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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 vector were transfected into A549 cells by Lipofectamine liposome method and divided into miR-149 transfection group and miR-149 control group. At the same time, untransfected A549 cells Control (untransfected group). The levels of miR-149 in each group were detected by real-time fluorescence quantitative PCR (QPCR) to evaluate the transfection efficiency. The proliferation and apoptosis of cells in each group were detected by MTT assay and Annexin V-FITC / PI flow cytometry. The expression of FOXM1 gene in each group was detected by QPCR. The expression of FOXM1 protein was detected by Western blotting. Dual luciferase reporter assay was used to validate the targeting effect between miR-149 and FOXM1. Results The relative expression of miR-149 in miR-149 transfected group was 2.493 ± 0.380 after 48 h transfection, which was higher than 1.077 ± 0.321 in untransfected group and 1.283 ± 0.273 in miR-149 control group (16.51 ± 2.49)%, (22.90 ± 3.65)% and (31.43 ± 5.27)%, respectively, in miR-149 transfected group at 24, 48, (P <0.05). The apoptosis rate of miR-149 transfected group was (29.17 ± 4.48)%, which was higher than that of untransfected group (5.34 ± 1.72)%, and (7.62 ± 1.59)% in miR-149 control group (P <0.05). The FOXM1 mRNA and protein levels in miR-149 transfected group were 0.624 ± 0.102 and 0.349 ± 0.065, respectively, which were all lower than 0.976 ± 0.076 and 0.654 ± 0.074 in untransfected group and 0.920 ± 0.117 and 0.718 ± 0.077 in miR-149 control group respectively (P <0.05) Luciferase reporter assay showed that miR-149 significantly inhibited the luciferase activity of wild-type FOXM1-3’UTR and had no effect on the luciferase activity of transfected cells. Conclusion miR-149 may be an effective target for molecular therapy of NSCLC by targeting FOXM1 to regulate the proliferation and apoptosis of lung cancer A549 cells.