微小RNA-149靶向调控FOXM1的表达及其对非小细胞肺癌细胞A549增殖和凋亡的影响

Effects of miRNA-149 on the targeted regulation of FOXM1 and proliferation and apoptosis of non-small cell lung cancer cell line A549

目的探讨微小RNA-149(miR-149)在非小细胞肺癌(NSCLC)中调控细胞增殖和凋亡的相关机制。方法采用Lipofectamine脂质体法将miR-149模拟物(mimics)及其对照载体转染至A549细胞并分为miR-149转染组和miR-149对照组,同时以未转染的A549细胞作对照(未转染组)。采用实时荧光定量PCR(QPCR)检测各组miR-149水平以评价转染的效果,采用MTT法和Annexin V-FITC/PI流式细胞术检测转染后各组细胞增殖和凋亡情况。QPCR检测各组FOXM1基因的表达情况,Western blotting检测FOXM1蛋白的表达情况。采用双荧光素酶报告基因实验验证miR-149与FOXM1之间靶向作用关系。结果QPCR检测转染48 h后miR-149转染组的miR-149相对表达量为2.493±0.380,高于未转染组的1.077±0.321和miR-149对照组的1.283±0.273,差异均有统计学意义(P<0.05);miR-149转染组转染24、48、72 h的增殖抑制率分别为(16.51±2.49)%、(22.90±3.65)%和(31.43±5.27)%,均高于其余两组,差异均有统计学意义(P<0.05);miR-149转染组转染48 h的凋亡率为(29.17±4.48)%,高于未转染组的(5.34±1.72)%和miR-149对照组的(7.62±1.59)%,差异均有统计学意义(P<0.05);miR-149转染组转染48 h后的FOXM1 mRNA和蛋白水平分别为0.624±0.102和0.349±0.065,均低于未转染组的0.976±0.076和0.654±0.074及miR-149对照组的0.920±0.117和0.718±0.077,差异均有统计学意义(P<0.05);双荧光素酶报告基因实验表明miR-149可显著抑制野生型FOXM1-3’UTR的荧光素酶活性,而对突变型质粒转染细胞的荧光素酶活性并无影响。结论 miR-149可能是通过靶向FOXM1调控肺癌A549细胞的增殖和凋亡,可作为NSCLC分子治疗的有效靶点。

Objective To investigate the mechanism of microRNA-149（ miR-149） in regulating cell proliferation and apoptosis in non-small cell lung cancer（ NSCLC）. Methods MiR-149 mimics and its control vectors were transfected into A549 cells by Lipofectamine liposome and assigned into miR-149 transfection group and miR-149 control group. The non-transfected A549 cells were served as controls（ non-transfected group）. The miR-149 levels of three groups were detected by real-time quantitative PCR（ QPCR）in order to evaluate the transfection efficiency. The MTT and Annexin V-FITC/PI flow cytometry were used to compare the proliferation and apoptosis among miR-149 transfection group,miR-149 control group and non-transfected group. QPCR was used to detect the expression of FOXM1 gene in each group,and the expression of FOXM1 protein was detected by Western blotting. Double luciferase reporter assay was applied to verify the targeting relationship between miR-149 and FOXM1. Results The QPCR detection showed that the relative expression of miR-149 in the miR-149 transfection group was 2. 493±0. 380,at 48 h after transfection,higher than 1. 077±0. 321 in the non-transfected group and 1. 283±0. 273 in the miR-149 control group（ P〈0. 05）. The proliferation inhibition rates of miR-149 transfection group were（ 16. 51±2. 49） %,（ 22. 90±3. 65） % and（ 31. 43 ±5. 27） % at 24,48 and 72 h after transfection,higher than other two group（ P〈0. 05）. The apoptotic rate of miR-149 transfection group was（ 29. 17± 4. 48） % at 48 h after transfection,higher than（ 5. 34±1. 72） % of the non-transfected group and（ 7. 62±1. 59） % of miR-149 control group（ P〈0. 05）. The mRNA and protein levels of FOXM1 in miR-149 transfection group were 0. 624±0. 102 and 0. 349 ±0. 065 at 48 h after transfection,lower than 0. 976±0. 076 and 0. 654±0. 074 in non-transfected group and 0. 920 ±0. 117 and 0. 718 ±0. 077 in miR-149 control group（ P〈0. 05）. Double luciferase reporter gene test showed that miR-149 could significantly inhibit the luciferase activity of wild type FOXM1-3 UTR,but had no effect on the luciferase activity of mutant plasmid transfected cells. Conclusion miR-149 may regulate the proliferation and apoptosis of lung cancer A549 cells by targeting FOXM1,and can be used as an effective target for the molecular therapy of NSCLC.