摘要目的 探讨长链非编码RNA钾电压门控通道亚家族Q成员1反向转录物1(KCNQ1OT1)在骨肉瘤顺铂(DDP)耐药发生中的作用及机制.方法 采用荧光定量聚合酶链反应(qPCR)方法检测KCNQ1OT1在亲代细胞及耐药细胞中表达.针对长链非编码RNA-KCNQ1OT1基因序列体外合成小干扰RNA (siRNA),脂质体法将siRNA转染至人骨肉瘤细胞,采用WST-1检测两组细胞顺铂半数抑制浓度(IC50),Transwell小室检测干扰KCNQ1OT1后对肿瘤迁徙及侵袭的影响情况,实时荧光定量聚合酶链式反应(qPCR)检测上皮-间质转化(EMT)相关分子的表达.所有结果采用GraphPad Prism 7.0(GraphPad Software Inc,La Jolla,CA)及SPSS 22.0软件(SPSS,Chicago,IL,USA)处理.Student's t检验进行统计分析.结果 骨肉瘤顺铂耐药细胞MG63/DDP及143B/DDP的IC50值分别为(17.960 ±0.153)、(7.725 ±0.183) μmol/L,显著高于亲代细胞的IC50值[(5.074±0.159)、(2.633 ±0.219) μmol/L,t=101.148、30.903,P＜0.01].MG63顺铂耐药细胞株(MG63/DDP)和143B顺铂耐药细胞株(143B/DDP)中KCNQ1OT1的相对表达量分别为6.830±0.105、6.284±0.465,与亲代细胞比较(3.081±0.167,2.014±0.216),差异有统计学意义(t=22.917、11.722,P＜0.01).干扰KCNQ1OT1表达后可抑制骨肉瘤细胞143B/DDP迁移及侵袭能力,WST-1检测干扰KCNQ1OT1后MG63/DDP及143B/DDP的IC50值分别为(8.363±0.089)、(3.947±0.093) μmol/L,与耐药细胞株比较,差异有统计学意义(t=-93.911、-31.878,P＜0.01).耐药细胞比较亲代细胞钙黏附蛋白E (E-cadherin)表达下调,钙黏附蛋白N(N-cadherin)、波形蛋白(Vimentin)、Slug表达上调,干扰KCNQ1OT1后,耐药细胞的E-cadherin表达上调,N-cadherin、Vimentin、Slug表达下调.结论 KCNQ1OT1可能通过调控EMT促进骨肉瘤耐药进程发生.

abstractsObjective To investigate the role and possible mechanism of long non-coding RNA KCNQ1 opposite strand transcript 1 (KCNQ1OT1) in the development of cisplatin (DDP) resistance in osteosarcoma (OS).Methods The expression of KCNQ1OT1 in parental cells and drug-resistant cells was detected by real-time polymerase chain reaction (qPCR).The small interfering RNA (siRNA) targeting lncRNA-KCNQ1OT1 was synthetized and transfected into osteosarcoma cells.The cisplatin sensitivity half maximal inhibitory concentration (IC50),migration and invasion of OS cells were assessed with WST-1 assays and Transwell assays,respectively.The expression of epithelial-mesenchymal transition (EMT)associated molecules was detected by qPCR.Results The IC50 values of cisplatin-resistant MG63/DDP and 143B/DDP in osteosarcoma were (17.960 ± 0.153) μmol/L and (7.725 ± 0.183) μmol/L,respectively,which were significantly higher than the IC50 value of the parental cells [(5.074 ± 0.159),(2.633±0.219) μmol/L] (t=101.148,30.903,P＜ 0.01).The relative expression levels of KCNQ1OT1 in MG63/DDP and 143B/DDP cells were 6.830 ± 0.105 and 6.284 ± 0.465,respectively,compared with parental cells (3.081 ± 0.167,2.014 ± 0.216),the difference was statistically significant (t =22.917,11.722,P ＜0.01).Knocking down the expression of KCNQ1OT1 can inhibit the migration and invasion of osteosarcoma DDP-resistant cells.After down-regulation of KCNQ1OT1,IC50 values of MG63/DDP and 143B/DDP were decreasedsignificantly.The expression of E-cadherin was down-regulated in the drug-resistant cells,and the expression of N-cadherin and Vimentin was up-regulated.After interference with KCNQ1OT1,the expression of E-cadherin was up-regulated and the expression of N-cadherin,Vimentin and Slug was down-regulated.Conclusion KCNQ1OT1 may promote osteosarcoma cisplatin resistance by regulating EMT.KCNQ1OT1 may be a potential therapeutic target for DDP-resistant osteosarcoma.