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

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（IC50）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IC50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。

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

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 IC50 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 IC50 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 549／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.