环状RNA circ_MTHFD2对mircoRNA-124的调控在肺癌培美曲塞耐药中的作用

The study on the mechanism by which circ_MTHFD2 promotes lung cancer resistance to pemetrexed through microRNA-124

目的探讨circ_MTHFD2调控microRNA-124(miR-124)的表达在肺癌培美曲塞耐药发生过程中的作用。方法采用高浓度反复间歇诱导法建立肺癌培美曲塞耐药细胞株A549/PEM。通过瘤内注射建立培美曲塞耐药的裸鼠移植瘤模型;采用Lipofectamine脂质体法将miR-124模拟物、miR-124抑制剂及阴性对照转染至A549和A549/PEM细胞,并分为miR-124上调组、miR-124抑制组及阴性对照组;采用CCK-8法和流式细胞术检测各组细胞的增殖及凋亡情况,比较各组细胞对培美曲塞的敏感性;采用实时荧光定量PCR(QPCR)和基因芯片检测miR-124和环状RNA的表达;通过测序技术检测耐药细胞及耐药裸鼠的肿瘤组织与对照组间环状RNA的表达,预测环状RNA上的miR-124结合位点。结果亲本细胞A549和耐药细胞的半数抑制浓度(IC_(50))分别为(30.78±1.97)μmol/L和(913.53±14.1)μmol/L,最终获得耐药指数(RI)为29.69±1.49的培美曲塞耐药细胞,命名为A549/PEM。耐药细胞A549/PEM中miR-124的表达显著降低,相比A549细胞,下降约145.952倍;下调miR-124表达的A549细胞对培美曲塞敏感性降低,miR-124抑制组及阴性对照组细胞的IC_(50)分别为(80.45±3.50)μmol/L和(9.94±1.90)μmol/L,差异有统计学意义(P<0.05);流式细胞术检测显示miR-124表达上调可以显著诱导A549、A549/PEM细胞的凋亡;通过高通量测序提示在耐药的细胞及组织中环状RNA表达明显上调,表达量上调的circ_MTHFD2可与miR-124结合。结论 circ_MTHFD2可能通过调控miR-124的表达,参与肺癌培美曲塞耐药的发生发展。

Objective To investigate the effect of microRNA-124（miR-124） mediated by circ_MTHFD2 on the resistance of lung cancer against pemetrexed.Methods The pemetrexed-resistant human lung cancer cell line was established by exposing pemetrexed repeatedly to high-level concentration.The xenograft model of pemetrexed resistant nude mice by intratumoral injection was established.MiR-124 mimics,miR-124 inhibitors and negative controls were transfected into A549 and A549/PEM cells by Lipofectamine liposome method,and divided into miR-124 up-regulation group,miR-124 inhibitory group and negative control group;then the sensitivity to pemetrexed was estimated by CCK-8 assay and flow cytometry.Real time fluorescent quantitative PCR（QPCR） and gene chip were used to detect the expression of miR-124 and circular RNA.Sequencing and bioinformatic analysis were used to detect the expression of circular RNA in different cells and tissue and to predict the binding sites between miR-124 and circular RNA.Results The IC_（50） of parental strains A549 was（30.78 ± 1.97） μmol/L,and the resistant strains A549/PEM was（913.53 ± 14.1）μmol/L.The resistance index（RI） of pemetrexed-resistant human lung cancer cell line A549 named A549/PEM was 29.69 ± 1.49.The expression of miR-124 in drug-resistant cell line A549/PEM was significantly decreased,which was about 145.952 times lower than that in A549 cells.Compared with the negative control group,the sensitivity to pemetrexed was significantly reduced in miR-124 inhibitor group,and the IC_（50） was（9.94 ± 1.90） μmol/L and（80.45 ± 3.50） μmol/L respectively,with significant difference between two groups（P 0.05）.Flow cytometry showed that up-regulation of miR-124 expression could induce the apoptosis of A549 and A549/PEM cells compared with the control group.The sequencing indicated that the expression of many circular RNAs were up-regulated in resistant cells and tissues,and up-regulated circ_MTHFD2 was proposed to bind miR-124.Conclusion Circ_MTHFD2 may be involved in the development of pemetrexed resistance in lung cancer by regulating the expression of miR-124.