The AP-1 transcription factor is a heterodimer protein that regulates gene expression in response to a variety of extrinsic stimuli through signal transduction. It is involved in processes including differentiation, proliferation, and apoptosis. Among the genes it regulates are transcription factors that contribute to the stemness phenotype. Cancer stem cells have the ability to self-renew and initiate differentiation into heterogenic cancer cells, which may cause metastasis and relapses. In the present study, we evaluated the effect of AP-1 complexes, as well as the
The AP-1 transcription factor consists of various proteins including
Recent experimental data indicated that C-JUN is important for the maintenance of the self-renewal and tumorigenicity of glioma stem-like cells [
Human colon cancer stem cells (36112-39P; Celprogen) were cultured in appropriate growth medium (M36112-39PS; Celprogen), supplemented with 10% FBS in 25 cm2 flasks (E36102-29P-T25; Celprogen) at 37°C in a 5% CO2 environment.
During the exponential phase of proliferation, cells were seeded in 24-well plates (E36112-39; Celprogen) and transfected with small interfering RNA (siRNA) specific for
Cells were tested in both cellular and molecular assays. The cellular assays were based on the ability of CSCs to form microspheres in semisuspension, using an inverted light microscope. The cultures have previously been evaluated by molecular analyses, including gene expression analysis for specific transcription factors [
RNA was extracted from cell cultures using an RNeasy Mini Kit (74105; Qiagen). The RNA samples were evaluated both spectrophotometrically and on agarose gel by checking the 18S-28S rRNA bands. Then, 1
Cells were stained with PE Annexin V and 7-Amino-Actinomycin (7AAD) (559763; BD Biosciences) for 15 min and then resuspended in 0.5 mL sheath fluid (8546859; Beckman Coulter) followed by flow cytometric analysis of more than 50,000 events. The data were analyzed with FCS Express Software (DeNovo). In each case, positive and negative controls were used.
The quantitative polymerase chain reaction (qPCR) results were tested according to the Kolmogorov-Smirnov test; all samples had normal distribution. Median values were used for the analysis. Finally, Mann-Whitney
Knockdown was up to 55% for
Relative gene expression analysis of stemness transcription factors, Nanog, Oct3/4, and Sox2, after
Relative gene expression analysis of stemness transcription factors, Nanog, Oct3/4, and Sox2, after
Relative gene expression analysis of stemness transcription factors, Nanog, Oct3/4, and Sox2, after AP-1 complex knockdown. Changes in gene expression caused by the suppression of AP-1. The percentage of knockdown reached 45%.
Relative gene expression analysis of stemness transcription factors, Nanog, Oct3/4, and Sox2, after AP-1 complex knockdown (II). Changes in gene expression caused by the suppression of AP-1. The percentage of knockdown reached 35%.
The number of cells undergoing apoptosis was three times higher following suppression of both genes by 45%. Under the same conditions, it was observed that there was an increase in dead cells of 1.5 times that of the control cells.
Percentage of dead cells and cells undergoing apoptosis before and after knockdown.
Cell line | Cells undergoing apoptosis (%) | Dead cells (%) |
---|---|---|
Control | 1.48 | 4.40 |
|
1.68 | 7.42 |
|
4.93 | 4.06 |
|
6.39 | 5.76 |
|
2.61 | 3.71 |
CSCs are defined by their ability to self-renew, differentiate, and proliferate. These cells are proposed to initiate tumor formation and propagate metastasis [
Although little is known about their origin, CSCs are a subpopulation of heterogeneous tumors that has the ability to enter the bloodstream and migrate to colonize secondary sites, thus resulting in metastases and relapses. The epithelial to mesenchymal transition (EMT) and the reverse process (mesenchymal to epithelial transition (MET)) contribute to this [
The AP-1 transcription factor consists mainly of the
Therefore, it is clear that there is a relationship between the AP-1 transcription factor and stemness. The present study aimed to clarify this relationship in colon CSCs. The contribution of the AP-1 complex has been shown in apoptosis, alongside that of the individual proteins. AP-1 seems to play a crucial role in the maintenance of stemness by controlling NANOG, OCT3/4, and SOX2. Suppression of AP-1 led to a reduction in the levels of
The present study indicated that the AP-1 transcription factor may be strongly related to the stemness phenotype in colon CSCs. The reduction of its expression leads to changes in the expression of major transcription factors that are essential for maintaining pluripotency and undifferentiation. Further studies need to be performed to further investigate this correlation.
Cancer stem cells
Small interfering RNA
Quantitative polymerase chain reaction
Embryonic stem cells
Epithelial to mesenchymal transition
Mesenchymal to epithelial transition
Nuclear factor kappa-light-chain-enhancer of activated B cells.
The authors declare that they have no competing interests.