Forkhead box L2 (FOXL2) is a transcription factor, which is involved in blepharophimosis, ptosis, and epicanthus in versus syndrome (BPES), premature ovarian failure (POF), as well as almost all stages of ovarian development and function. FOXL2 has various target genes, which are implicated in numerous processes, including sex determination, cell cycle regulation and apoptosis and stress response regulation in mammals. However, studies regarding the upstream regulation of
Forkhead box L2 (
However, the regulation of
Signal transducer and activator of transcription 3 (STAT3) is a clinically significant latent transcription factor that is activated by multiple extracellular and intracellular signals, including the Janus kinase (JAK) family and the receptor tyrosine kinase (
The present study predominantly focuses on the upstream transcriptional regulation of
The HeLa cell line, which was obtained from the Provincial Key Laboratory of Plastic and Microscopic Reconstructive Surgery Techniques of Shandong (Shandong, China), was cultured in high-glucose Dulbeccos modified Eagles medium (DMEM; Hyclone; GE Healthcare Life Sciences, Logan, UT, USA) supplemented with 10% fetal bovine serum (Hyclone; GE Healthcare Life Sciences) at 37°C in a humidified environment with 5% CO2.
The STAT3 inhibitors, C188–9 and WP1066, were synthesized and supplied by Dr. Shaopeng Yuan (Institute of Materia Medica, Chinese Academy of Medical Sciences, Beijing, China), and dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) as stock solution. This was serially diluted to the desired concentration using DMEM. The final concentration of DMSO in the culture systems was <0.05%.
Based on the human mRNA sequence of
The promoter sequence of
For transient transfection using Invitrogen Lipofectamine® 3000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA), the Lipofectamine® 3000 Reagent was diluted in Opti-MEM medium (Thermo Fisher Scientific, Inc.), and the master plasmid was prepared by diluting the recombinant pGL3-ProFOXL2 plasmid in Opti-MEM medium and adding P3000 Reagent (Thermo Fisher Scientific, Inc.). Subsequently, diluted plasmid was added to each tube of diluted Lipofectamine® 3000 Reagent (1:1 ratio) and incubated for 5 min at room temperature. Different plasmid-lipid complexes (0.25 µg/well) were then added to HeLa cells plated in 12-well plates (70–90% confluent) at 37°C separately. All cells were co-transfected with the
HeLa cells (1.5×106 cells/well) in 6-well plates, which had been grown under normal conditions, were treated with different concentrations (1, 2.5, 5 or 10 µM) WP1066 for 48 h; cells treated with DMSO to a final concentration at 1‰ served as the control group. Proteins were collected using 150 µl/well radioimmunoprecipitation assay lysis buffer (Thermo Fisher Scientific, Inc.), frozen for 30 min and stored at −20°C.
For western blot analysis, proteins that had been extracted from HeLa cells were boiled in 1X SDS-PAGE loading buffer (Takara Bio, Inc.). Protein concentrations were measured using a bicinchoninic acid protein assay and equal amounts of extracted protein samples (30 µl lysate/lane) were separated on 10% SDS-PAGE, as previously described (
The cytotoxic effect of C188-8 or WP1066 was determined using the xCELLigence system (Roche Diagnostics, Basel, Switzerland), which measures cell status (provided as cell index) utilizing an electric current to determine cellular attachment to an electrode-containing plate. Cells (5×103 cells/well; 100 µl) were seeded in the electrode-containing plate (e-plate) and incubated for 24 h at 37°C in a humidified environment with 5% CO2. The inhibitors of STAT3, C188-9 or WP1066, were added (at a concentration of 5 or 10 µM) and the electrical impedance, caused by cell adhesion to the plate (which is directly proportional to cellular viability) was numerically reported. Cells treated with DMSO served as a negative control. Data from the xCELLigence system was collected using the software provided with the system, and values were normalized at the point of treatment with compounds for all cell lines.
Statistical analysis was performed using the Data Processing System software version 7.0 developed by Zhejiang University (Hangzhou, China). Data are expressed as the mean ± standard error of the mean of at least 3 independent experiments. The statistical significance of the differences between groups was evaluated using one-way analysis of variance followed by a post hoc Duncans test for multiple comparisons. P<0.05 was considered to indicate a statistically significant difference.
In order to characterize the transcriptional regulation mechanism of
To assess whether the cloned 5 flanking region was actually the promoter of
Then the constructed vectors were transient transfected into HeLa cells, and the LUC activity was measured using a DLR assay system. As shown in
Using TFSERACH and Nsite, the Cis-regulatory elements in the promoter of
Subsequently, further detection was performed using western blotting to verify the regulation of
For further analysis of the influence of the STAT3 inhibitor, HeLa cells, which were plated onto an e-plate, were treated with different concentration inhibitors of STAT3, and the real-time cell viabilities (cell index) were detected (
As a transcription factor,
In the present study, the promoter region of
As a transcription factor, STAT3 is constitutively expressed in a wide variety of tissue types, and is activated by various cytokines and growth factors, as well as proto-oncogenes and oncogenes (
Although the expression level of the
DMSO is an important aprotic solvent that solubilizes a wide variety of otherwise poorly soluble polar and nonpolar molecules (
A previous study demonstrated that FOXL2 regulates certain genes, which are directly or indirectly involved in cell proliferation and apoptosis, such as tumor necrosis factor receptor 1 (
In conclusion, the promoter of FOXL2 was successfully cloned and registered in Genbank (GenBank accession no. KR030055), and LUC activity was observed to be significantly induced by the promoter. Notably, the current results demonstrated for the first time that FOXL2 was regulated by STAT3, according to the DLR and western blot analysis. In addition, cell viability was revealed to be suppressed by DMSO even at a low concentration.
The authors would like to thank Dr Shaopeng Yuan for kindly providing the inhibitors of STAT3. The present study was supported by the National Natural Foundation of China (grant nos. 81501683 and 81471880), Natural Science Foundation of Shandong Province (grant no. ZR2015HL057) and the Doctoral Foundation of Weifang Medical University (Weifang, China).
blepharophimosis, ptosis, and epicanthus inversus syndrome
dual luciferase reporter
forkhead box L2
granulose cell
luciferase
real-time cellular analysis
signal transducer and activator of transcription 3
Cloning of
Construction of vectors containing the promoter of
Luciferase activity was induced significantly by the promoter of
FOXL2 was downregulated by the STAT3 inhibitor, WP1066. (A) HeLa cells in 24-well plates were transfected with pGL3-Basic or pGL3-Enhancer vector plasmids containing the fusion promoter of
Real-time cell viability was detected following treatment with STAT3 inhibitor. Normal HeLa cells were plated onto an electrode-containing plate (e-plate) where they adhered for 24 h, and then were treated with various STAT3 inhibitors (5 or 10 µM); (A) C188-8 and (B) WP1066, while cells treated with DMSO served as the negative control. Cell viability was tracked using the xCelligence real-time viability system and each experiment was repeated at least two times. STAT3, signal transducer and activator of transcription 3; DMSO, dimethyl sulfoxide.