[Role of eukaryotic initiation factor-4E (eIF-4E) in regulation of expression of NF-kappaB and its subsequent influence on transcription and activity of heparanase in human colon adenocarcinoma cell line].
OBJECTIVE
Recent studies have revealed that some transcription factors may be translationally regulated by eukaryotic initiation factor-4E (eIF-4E) in human cancer. These modifications in the expression levels of the key transcription factors will alter the expression of some malignancy-related gene products at the transcriptional levels. The current study was designed to investigate the effect of eIF-4E on the expression and activity of NF-kappaB and observe how the activity level of NF-kappaB contributes, as a secondary effect, to the transcription of heparanase in human colon adenocarcinoma LS-174T cells.


METHODS
In order to repress the expression of eIF-4E, a 20-mer antisense oligodeoxynucleotide (ASODN) targeted against the translation start site of eIF-4E mRNA was transfected into human colorectal cancer cell line LS-174T via liposome reagent, followed by assessment of the activity and the protein expression of NF-kappaB by an electrophoretic gel mobility shift assay (EMSA) and Western blot analysis respectively. The alterations of heparanase expression were examined by RT-PCR and Western blot analysis, and heparanase activity in LS-174T cells was measured by specific enzymatic activity test using radiolabeled heparan sulfate as the substrate and gel filtration chromatography for the analysis of the degradation product.


RESULTS
The 20-mer ASODN against eIF-4E specifically and significantly inhibited eIF-4E expression at both transcriptional and translational levels, and the repression of eIF-4E gene expression was correlated with decreased expression levels and activity of NF-kappaB protein. Furthermore, this down regulation of the ubiquitous transcription factor NF-kappaB led to reduced transcription of the heparanase gene, and the transfected cells also showed a considerable decrease in heparanase protein and activity.


CONCLUSIONS
These results suggest that eIF-4E play an important role in translational regulation of NF-kappaB expression in LS-174T cells.NF-kappaB is an essential factor in the regulatory mechanisms of heparanase gene transcription, and the suppressed NF-kappaB activity in LS-174T cells may significantly reduce the transcriptional expression of heparanase that consequently leads to decreased heparanase protein expression and enzymatic activity.
Yang
Yu-Jie
YJ
PLA Institute of Digestive Disease, Nanfang Hospital, The First Military Medical University, Guangzhou, Guangdong, 510515, PR China. yujiey@fimmu.edu.cn
Zhang
Ya-Li
YL
Wang
Ji-De
JD
Lai
Zhuo-Sheng
ZS
Wang
Qun-Ying
QY
Cui
Hai-Hong
HH
D000230:Adenocarcinoma / Q000378:metabolism*; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003110:Colonic Neoplasms / Q000378:metabolism*; D039561:Eukaryotic Initiation Factor-4E / Q000502:physiology*; D015972:Gene Expression Regulation, Neoplastic; D005966:Glucuronidase / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D016328:NF-kappa B / Q000235:genetics*; D014158:Transcription, Genetic; D000099175:Heparanase
D039561:Eukaryotic Initiation Factor-4E; D005966:Glucuronidase; D016328:NF-kappa B; D000099175:Heparanase