食管鳞癌中转录因子IRF4对miR-133b表达的调控作用研究

Transcription factor IRF4 regulates the expression of miR-133b in esophageal squamous cell carcinoma

目的食管鳞癌中miR-133b通过调控EGFR/ITGB4/FAK信号通路,发挥抑制肿瘤增殖、侵袭、转移的作用。但目前对于miR-133b的上游调控机制尚不完全清楚,故展开探索。方法采用生信分析预测参与调控miR-133b表达的上游转录因子;采用qRT-PCR、Western blotting验证转染IRF4过表达质粒对IRF4和miR-133b在转录和蛋白水平表达的影响;采用染色质免疫共沉淀技术确认IRF4与miR-133b的相互作用;双荧光素酶报告基因实验验证IRF4对miR-133b表达的调控作用机制。结果使用ConTra V3预测发现转录因子IRF4能够与miR-133b结合;qRT-PCR、Western blotting实验证实转染IRF4过表达质粒可显著上调IRF4在mRNA(t=-18.950,P=0.010)和蛋白水平的表达(t=-4.061,P=0.042),并促进miR-133b的表达(t=-8.681,P=0.005);染色质免疫共沉淀实验(引物1:t=-17.391,P=0.003;引物2:t=-14.421,P=0.004)和双荧光素酶报告基因实验(t=-22.112,P=0.010)证实转录因子IRF4可直接靶向作用于miR-133b。结论验证IRF4对miR-133b的直接靶向调控作用,有助于构建miR-133b的信号调控网络,为食管鳞癌的诊疗提供新靶点。

Objective To research the upstream regulation mechanism of miR-133 b in esophageal squamous cell carcinoma. Methods The upstream transcription factors involved in the regulation of miR-133 b expression were predicted by bioinformatics analysis. The effects of IRF4 overexpression plasmid on the expression of IRF4 and miR-133 b were verified by qRT-PCR and Western blotting. The mechanisms of IRF4 regulating miR-133 b expression were verified by chromatin immunoprecipitation(ChIP) and dual-luciferase reporter assay. Results ConTra V3 predicted that the transcription factor IRF4 could bind to miR-133 b. The transfection of IRF4 overexpression plasmid could significantly up-regulate the expression of IRF4 at mRNA(t=-18.950, P=0.010) and protein(t=-4.061, P=0.042) levels, and promote the expression of miR-133 b(t=-8.681, P=0.005). ChIP( primer 1: t=-17.391, P=0.003;primer 2: t=-14.421, P=0.004) and dual-luciferase reporter assay(t=-22.112, P=0.010) confirmed that the transcription factor IRF4 could directly target miR-133 b. Conclusion IRF4 can directly target miR-133 b, which is helpful to construct the signal regulatory network of miR-133 b and provide a new target for the diagnosis and treatment of esophageal squamous cell carcinoma.