Impact of biological function on ovarian clear cell carcinoma ES2 cell line with ARID1A gene expression down-regulating in vitro

Abstract

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. (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. (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). 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.