LncRNA KCNQ1OT1在卵巢癌组织中的表达及对卵巢癌细胞顺铂耐药的作用

Expression of LncRNA KCNQ1OT1 in ovarian cancer tissues and its role in cisplatin resistance of ovarian cancer cells

目的:探讨卵巢癌组织中长链非编码RNA(long non-coding RNA,LncRNA)KCNQ1OT1(KCNQ1 opposite strand/antisense transcript 1)的表达及对卵巢癌细胞顺铂(cisplatin,DDP)耐药的影响。方法 :应用实时荧光定量PCR法检测DDP敏感或不敏感患者卵巢癌组织、卵巢癌组织及对应的癌旁组织、DDP耐药的卵巢癌SKOV3/DDP细胞及亲本细胞SKOV3、不同卵巢癌细胞(OVCAR3、OVCAR5、OVCAR8、SKOV3、A2780和PA-1)和永生化卵巢上皮细胞(OSE、IOSE120T和IOSE398)中LncRNA KCNQ1OT1的表达。合成2对靶向LncRNA KCNQ1OT1的shRNA序列(shRNA1和shRNA2)及阴性对照(negative control,NC)shRNA,并克隆到慢病毒表达载体PLKO.1/puro中,将其转染到293T细胞,包装出慢病毒,再用慢病毒感染卵巢癌耐药细胞SKOV3/DDP。然后,应用实时荧光定量PCR法检测各组细胞中LncRNA KCNQ1OT1的表达,MTT法和FCM法检测各组细胞的细胞增殖及细胞周期分布情况;计算LncRNA KCNQ1OT1表达抑制后,DDP对SKOV3/DDP细胞的最大半数抑制浓度(halfmaximalinhibitoryconcentration,IC50)值,蛋白质印迹法及实时荧光定量PCR法分别检测各组细胞中多药耐药1(multidrug resistance 1,MDR1)蛋白及mRNA的表达。结果 :DDP不敏感患者卵巢癌组织中LncRNA KCNQ1OT1的表达水平高于敏感患者(P <0.05),而且卵巢癌组织中LncRNA KCNQ1OT1的表达水平高于相应的癌旁组织(P <0.05);DDP耐药的卵巢癌SKOV3/DDP细胞中LncRNAKCNQ1OT1的表达水平高于亲本细胞SKOV3(P <0.05),卵巢癌OVCAR3、OVCAR5、OVCAR8、SKOV3、A2780和PA-1细胞中LncRNAKCNQ1OT1的表达水平高于永生化卵巢上皮细胞(OSE、IOSE120T和IOSE398)(P值均<0.05)。成功构建靶向LncRNA KCNQ1OT1的shRNA慢病毒表达载体,并包装出慢病毒。该慢病毒感染SKOV3/DDP细胞后,能明显下调细胞中LncRNA KCNQ1OT1的表达水平(P <0.05),抑制细胞增殖(P <0.05),并将细胞周期阻滞于G0/G1期(P <0.05);LncRNA KCNQ1OT1表达抑制能降低DDP对SKOV3/DDP细胞的IC50值(P <0.05),并可显著下调SKOV3/DDP细胞中MDR1蛋白及mRNA的表达水平(P值均<0.05)。结论:DDP不敏感患者卵巢癌组织中LncRNA KCNQ1OT1的表达水平较高。LncRNA KCNQ1OT1敲低可提高卵巢癌耐药细胞SKOV3/DDP对DDP的敏感性。

Objective:To investigate the expression of long non-coding RNA(LncRNA)KCNQ1 opposite strand/antisense transcript 1(KCNQ1 OT1)in ovarian cancer tissues,and to explore the effect of LncRNA KCNQ1 OT1 on the resistance of ovarian cancer cells to cisplatin(DDP).Methods:The expression of LncRNA KCNQ1 OT1 in ovarian cancer specimens from the DDP sensitive and insensitive patients,ovarian cancer tissues and their matched paracancerous normal tissues,DDP-resistant SKOV3/DDP cells and parental SKOV3 cells,different human ovarian cancer cell lines(OVCAR3,OVCAR5,OVCAR8,SKOV3,A2780 and PA-1)and immortalized ovarian epithelial cell lines(OSE,IOSE120 T and IOSE398)was detected by real-time fluorescent quantitative PCR.Two pairs of short hairpin RNA(shRNA)targeting LncRNA KCNQ1 OT1(shRNA1 and shRNA2)and their negative control shRNA(NC shRNA)were synthesized and cloned into lentivirus vector PLKO.1/puro,respectively.The recombinant plasmids were transfected into 293 T cells,and the lentiviruses were packaged and infected into the ovarian cancer SKOV3/DDP cells.Then the expression of LncRNA KCNQ1 OT1 was detected by real-time fluorescent quantitative PCR.The proliferation and cell cycle of SKOV3/DDP cells after infection with lentivirus were detected by MTT and FCM method,respectively.The half maximal inhibitory concentration(IC50)value of DDP in SKOV3/DDP cells was calculated.The expressions of multidrug resistance 1(MDR1)protein and mRNA in SKOV3/DDP cells after infection with lentivirus were detected by real-time fluorescent quantitative PCR and Western blotting,respectively.Results:The expression level of LncRNA KCNQ1 OT1 in ovarian cancer tissues of DDPinsensitive patients was higher than that of DDP-sensitive patients(P<0.05).The expression level of LncRNA KCNQ1 OT1 in ovarian cancer tissues was higher than that in the matched paracancerous tissues(P<0.05).The expression level of LncRNA KCNQ1 OT1 in SKOV3/DDP cells was higher than that in SKOV3 cells(P<0.05).The expression level of LncRNA KCNQ1 OT1 in human ovarian cancer cell line OVCAR3,OVCAR5,OVCAR8,SKOV3,A2780 and PA-1 was higher than that in immortalized ovarian epithelial cell line OSE,IOSE120 T and IOSE398(all P<0.05).After the lentivirus containing LncRNA KCNQ1 OT1 shRNA was constructed and infected into SKOV3/DDP cells,the expression of LncRNA KCNQ1 OT1 was obviously decreased(P<0.05).LncRNA KCNQ1 OT1 knockdown inhibited the proliferation of SKOV3/DDP cells(P<0.05),and blocked cell cycle in G0/G1 phase(P<0.05),decreased the IC50 value of DDP in SKOV3/DDP cells(P<0.05),and down-regulated the expression levels of MDR1 protein and mRNA in SKOV3/DDP cells(both P<0.05).Conclusion:The expression level of LncRNA KCNQ1 OT1 in ovarian cancer tissues from DDPinsensitive patients is obviously high.LncRNA KCNQ1 OT1 knockdown can significantly enhance the sensitivity of SKOV3/DDP cells to DDP.