。ESCC组织中miR-17-5p水平与RBL2水平呈负相关(r=-0.462,<0.001)。下调RBL2可逆转miR-17-5p抑制剂诱导的细胞增殖和侵袭能力抑制。miR-17-5p参与ESCC的发生发展,可能是ESCC患者潜在的治疗靶点。