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（westemblot）法检测3种不同的ARIDIA基因siRNA瞬时转染后ES2细胞中ARID1AmRNA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、96h）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；westernblot法检测转染后ES2细胞中核因子KB（NF．KB）、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ARID1AmRNA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）；westernblot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6h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、96h后，siN3转染组ES2细胞的增殖活性明显高于阴�
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-KB）, 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 （
出处
《中华妇产科杂志》
CAS
CSCD
北大核心
2016年第3期209-215,共7页
Chinese Journal of Obstetrics and Gynecology
关键词
卵巢肿瘤
腺癌
透明细胞
细胞系
肿瘤
核蛋白质类
转录因子
RNA
小分子干扰
Ovarian neoplasms
Adenocarcinoma, clear cell
Cell line, tumor
Nuclear proteins
Transcription factors
RNA, small interfering
