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[Impact of biological function on ovarian clear cell carcinoma ES2 cell line with ARID1A gene expression
1Department of Obstetrics and Gynecology, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100730, China.
Objective:
To investigate the efficiency of biological function of AT rich interaction domain 1A (ARID1A) gene silenced by small interfering RNA (siRNA) on ovarian clear cell carcinoma ES2 cell line.
Methods:
(1) The three pairs ARID1A gene siRNA interference fragments siN1 (ARID1A-705), siN2 (ARID1A-1513), siN3 (ARID1A-2282) and one pair negative control were respectively designed, and transfected into ES2 cells by RNA interference max reagent transiently. Reverse transcription (RT)-PCR and western blot methods were used to detect the expression of ARID1A mRNA and protein in ES2 cells transfected with interference fragments respectively. So as to select the best silencing effect of siRNA interference fragment(that was siN3), and then was used in the following experiment. (2) The following experiment were divided into three groups, namely siN3 transfection group, negative control group and blank control group. The proliferative activity of three groups of cells after transient transfection (6, 24, 48, 72, 96 hours) was assessed by cell counting kit-8 (CCK-8) assay and expressed as absorbance (A) value; the apoptosis rate of three groups of cells transfected transiently with interference fragment was measured by flow cytometry with annexin V/propidium iodide (PI) staining; the ability of cellular invasion of three groups of cells transfected transiently with interference fragment was tested by transwell experiment; the expression of nuclear factor-kappa B (NF-κB), membrane type-1 matrix metalloproteinase (MT1-MMP) and matrix metalloproteinase-2 (MMP2) protein in ES2 cells transfected transiently with interference fragment was measured by western blot.
Results:
(1) The RT-PCR results showed that the ARID1A mRNA relative expression levels in ES2 cells after transfected transiently with siN1, siN2 and siN3 were 0.007 8±0.005 7, 0.006 8±0.000 3 and 0.002 8±0.000 3 respectively. They were all apparently lower than that in the negative control group (0.034 6±0.001 3; all P<0.01). The western blot results showed that the expression levels of ARID1A protein were 0.439 4±0.000 7, 0.424 4±0.005 0 and 0.386 0±0.005 8 respectively. They were also lower than that in the negative control group (0.732 4 ±0.030 3; all P<0.01). The siN3 with the highest transfection efficiency was selected to use in the following experiment. (2) The CCK-8 method showed that the proliferative activity of siN3 transfection group cells after transfected transiently at 6 hours was not statistically significant difference compared with those in negative control group and blank control group (0.506±0.010, 0.491±0.006, 0.498±0.009, respectively; all P>0.05). However, the proliferative activity of siN3 transfection group cells after transfected transiently at 24, 48, 72, 96 hours were higher than those in negative control group and blank control group (all P<0.01). The flow cytometry results showed that the apoptosis rate of siN3 transfection group cells was (20.0±3.9)%, which was significantly lower than those in negative control group and blank control group [(31.5±5.0)%, (34.0±4.2)%, respectively; all P<0.05]. The transwell experiment showed that the penetrated cell counts of siN3 transfection group was 60.4±2.9, which was apparently higher than those in negative control group and blank control group (54.2±3.5, 52.1±3.8, respectively; all P<0.01). Western blot experiment showed that the relative expression levels of NF-κB, MT1-MMP and MMP2 protein in siN3 transfection group were respectively 1.85±0.16, 0.37±0.08, 1.38± 0.11, which were apparently higher than those in negative control group (0.93±0.11, 0.17±0.05, 0.86±0.06; all P<0.05) and blank control group (0.94 ± 0.04, 0.15 ± 0.08, 0.85 ± 0.10, respectively; all P<0.01).
Conclusions:
It would be to promote the cell doubling time, reduce cell apoptosis and increase the invasive capability in ES2 cells that ARID1A expression was down-regulating by ARID1A mRNA interference. The invasion mechanism may be related to the activation of NF-κB signal transduction pathway, up-regulation of MT1-MMP expression and then promoting the invasion of tumor cells via the up-regulation of MMP2 expression.
Insights
Silencing the ARID1A gene in ovarian cancer cells promotes proliferation and invasion while reducing apoptosis. This suggests ARID1A plays a role in ovarian clear cell carcinoma progression, potentially via the NF-κB pathway.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- Ovarian clear cell carcinoma (OCCC) is an aggressive gynecological malignancy.
- The AT-rich interaction domain 1A (ARID1A) gene is frequently mutated in OCCC, suggesting its role as a tumor suppressor.
- Understanding ARID1A's function is crucial for developing targeted therapies.
Purpose of the Study:
- To investigate the functional impact of ARID1A gene silencing in the ES2 ovarian clear cell carcinoma cell line.
- To elucidate the potential molecular mechanisms underlying ARID1A's role in OCCC progression.
Main Methods:
- Small interfering RNA (siRNA) was used to silence ARID1A expression in ES2 cells.
- Cell proliferation was assessed using CCK-8 assays.
- Apoptosis rates were measured by flow cytometry.
- Cell invasion was evaluated using Transwell assays.
- Expression levels of NF-κB, MT1-MMP, and MMP2 were determined by Western blot.
Main Results:
- ARID1A mRNA and protein expression were significantly reduced by siRNA transfection (siN3).
- ARID1A silencing increased ES2 cell proliferation and invasion.
- Apoptosis rates were significantly decreased in ARID1A-silenced cells.
- Silencing ARID1A led to increased expression of NF-κB, MT1-MMP, and MMP2.
Conclusions:
- Down-regulation of ARID1A promotes cell proliferation, reduces apoptosis, and enhances invasion in ES2 cells.
- The observed effects are potentially mediated by the activation of the NF-κB signaling pathway, leading to increased MT1-MMP and MMP2 expression.
