lncRNA MAGI2-AS3下调miR-31-5p抑制肺腺癌细胞的增殖、迁移、侵袭并促进凋亡

Effects of lncRNA MAGI2-AS3 targeting miR-31-5p on the inhibition of proliferation, migration,invasion and promotion of apoptosis of lung adenocarcinoma cancer cells

目的研究lncRNA MAGI2-AS3对肺癌A549细胞增殖、迁移、侵袭和凋亡的影响和潜在的分子机制。方法根据转染载体不同将A549细胞分为pcDNA3.1组(转染pcDNA3.1)、pcDNA3.1-MAGI2-AS3组(转染pcDNA3.1-MAGI2-AS3)、anti-miR-NC组(转染anti-miR-NC)、anti-miR-31-5p组(转染anti-miR-31-5p)、pcDNA3.1-MAGI2-AS3+miR-NC组(共转染pcDNA3.1-MAGI2-AS3和miR-NC)、pcDNA3.1-MAGI2-AS3+miR-31-5p组(共转染pcDNA3.1-MAGI2-AS3和miR-31-5p mimics)。实时荧光定量PCR(qRT-PCR)检测miR-31-5p和MAGI2-AS3 RNA的表达,四氮唑蓝(MTT)法测定A549细胞增殖活性,Transwell实验检测细胞迁移和侵袭能力,双荧光素酶报告系统验证MAGI2-AS3与miR-31-5p的调控关系,流式细胞术检测细胞凋亡与周期。两组间比较采用独立样本t检验进行分析;多组间比较采用单因素方差分析,组内多重比较采用SNK-q检验。结果与人正常肺细胞HBE相比,肺癌细胞A549中的MAGI2-AS3表达量(0.48±0.03比1.29±0.06)降低,miR-31-5p表达量(1.01±0.05比0.25±0.02)升高;与pcDNA3.1组比较,pcDNA3.1-MAGI2-AS3组A549细胞活力(0.48±0.04比0.77±0.06)、迁移[(81.33±2.87)个比(124.33±3.09)个]和侵袭[(32.00±2.83)个比(53.00±3.27)个]细胞数、S期细胞所占比例(23.01﹪±1.00﹪比32.95﹪±1.06﹪)均降低,凋亡率(19.95﹪±1.25﹪比7.23﹪±0.51﹪)、G0-G1期细胞所占比例(43.58﹪±2.15﹪比33.56﹪±1.23﹪)均升高;与anti-miR-NC组比较,anti-miR-31-5p组A549细胞活力(0.53±0.04比0.78±0.06)、迁移[(76.00±3.74)个比(108.33±2.87)个]和侵袭[(30.00±1.63)个比(42.33±2.05)个]细胞数、S期细胞所占比例(24.43﹪±1.13﹪比32.91﹪±1.08﹪)降低,凋亡率(18.21﹪±1.24﹪比7.29﹪±0.51﹪)、G0-G1期细胞所占比例(41.56﹪±2.19﹪比33.53﹪±1.27﹪)升高,差异有统计学意义(P均<0.05);双荧光素酶报告系统结果显示,MAGI2-AS3靶向负调控miR-31-5p的表达。与pcDNA3.1-MAGI2-AS3+miR-NC组比较,pcDNA3.1-MAGI2-AS3+miR-31-5p组A549细胞活力(0.68±0.06比0.50±0.04)、迁移[(91.00±1.63)个比(52.67±2.62)个]和侵袭[(62.67±2.49)个比(31.67±4.03个)]细胞数升高,凋亡率(10.59﹪±1.0﹪比21.11﹪±1.14﹪)降低,差异有统计学意义(P均<0.05)。结论lncRNA MAGI2-AS3通过靶向miR-31-5p抑制A549细胞的增殖、迁移和侵袭,促进细胞凋亡。lncRNA MAGI2-AS3是肺癌潜在分子治疗靶点。

Objective To investigate the effects and mechanisms of lncRNA MAGI2-AS3 on the proliferation,migration,invasion and apoptosis of lung cancer A549 cells.Methods A549 cells were divided into pcDNA3.1 group(transfected with pcDNA3.1),pcDNA3.1-MAGI2-AS3 group(transfected with pcDNA3.1-MAGI2-AS3),anti-miR-NC group(transfected with anti-miR-NC),anti-miR-31-5p group(transfected with anti-miR-31-5p),pcDNA3.1-MAGI2-AS3+miR-NC group(co-transfected with pcDNA3.1-MAGI2-AS3 and miR-NC),pcDNA3.1-MAGI2-AS3+miR-31-5p group(co-transfection with pcDNA3.1-MAGI2-AS3 and miR-31-5p mimics).The RNA expression levels of miR-31-5p and MAGI2-AS3 were detected by qRT-PCR.The proliferation rate of A549 cells was measured by MTT assay.Migration and invasion abilities of A549 cells were determined by Transwell assay.The relationship between lncRNA MAGI2-AS3 and miR-31-5p was verified by dual-luciferase reporter assay system.Apoptosis and cell cycle of A549 cells were detected by flow cytometry.The differences between groups were compared by t-test,and the differences among groups were compared by ANOVA and SNK-q test.Results Compared with normal lung cell HBE,the expression levels of MAGI2-AS3 in lung cancer cell A549 was significantly decreased(0.48±0.03 vs 1.29±0.06),and the expression levels of miR-31-5p was remarkably increased(1.01±0.05 vs 0.25±0.02).Compared with pcDNA3.1 group,the viability(0.48±0.04 vs 0.77±0.06),the cell numbers of migration(81.33±2.87 vs 124.33±3.09)and invasion(32.00±2.83 vs 53.00±3.27),proportion of S-phase cells(23.01﹪±1.00﹪vs 32.95﹪±1.06﹪)in A549 cells of pcDNA3.1-MAGI2-AS3 group were significantly reduced,while the apoptosis rate(19.95﹪±1.25﹪vs 7.23﹪±0.51﹪)and proportion of cells in G0-G1 phase(43.58﹪±2.15﹪vs 33.56﹪±1.23﹪)were significantly increased.Compared with anti-miR-NC group,the viability(0.53±0.04 vs 0.78±0.06),the cell numbers of migration(76.00±3.74 vs 108.33±2.87)and invasion(62.67±2.49 vs 31.67±4.03),proportion of cells in S phase(24.43﹪±1.13﹪vs 32.91﹪±1.08﹪)in A549 cells of anti-miR-31-5p group were significantly reduced,while the apoptosis rate(18.21﹪±1.24﹪vs 7.29﹪±0.51﹪)and proportion of cells in G0-G1 phase(41.56﹪±2.19﹪vs 33.53﹪±1.27﹪)were significantly increased,and the difference is statistically significant(all P<0.05).The results of dual-luciferase reporter assay system showed that MAGI2-AS3 targeted and negatively regulated the expression of miR-31-5p.Compared with pcDNA3.1-MAGI2-AS3+miR-NC group,the viability(0.68±0.06 vs 0.50±0.04),the cell numbers of migration(91.00±1.63 vs 52.67±2.62)and invasion(62.67±2.49 vs 31.67±4.03)in A549 cells of pcDNA3.1-MAGI2-AS3+miR-31-5p group were significantly increased,and the apoptosis rate(10.59﹪±1.01﹪vs 21.11﹪±1.14﹪)was decreased significantly,and the difference is statistically significant(all P<0.05).Conclusions LncRNA MAGI2-AS3 could inhibite the proliferation,migration and invasion of A549 cells and promoted cell apoptosis by targeting miR-31-5p.LncRNA MAGI2-AS3 is a potential molecular therapeutic target for lung cancer.