微小RNA-124靶向调控丙酮酸激酶同工酶2对喉癌Hep-2细胞增殖与迁移及侵袭的影响

Effect of miR-124 on the proliferation,migration and invasion of laryngeal carcinoma Hep-2 cells through targeted regulation of pyruvate kinase isozyme type M2

目的观察微小RNA-124(miR-124)靶向调控肌肉丙酮酸激酶同工酶2(PKM2)对喉癌Hep-2细胞增殖、迁移及侵袭能力的影响,并探讨其作用机制。方法将Hep-2喉癌细胞分为3组:空白组、mimics组和抑制组。将miR-124类似物(miR-124 mimics)转染到mimics组细胞中,miR-124抑制物转染至抑制组,空白组不做任何干预。其中转染对应DNA均为0.2μg,室温孵育5 min。用双荧光素酶活性检测验证miR-124与PKM2靶向关系;用荧光定量PCR检测细胞miR-124的表达;用蛋白质印迹法检测PKM2蛋白的表达;用CCK-8法、细胞划痕实验、Transwell侵袭实验检测细胞增殖水平(OD值)、迁移和侵袭能力(转染细胞穿过基质膜至下室的细胞数目)。结果与野生型PKM2的3’UTR(PKM2-WT)与miR-124-NC共转染的Hep-2细胞比较,PKM2-WT与miR-124 mimics共转染的Hep-2细胞的荧光酶活性分别为0.97±0.01,0.43±0.05。空白组、mimics组和抑制组的miR-124相对表达量为0.20±0.03,0.43±0.05和0.07±0.02;PKM2蛋白的相对表达量为0.44±0.06,0.19±0.04和0.55±0.04;在12 h的增殖水平分别为0.21±0.04,0.18±0.04和0.26±0.04;在24 h的细胞迁移能力分别为0.37±0.04,0.13±0.03和0.44±0.04;侵袭能力分别为(132±5),(98±5)和(154±6)个。上述指标:与PKM2-WT共转染的miR-124-NC与miR-124 mimics比较差异有统计学意义(P<0.05);抑制组与空白组和mimics组比较差异均有统计学意义(均P<0.05)。结论miR-124的表达能够影响喉癌Hep-2细胞的增殖、迁移和侵袭能力,其作用机制可能是miR-124靶向调控PKM2实现。

Objective To observe the effect and mechanism of miR-124 on the proliferation,migration and invasion of laryngeal cancer Hep-2 cells through targeted regulation of muscle pyruvate kinase isozyme type M2(PKM2).Methods Hep-2 laryngeal cancer cells were divided into 3 groups:blank group,mimics group and inhibitor group,and the corresponding transfection reagent were transfected into the cells.The blank group did not undergo any intervention.The transfection corresponding DNA is 0.2μg,and incubated at room temperature for 5 min.The targeting relationship between miR-124 and PKM2 were verified by dual luciferase activity detection.The relative expression of miR-124 in transfected cells were detected by real-time quantitative PCR.The relative protein expression of PKM2 were detected by Western blot.The proliferation level(OD value),migration and invasion abilities(transfected cells passed through the matrix membrane to the number of cells)of transfected cells were tested by CCK-8 method,cell scratch test and Transwell invasion test.Results The luciferase activity of miR-124-NC cells co-transfected with 3’UTR of wild-type PKM2(PKM2-WT)were higher than that of miR-124-mimics cells.The relative expression of miR-124 in the blank group,mimics group and inhibitor group of cells were 0.20±0.03,0.43±0.05 and 0.07±0.02,respectively;the relative expression of PKM2 protein were 0.44±0.06,0.19±0.04 and 0.55±0.04,respectively;the proliferation level were 0.21±0.04,0.18±0.04 and 0.26±0.04 at 12 h,respectively;cell migration ability were 0.37±0.04 at 24 h;the invasion abilities were(132±5),(98±5),(154±6)cell,respectively.Comparison between miR-12-NC co-transfected with PKM2-WT and miR-12 mimics,the difference was significant(P<0.05);Comparison between inhibitor group and blank group,mimics group,the difference of the factors were significant(all P<0.05).Conclusion The expression of miR-124 can affect the proliferation,migration and invasion of laryngeal cancer Hep-2 cells,and its possible mechanism is that miR-124 targets and regulates PKM2.