摘要目的：探讨AT丰富结合域1A（ARID1A）基因表达下调对卵巢透明细胞癌细胞系ES2细胞生物学功能的影响。方法（1）设计3对ARID1A基因小分子干扰RNA（siRNA）序列，分别命名为siN1（即ARID1A-705）、siN2（即ARID1A-1513）、siN3（即ARID1A-2282），以及1对与ARID1A基因无关（阴性对照）的siRNA序列。分别采用逆转录（RT）-PCR技术和蛋白质印迹（western blot）法检测3种不同的ARID1A基因siRNA瞬时转染后ES2细胞中ARID1A mRNA和蛋白的表达水平，选择最佳沉默效应的siRNA干扰片段（即siN3）用于后续实验。（2）实验分为3组，即siN3转染组、阴性对照组和空白对照组，采用活细胞计数（CCK-8）法检测转染不同时间（6、24、48、72、96 h）后3组细胞的增殖活性，以吸光度（A）值表示；流式细胞仪检测转染后3组细胞的凋亡情况；体外侵袭实验检测转染后3组细胞的侵袭能力；western blot法检测转染后ES2细胞中核因子κB（NF-κB）、膜型基质金属蛋白酶1（MT1-MMP）及基质金属蛋白酶2（MMP2）蛋白的表达水平。结果（1）RT-PCR技术检测显示，siN1、siN2、siN3瞬时转染后ES2细胞中ARID1A mRNA的表达水平分别为0.0078±0.0057、0.0068±0.0003、0.0028±0.0003，均明显低于阴性对照（0.0346±0.0013；P均<0.01）；western blot法检测显示，siN1、siN2、siN3瞬时转染后ES2细胞中ARID1A蛋白的表达水平分别为0.4394±0.0007、0.4244±0.0050、0.3860±0.0058，均明显低于阴性对照（0.7324±0.0303；P均<0.01）。故用于后续实验的最佳沉默效应的siRNA干扰片段即为siN3。（2）CCK-8法检测显示，转染6 h后，siN3转染组ES2细胞的增殖活性分别与阴性对照组及空白对照组（分别为0.506±0.010、0.491±0.006、0.498±0.009）比较，差异均无统计学意义（P>0.05）；而转染24、48、72、96 h后，siN3转染组ES2细胞的增殖活性明显高于阴性对照组及空白对照组（P<0.01）。流式细胞仪检测显示，siN3转染组ES2细胞的凋亡率为（20.0±3.9）%，明显低于阴性对照组和空白对照组[分别为（31.5±5.0）%、（34.0±4.2）%；P<0.05]。体外侵袭实验检测显示，siN3转染组穿膜细胞数为（60.4±2.9）个，明显高于阴性对照组和空白对照组[分别为（54.2±3.5）、（52.1±3.8）个；P<0.01]。western blot法检测显示，转染72 h后siN3转染组ES2细胞中NF-κB、MT1-MMP及MMP2蛋白的表达水平分别为1.85±0.16、0.37±0.08、1.38±0.11，明显高于阴性对照组（分别为0.93±0.11、0.17±0.05、0.86±0.06；P<0.05）和空白对照组（分别为0.94±0.04、0.15±0.08、0.85±0.10；P<0.01）。结论 ARID1A基因表达下调使卵巢透明细胞癌ES2细胞的增殖活性增高，细胞凋亡减少，侵袭能力增强；其侵袭作用机制可能与激活NF-κB信号传导通路，上调MT1-MMP蛋白的表达，进而通过提高MMP2蛋白的表达，促进肿瘤细胞的侵袭有关。

abstractsObjective To investigate the efficiency of biological function of AT rich interaction&nbsp;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.