Abstract
AimsThe transcription factor CCAAT/enhancer binding protein α (C/EBPα) and microRNA (miRNA) let-7a-1 act as tumor suppressors in many types of cancers including lung cancer. In the present study, we aim to investigate whether let-7a-1 is a novel important target of C/EBPα in lung cancer cells.MethodsThe DNA sequence of the 2.1 kb let-7a-1 promoter was analyzed with MatInspector 4.1 (http://www.genomatix.de). Human lung cancer cell lines A549 and H1299, and human cervical cancer cell line Hela were used for transfection. Total RNA was extracted from cells using Trizol reagent and pri-let-7a-1 mRNA expression was measured using quantitative real-time polymerase chain reaction. Western blotting was performed to detect C/EBPα protein expression. To test whether C/EBP-α could up-regulate the expression level of let-7a at transcription level, dual-luciferase reporter gene assay was carried out. To determine whether C/EBPα could bind let-7a-1 promoter, electrophoretic mobility shift assay was employed. To further confirm the direct targeting let-7a-1 promoter by C/EBPα, chromatin immunoprecipitation was used.ResultsBoth C/EBPα and let-7a-1 were down-regulated in lung cancer A549 and H1299 cells, but up-regulated in Hela cells. Transfection and reporter gene assay showed that C/EBPα increased the expression of let-7a-1 at transcription level. Bioinformatics assay identified four putative C/EBP elements within let-7a-1 promoter. Dual-luciferase reporter gene, electrophoretic mobility shift assay and chromatin immunoprecipitation assays demonstrated that these four elements mediated the up-regulation effect of C/EBPα on let-7a-1.ConclusionsThe present study reveals that decreased C/EBPα contributes to the down-regulation of miRNA let-7a-1 in lung cancer cells.
Highlights
MicroRNA let-7 family plays pivotal roles in regulating carcinogenesis [1]
We investigate the influence of the CCAAT/enhancer binding protein α (C/EBPα) on let-7a-1 regulation in lung cancer cells
Positive correlation may exist between the expression levels of C/EBPα and pri‐let‐7a‐1 To determine the expression levels of C/EBPα and prilet-7a-1, western blotting and qRT-PCR were used, respectively
Summary
MicroRNA (miRNA) let-7 family plays pivotal roles in regulating carcinogenesis [1]. The let-7 family functions as tumor suppressors in multiple tumor types. They can inhibit the expressions of multiple oncogenes, including rat sarcoma viral oncogene homolog (RAS), high mobility group AT-hook 2 (HMGA2) and v-myc avian myelocytomatosis viral oncogene homolog (MYC) [2,3,4]. Down-regulation of let-7 family members is observed in multiple carcinomas, especially in lung cancer [5,6,7]. The mechanism of down-regulation of let-7 family members in various cancers needs to be elucidated, and will help understand the regulation mechanism of let-7 in carcinogenesis.
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