Backgrounds: ESCC is a life-threatening disease due to invasion and metastasis in the early stage. Great efforts had been made to detect the molecular mechanisms which led to the invasion and metastasis in ESCC. Recent evidence had suggested that deregulation of miR-424-5p took an important role in cancers. However, its role and functional mechanism in ESCC had seldom been elucidated.
Methods: The expression levels of miR-424-5p were detected in ESCC tissues and cell lines by real-time PCR methods. Then, the invasion, metastasis and proliferation ability of ESCC cell lines transfected with miR-424-5p mimics were analyzed separately by transwell invasion assay, wound healing assay and cell proliferation assay. Finally, the target gene of miR-424-5p was studied and verified by luciferase activity assay. And the role of miR-424-5p in EMT was also investigated by real-time PCR and western blot assay.
Results: We showed that the expression levels of miR-424-5p were decreased both in ESCC tissues and cell lines. Furthermore, the expression levels of miR-424-5p were negatively linked to lymph node metastasis in ESCC tissues. Restoration of miR-424-5p in EC-1 cells by using miR-424-5p mimics could decrease the invasion, metastasis and proliferation of EC-1 cells, indicating its role in inhibition on the invasion and metastasis ability of ESCC cells and tissues. In addition, we demonstrated that SMAD7 was a specific target gene for miR-424-5p by luciferase activity assay and miR-424-5p could not only negatively regulate SMAD7 expression but also participate in EMT via SMAD7, because overexpression of SMAD7 could partly enhance the miR-424-5p anti-EMT function.
Conclusions: Our results described that miR-424-5p -SMAD7 pathway contributed to ESCC invasion and metastasis and up-regulation of miR-424-5p perhaps provided a strategy for preventing tumor invasion, metastasis.
目的:食管癌因为早期侵袭转移的发生所以预后不佳。最近研究表明miR-424-5p表达下调在多种肿瘤的发生、发展过程中发挥重要作用。但其在食管癌发生、发展中的机制国内外未见报道。
方法:实时定量PCR法检测食管癌组织和细胞系中miR-424-5p的表达;合成miR-424-5p拟似物转染食管癌细胞系,侵袭小室、划痕试验和MTT试验检测miR-424-5p表达升高对食管癌细胞侵袭转移和增殖能力的影响;荧光素酶报告试验检测miR-424-5p下游靶基因;并进一步使用实时定量PCR和Western blot法检测miR-424-5p在食管癌上皮间质转化发生发展中的作用。
结果:miR-424-5p在食管癌组织和细胞系中的表达均明显下降,且其表达下降与食管癌组织的淋巴结转移密切相关。通过miR-424-5p拟似物增高miR-424-5p的表达可以降低食管癌细胞的体外侵袭转移和增殖能力。说明miR-424-5p在食管癌侵袭转移中发挥重要作用。荧光素梅报告试验证实SMAD7是miR-424-5p下游靶基因,miR-424-5p可以通过调节SMAD7的表达在食管癌的EMT发生发展中发挥作用。
结论:miR-424-5p -SMAD7途径在食管癌侵袭转移中发挥重要作用,升高miR-424-5p 的表达有可能成为食管癌治疗的手段之一。
Keywords: Epithelial-mesenchymal transition; Esophageal squamous cell carcinoma; SMAD7; miR-424-5p.