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中华细胞与干细胞杂志(电子版)

中华细胞与干细胞杂志(电子版) ›› 2020, Vol. 10 ›› Issue (04) : 204 -212. doi: 10.3877/cma.j.issn.2095-1221.2020.04.002

所属专题： 文献；

论著

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
龚年金1, 李光才1,†(), 张明华1, 刘培俊1, 向悦华1   
  1. 1. 445000　恩施，湖北省恩施土家族苗族自治州中心医院呼吸内科
  • 收稿日期:2019-10-17 出版日期:2020-08-01
  • 通信作者: 李光才

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

Nianjin Gong1, Guangcai Li1,†(), Minghua Zhang1, Peijun Liu1, Yuehua Xiang1   

  1. 1. Department of Respiratory Medicine, Enshi Tujia and Miao Autonomous Prefecture Central Hospital, Enshi 445000, China
  • Received:2019-10-17 Published:2020-08-01
  • Corresponding author: Guangcai Li
  • About author:
    Corresponding author:Li Guangcai, Email:
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引用本文：

龚年金, 李光才, 张明华, 刘培俊, 向悦华. 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[J]. 中华细胞与干细胞杂志(电子版), 2020, 10(04): 204-212.

Nianjin Gong, Guangcai Li, Minghua Zhang, Peijun Liu, Yuehua Xiang. 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[J]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2020, 10(04): 204-212.

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。

关键词: 肺癌, A549细胞, MAGI2-AS3, MiR-31-5p, 增殖, 迁移, 侵袭, 凋亡
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.

