Search

Aims: The transcription factor CCAAT/enhancer binding protein a (C/EBPa) 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/EBPa in lung cancer cells. Methods: The 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. Results: Both 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. Conclusions: The present study reveals that decreased C/EBPα contributes to the down-regulation of miRNA let-7a-1 in lung cancer cells. [ABSTRACT FROM AUTHOR]

Background: Fatty acid (FA) composition is the most important parameter affecting the flavor and nutritional value of the meat. The final and the only committed step in the biosynthesis of triglycerides is catalyzed by diacylglycerol acyltransferase 2 (DGAT2). The role of DGAT2 in lipid accumulation has been demonstrated in adipocytes, However, little is known about the effect of DGAT2 on the FA composition of these cells. Methods: To investigate the role of DGAT2 in regulating lipid accumulation, FA composition and the expression of adipogenic genes, we cloned the open reading frame of the porcine DGAT2 gene and established 3T3-L1 cells that overexpressed DGAT2. Cells were then cultured in differentiation medium (DM) without FA, with a mixture of FAs (FA-DM), or containing a 13C stable isotope-labeled FA mixture (IFA-DM). The FA composition of adipocytes was analyzed by gas chromatography-mass spectrometry and gas chromatography-isotope ratio mass spectrometry. Quantitative PCR and western blotting were employed to detect expression of adipogenic genes in 3T3-L1 adipocytes cultured with FA-DM for 12 d. Results: The triacylglyceride (TAG) content was significantly higher in 3T3-L1 adipocytes overexpressing DGAT2 than in control cells. When cultured in DM or FA-DM for 12 d, cells overexpressing DGAT2 showed a higher proportion of unsaturated FAs (C16:1 and C18:1). However, when cells overexpressing DGAT2 were cultured with FA-DM for 30 min, the FA composition was almost identical to that of controls. Further, the proportion of stable isotopelabeled FAs were similar in 3T3-L1 adipocytes overexpressing DGAT2 and control cells cultured in IFA-DM for 12 d. These results collectively indicate that the higher proportion of mono-unsaturated FAs, C16:1 and C18:1, may originate from de novo FA synthesis but not from the uptake of specific FAs from the medium. This hypothesis is further supported by evidence that both mRNA and protein expression of genes involved in FA synthesis (ACACA, FASN, SCD1, and A-FABP) were significantly higher in cells overexpressing DGAT2 than in control cells. Conclusions: In conclusion, our study revealed that TAG accumulation, the proportion of MUFAs, and the expression of adipogenic genes were higher in 3T3-L1 cells overexpressing DGAT2 than in control cells. [ABSTRACT FROM AUTHOR]