Identification and characterization of NF-kappaB binding sites in human miR-1908 promoter.
BACKGROUND
Previous studies reported that miR-1908 was highly expressed in mature human adipocytes. Adipokines tumor necrosis factor α (TNF-α) that was known to activate NF-kappaB signaling could affect the expression of miR-1908 in adipocytes. However, little is known about the underlying mechanisms.


METHODS
In this study, we identified miR-1908 promoter using polymerase chain reaction (PCR) from human genomic DNA. Bioinformatic analysis was applied to predict the NF-kappaB binding sites in miR-1908 promoter. Real-time PCR, dual luciferase reporter assay, Mutagenesis analysis and electrophoretic mobility shift assay (EMSA) were performed to demonstrate the function of NF-kappaB binding sites in miR-1908 promoter.


RESULTS
1243bp miR-1908 promoter located in the intron of host gene fatty acid desaturase 1 (FADS1). Bioinformatic analysis revealed the presence of two putative NF-kappaB binding sites. TNF-α restricts miR-1908 expression in preadipocytes, and TNF-α decreases miR-1908 promoter activity in HEK293T cells. In addition, those two NF-kappaB transcription factor binding sites in miR-1908 promoter were functional.


CONCLUSIONS
Our findings demonstrated that miR-1908 has its own transcription unit, and revealed the transcriptional mechanisms of miR-1908 expression based on NF-kappaB signaling. This study offers a theoretical basis for understanding the transcriptional mechanism of miR-1908 expression and may provide a new strategy for obesity clinical therapy.
Nanjing Maternal and Child Health Institute, Nanjing Maternal and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing,; Department of Pediatrics, Affiliated Hospital of Nantong University, Nantong, China. Electronic address: kuangqianhuining@126.com.
Qianhuining
Q
Kuang
Nanjing Maternal and Child Health Institute, Nanjing Maternal and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing.
Jingyun
J
Li
Nanjing Maternal and Child Health Institute, Nanjing Maternal and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing.
Lianghui
L
You
Nanjing Maternal and Child Health Institute, Nanjing Maternal and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing.
Chunmei
C
Shi
Nanjing Maternal and Child Health Institute, Nanjing Maternal and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing.
Chenbo
C
Ji
Nanjing Maternal and Child Health Institute, Nanjing Maternal and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing.
Xirong
X
Guo
Department of Pediatrics, Affiliated Hospital of Nantong University, Nantong, China.
Meiyu
M
Xu
Nanjing Maternal and Child Health Institute, Nanjing Maternal and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing.
Yuhui
Y
Ni
D017667:Adipocytes / Q000378:metabolism*; D001665:Binding Sites; D000090943:Delta-5 Fatty Acid Desaturase; D024202:Electrophoretic Mobility Shift Assay; D057809:HEK293 Cells; D006801:Humans; D035683:MicroRNAs / Q000235:genetics*; D016328:NF-kappa B / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000235:genetics; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism*
D000090943:Delta-5 Fatty Acid Desaturase; C577645:MIRN1908 microRNA, human; D035683:MicroRNAs; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha; C000716847:FADS1 protein, human
NF-kappaB binding sites; Promoter; miR-1908