Key words: Lung cancer, A549 cell, MAGI2-AS3, miR-31-5p, Proliferation, Migration, Invasion, Apoptosis
表1 引物序列信息
基因 序列（5'-3'） 扩增长度（bp）
MAGI2-AS3 上游CACCTTGCTTGACAACTTGA 121
? 下游CATTACAGCTCGGCTACTGC ?
miR-31-5p 上游GCGCAGGCAAGATGCTGG 276
? 下游GTGCAGGGTCCGAGGT ?
U6 上游CTCGCTTCGGCAGCACA 94
? 下游AACGCTTCACGAATTTGCGT ?
GAPDH 上游AATGGGCAGCCGTTAGGAAA 168
? 下游GCGCCCAATACGACCAAATC ?
表2 MAGI2-AS3和miR-31-5p在细胞A549和HBE中的表达（±s，n = 3）
分组 MAGI2-AS3 miR-31-5p
HBE组 1.29±0.06 0.25±0.02
A549组 0.48±0.03 1.01±0.05
t值 20.914 24.444
P值 < 0.001 < 0.001
图2 光学显微镜下观察过表达MAGI2-AS3对细胞A549迁移、侵袭细胞数的影响（结晶紫染色，×200）。pcDNA3.1-MAGI2-AS3组（2b图）细胞迁移数目较pcDNA3.1组（2a图）减少；pcDNA3.1-MAGI2-AS3组（2d图）细胞侵袭数目录较pcDNA3.1组（2c图）减少
表3 过表达MAGI2-AS3对细胞A549增殖凋亡的影响（±s，n = 3）
分组 MAGI2-AS3 OD值 凋亡率（﹪）
24 h 48 h 72 h
pcDNA3.1 0.42±0.03 0.46±0.03 0.77±0.06 1.03±0.08 7.23±0.51
pcDNA3.1-MAGI2-AS3 0.85±0.07 0.41±0.03 0.48±0.04 0.68±0.05 19.95±1.25
t值 9.779 2.041 6.966 6.426 16.319
P值 < 0.001 0.111 0.002 0.003 < 0.001
表4 过表达MAGI2-AS3对细胞A549细胞周期的影响（﹪，±s，n = 3）
分组 G0-G1 S G2-M
pcDNA3.1 33.56±1.23 32.95±1.06 33.49±1.61
pcDNA3.1-MAGI2-AS3 43.58±2.15 23.01±1.00 33.41±1.60
t值 7.007 11.814 0.061
P值 0.002 < 0.001 0.954
表5 过表达MAGI2-AS3对细胞A549迁移、侵袭的影响（个，±s，n = 3）
分组 迁移细胞数 侵袭细胞数
pcDNA3.1 124.33±3.09 53.00±3.27
pcDNA3.1-MAGI2-AS3 81.33±2.87 32.00±2.83
t值 17.660 8.411
P值 < 0.001 0.001
图4 光学显微镜下观察抑制miR-31-5p对A549细胞迁移和侵袭的影响（结晶紫染色，×200）。anti-miR-31-5p组（4b图）细胞迁移数目较anti-miR-NC组（4a图）减少；anti-miR-31-5p组（4d图）细胞侵袭数目较anti-miR-NC组（4c图）减少
表6 抑制miR-31-5p对细胞A549增殖、凋亡的影响（±s，n = 3）
分组 miR-31-5p ? OD值 ? 凋亡率（﹪）
24 h 48 h 72 h
anti-miR-NC组 1.02±0.06 0.47±0.03 0.78±0.06 1.05±0.08 7.29±0.51
anti-miR-31-5p组 0.48±0.03 0.42±0.03 0.53±0.04 0.76±0.06 18.21±1.24
t值 13.943 2.041 6.005 5.023 14.107
P值 < 0.001 0.111 0.004 0.007 < 0.001
表7 抑制miR-31-5p对细胞A549迁移、侵袭及细胞周期的影响（±s，n = 3）
分组 G0-G1 （﹪） S （﹪） G2-M （﹪） 迁移细胞数（个） 侵袭细胞数（个）
anti-miR-NC组 33.53±1.27 32.91±1.08 33.56±1.68 108.33±2.87 42.33±2.05
anti-miR-31-5p组 41.56±2.19 24.43±1.13 34.01±1.23 76.00±3.74 30.00±1.63
t值 5.494 9.397 0.374 11.878 8.154
P值 0.005 0.001 0.727 < 0.001 0.001
图5 MAGI2-AS3靶向miR-31-5p
表8 双荧光素酶报告实验（±s，n = 3）
分组 WT-MAGI2-AS3 MUT-MAGI2-AS3
miR-NC组 1.01±0.06 1.01±0.05
miR-31-5p组 0.23±0.02 1.00±0.05
t值 21.361 0.245
P值 <0.001 0.819
图6 流式细胞仪检测过表达miR-31-5p能逆转MAGI2-AS3对细胞A549的凋亡影响
图7 光学显微镜下观察过表达miR-31-5p能逆转MAGI2-AS3对细胞A549迁移、侵袭的影响（结晶紫染色，×200）
表9 过表达miR-31-5p能逆转lncRNA MAGI2-AS3对细胞A549的凋亡、增殖及迁移、侵袭的影响（±s，n = 3）
分组 miR-31-5p OD值 凋亡率（﹪） 迁移细胞数 侵袭细胞数
24 h 48 h 72 h
pcDNA3.1组 1.02±0.08 0.47±0.04 0.76±0.07 1.04±0.09 7.23±0.51 125.00±2.16 81.67±2.49
pcDNA3.1-MAGI2-AS3组 0.34±0.02a 0.42±0.04 0.49±0.04a 0.69±0.05a 20.59±1.11a 53.33±2.87a 32.33±2.35a
pcDNA3.1-MAGI2-AS3+miR-NC组 0.35±0.02a 0.43±0.04 0.50±0.04a 0.71±0.05a 21.11±1.14a 52.67±2.62a 31.67±4.03a
pcDNA3.1-MAGI2-AS3+miR-31-5p组 0.78±0.06abc 0.44±0.04 0.68±0.06abc 0.95±0.07abc 10.59±1.01abc 91.00±1.63abc 62.67±2.49abc
F值 558.665 2.625 55.256 60.850 155.664 642.749 207.998
P值 < 0.001 0.067 < 0.001 < 0.001 < 0.001 < 0.001 < 0.001
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