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中国癌症防治杂志

中国癌症防治杂志 ›› 2023, Vol. 15 ›› Issue (2): 156-162.doi: 10.3969/j.issn.1674-5671.2023.02.07

• 基础研究 • 上一篇    下一篇

miR⁃93⁃5p靶向PKMYT1对肺腺癌A549细胞增殖、迁移及侵袭的影响

  

  1. 青海省第五人民医院肿瘤内科
  • 出版日期:2023-04-25 发布日期:2023-05-05
  • 通讯作者: 马海军 E?mail：niko3143@163.com
  • 基金资助:
    青海省2017年基础研究计划项目（2017?ZJ?707）

Effect of miR-93-5p targeting PKMYT1 on the proliferation,migration and invasion of lung adenocarcinoma A549 cells

  • Online:2023-04-25 Published:2023-05-05

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摘要： 目的 探讨miR⁃93⁃5p靶向蛋白激酶膜相关酪氨酸/苏氨酸1（protein kinase membrane associated tyrosine/threonine 1，PKMYT1）对肺腺癌细胞增殖、迁移及侵袭的影响及其作用机制。方法 使用LipofectamineTM 2000分别将质粒inhibitor NC、miR⁃93⁃5p inhibitor、pcDNA、pc⁃PKMYT1转染至A549细胞中，记为inhibitor NC组、miR⁃93⁃5p inhibitor组、pcDNA组和pc⁃PKMYT1组，同时将未转染的细胞记为control组。利用qRT⁃PCR检测细胞中miR⁃93⁃5p、PKMYT1 mRNA的表达水平。采用双荧光素酶报告基因实验验证miR⁃93⁃5p与PKMYT1的靶向关系；CCK⁃8法检测细胞增殖能力；流式细胞术检测细胞周期情况；Transwell实验检测细胞迁移和侵袭能力；Western blot法检测细胞中PKMYT1、细胞周期素D1（Cyclin D1）、细胞周期素B1（Cyclin B1）的蛋白表达水平。结果 与BEAS⁃2B细胞相比，SPC⁃A⁃1细胞、A549细胞、LTEP⁃α⁃2细胞中miR⁃93⁃5p表达水平均升高（均P<0.01），PKMYT1 mRNA和蛋白表达水平均降低（均P<0.001）。转染24 h后，分别与control组、inhibitor NC组相比，miR⁃93⁃5p inhibitor组A549细胞增殖活性、S期细胞比例、迁移细胞数目、侵袭细胞数目、Cyclin D1和Cyclin B1蛋白水平均降低（均P<0.05），G2/M期细胞比例升高（P<0.001）。双荧光素酶报告基因实验结果显示，与miR⁃93⁃5p NC+3'UTR⁃WT组相比，miR⁃93⁃5p mimic+3'UTR⁃WT组细胞相对荧光素酶活性下降（P<0.001）。转染24 h后，与pcDNA组相比，pc⁃PKMYT1组A549细胞增殖活性、S期细胞比例、迁移细胞数目、侵袭细胞数目、Cyclin D1和Cyclin B1蛋白水平均降低（均P<0.001），G2/M期细胞比例升高（P<0.001）。结论 沉默miR⁃93⁃5p可能通过上调PKMYT1表达水平抑制肺腺癌细胞增殖、迁移及侵袭。

关键词:  , 肺腺癌；miR?93?5p；蛋白激酶膜相关酪氨酸/苏氨酸1；增殖；迁移；侵袭

Abstract: Objective To investigate the effect of miR⁃93⁃5p targeting protein kinase membrane associated tyrosine/threonine 1 (PKMYT1) on the proliferation, migration and invasion of lung adenocarcinoma cells and its mechanism. Methods Plasmids inhibitor NC, miR⁃93⁃5p inhibitor, pcDNA, and pc⁃PKMYT1 were transfected into A549 cells by using LipofectamineTM 2000 and recorded as inhibitor NC group, miR⁃93⁃5p inhibitor group, pcDNA group, and pc⁃PKMYT1 group, respectively, while the untransfected cells were recorded as the control group. The expression level of miR⁃93⁃5p and PKMYT1 mRNA were detected by qRT⁃PCR. The targeting relationship between miR⁃93⁃5p and PKMYT1 was verified by using the double luciferase reporter gene assay. The cell proliferation ability was detected by CCK⁃8; the cell cycle was measured by the flow cytometry; the cell migration and invasion were detected by Transwell assay; and the expression levels of PKMYT1, Cyclin D1 and Cyclin B1 were detected by Western blot. Results Compared with BEAS⁃2B cells, the expression levels of miR⁃93⁃5p were increased in SPC⁃A⁃1 cells, A549 cells and LTEP⁃α⁃2 cells (all P<0.01), while the expressions of PKMYT1 mRNA and protein were decreased (all P<0.001). After 24 h transfection, compared with those of the control group and the inhibitor NC group, the proliferative activity of A549 cells, the proportion of S phase cells, the numbers of migrating cells, the numbers of invading cells and the protein levels of Cyclin D1 and Cyclin B1 in the miR⁃93⁃5p inhibitor group were decreased (all P<0.05), while the proportion of G2/M phase cells was increased (P<0.001). Double luciferase report gene assay showed that compared with the miR⁃93⁃5p NC+ 3'UTR⁃WT group, the relative activity of luciferase in the miR⁃93⁃5p mimic + 3'UTR⁃WT group was decreased (P<0.001). After 24 h transfection, compared with the pcDNA group, the proliferative activity of A549 cells, the proportion of S phase cells, the number of migrating cells, the number of invading cells and the protein levels of Cyclin D1 and Cyclin B1 in the pc⁃PKMYT1 group were decreased (all P<0.001), while the proportion of G2/M phase cells was increased (P<0.001). Conclusions Silence of miR⁃93⁃5p may inhibit the proliferation, migration and invasion of lung adenocarcinoma cells by up⁃regulating the PKMYT1 expression.

Key words: Lung adenocarcinoma, miR?93?5p, Protein kinase membrane associated tyrosine/threonine 1, Proliferation, Migration, Invasion

中图分类号: 

  • R734.2