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沉默XIST通过上调miR-488-3p抑制宫颈鳞状细胞癌细胞的顺铂耐药性
作者:吴莉敏1  吴莉娜2  付丽丽1  李宝娣1  刘婷婷1  寇卫华3  李荣4 
单位:1. 西安交通大学第二附属医院 妇产科, 陕西 西安 710004;
2. 西安交通大学第二附属医院 手术室, 陕西 西安 710004;
3. 西安交通大学第二附属医院 肿瘤科, 陕西 西安 710004;
4. 西安交通大学第二附属医院 小儿外科, 陕西 西安 710004
关键词:宫颈鳞状细胞癌细胞 顺铂耐药性 长链非编码RNA XIST 微小RNA-488-3p RNA拉下实验 
分类号:R737.33
出版年·卷·期(页码):2019·47·第四期(415-421)
摘要:

目的:探索长链非编码RNA XIST对宫颈鳞状细胞癌细胞顺铂耐药性的作用及机制。方法:建立宫颈鳞状细胞癌细胞顺铂耐药系CASKI/cDDP;构建沉默XIST的慢病毒载体(si-XIST)及miR-488-3p沉默载体miR-inhibitor;将si-XIST单独稳定转入CASKI/cDDP细胞或将si-XIST与miR-inhibitor共转入CASKI/cDDP细胞,qRT-PCR检测XIST、miR-488-3p水平变化;MTT法检测细胞存活率变化,蛋白质印迹法检测多药耐药基因1(MDR1)表达变化,RNA拉下实验确定miR-488-3p与XIST的结合关系。结果:与CASKI相比,CASKI/cDDP中XIST表达升高(P<0.05);si-XIST转染CASKI/cDDP细胞48 h后,XIST的水平显著下调(P<0.05)、细胞的存活率下降(P<0.05)、MDR1表达水平下降(P<0.05)。CASKI/cDDP细胞XIST沉默可增加miR-488-3p水平(P<0.05);生物信息学预测宫颈癌关键标志物microRNA-488-3p与XIST结合;RNA拉下实验确认XIST直接结合miR-488-3p。CASKI/cDDP细胞共转染si-XIST与miR-488-3p-inhibitor;miR-488-3p-inhibitor部分逆转si-XIST的功能,提高CASKI/cDDP在顺铂处理下的细胞活性(P<0.05)。结论:沉默XIST通过上调miR-488-3p抑制宫颈鳞状细胞癌细胞的顺铂耐药性。

Objective:To investigate the effect and mechanism of long non-coding(lnc) RNA XIST on the cisplatin(cDDP) resistance in cervical carcinoma cells. Methods:CASKI/cDDP, the cDDP resistant cell line of cervix squamous cell carcinoma was constructed. Additionally, the lentivirus vector for silencing XIST(si-XIST) and miR-488-3p silencing vector(miR-inhibitor) were constructed. Then, si-XIST was stably transfected into CASKI/cDDP cell alone or si-XIST and miR-inhibitor were co-transfected into CASKI/cDDP cell. The changes of XIST and miR-488-3p levels were tested by qRT-PCR. Cell proliferation activity was detected by MTT. The expression of multidrug resistance gene 1(MDR1) was detected by western blot. The relationship between miR-488-3p and XIST was studied by RNA pull down assay. Results:The expression of XIST in CASKI/cDDP was higher than that in CASKI(P<0.05). The expression of XIST and MDR1 and the cell proliferation activity in si-XIST-transfected CASKI/cDDP cells were downregulated(P<0.05). MiR-488-3p, a key marker of cervical cancer, was predicted to bind with XIST by bioinformatics prediction. RNA pull down assay confirmed that XIST binded directly to miR-488-3p. Co-transfection of si-XIST and miR-inhibitor into CASKI/cDDP cells partially reversed the function of si-XIST, and increased the cell viability of CASKI/cDDP treated with cisplatin(P<0.05). Conclusion:Silencing XIST inhibits the cisplatin resistance in cervical squamous cell carcinoma by up-regulating miR-488-3p.

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