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中华细胞与干细胞杂志(电子版) ›› 2023, Vol. 13 ›› Issue (06) : 346 -354. doi: 10.3877/cma.j.issn.2095-1221.2023.06.004

论著

miR-340-5p调控PDE4D表达对胃癌细胞顺铂耐药性的影响
吴亚萍1, 吴颖1,()   
  1. 1. 226500 南通,江苏南通大学附属如皋医院普外科
  • 收稿日期:2024-01-18 出版日期:2023-12-01
  • 通信作者: 吴颖

Effect of miR-340-5p regulating PDE4D expression on cisplatin resistance of gastric cancer cells

Yaping Wu1, Ying Wu1,()   

  1. 1. Department of General Surgery, Nantong University Affiliated Rugao Hospital, Nantong, Jiangsu 226500, China
  • Received:2024-01-18 Published:2023-12-01
  • Corresponding author: Ying Wu
引用本文:

吴亚萍, 吴颖. miR-340-5p调控PDE4D表达对胃癌细胞顺铂耐药性的影响[J/OL]. 中华细胞与干细胞杂志(电子版), 2023, 13(06): 346-354.

Yaping Wu, Ying Wu. Effect of miR-340-5p regulating PDE4D expression on cisplatin resistance of gastric cancer cells[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2023, 13(06): 346-354.

目的

探讨miR-340-5p调控磷酸二酯酶4D(PDE4D)表达对胃癌细胞顺铂(DDP)耐药性的影响。

方法

将胃癌细胞AGS连续暴露于0.1 ~ 1 μg/mL DDP中来构建耐DDP的胃癌细胞系AGS/DDP,1、3、5、7 μg/mL DDP处理AGS、AGS/DDP细胞,CCK-8检测细胞活力及IC50;qRT-PCR检测miR-340-5p表达量;Western blot检测PDE4D蛋白表达水平;验证miR-340-5p与PDE4D靶向关系;分别将miR-340-5p mimics、siRNA PDE4D转染于AGS/DDP细胞,miR-340-5p mimics和pcDNA-PDE4D共转染于AGS/DDP细胞,将转染成功的AGS/DDP细胞以及未转染AGS/DDP细胞分别用5 μg/mL DDP处理,观察AGS/DDP细胞增殖、迁移和侵袭变化。两组间比较采用独立样本t检验,多组间比较采用单因素方差分析和SNK-q检验。

结果

与AGS细胞相比,AGS/DDP细胞在1、3、5、7 μg/mL DDP处理时的细胞活力升高,IC50值[(5.89 ± 0.27)比(1.46 ± 0.11)μg/mL]升高;与AGS细胞相比,AGS/ DDP细胞中miR- 340- 5p的表达(0.34 ± 0.17比1.00 ± 0.00)降低,而PDE4D蛋白水平(1.83 ± 0.11比0.25 ± 0.01)升高;PDE4D为miR-340-5p靶基因;过表达miR-340-5p或敲低PDE4D可抑制AGS/ DDP细胞增殖、迁移[(54.17 ± 4.12)比(26.00 ± 2.28)个、(55.00 ± 4.43)比(24.50 ± 2.07)个]与侵袭[(37.17 ± 3.19)比(19.33 ± 1.63)个、(38.33 ± 3.14)比(16.33 ± 1.63)个];上调PDE4D表达可逆转过表达miR-340-5p对AGS/DDP细胞增殖、迁移和侵袭的抑制作用(P均< 0.05)。

结论

miR-340- 5p通过靶向下调PDE4D表达抑制AGS/DDP细胞增殖、迁移和侵袭,降低其对DDP的耐药性。

Objective

To investigate the effect of miR-340-5p regulating the expression of cAMPspecific 3', 5'-cyclic phosphodiesterase 4D (PDE4D) on the drug resistance of gastric cancer cells to cisplatin (DDP) .

Methods

The gastric cancer cell line AGS was continuously exposed to 0.1-1 μg/mL DDP to construct a DDP-resistant gastric cancer cell line named AGS/DDP. AGS and AGS/DDP cells were treated with 1, 3, 5, 7 μg/mL DDP, and cell viability and IC50 were detected by CCK-8; qRT-PCR was used to detect the expression of miR-340-5p; and Western blot was used to detect the PDE4D protein expression level; the targeting relationship between miR- 340- 5p and PDE4D was verified. At the same time, the miR-340-5p mimics and siRNA PDE4D were transfected into AGS/DDP cells respectively. The miR-340-5p mimics and pcDNA-PDE4D were co-transfected into AGS/DDP cells, the successfully transfected AGS/DDP cells and untransfected AGS/DDP cells were treated with 5 μg/mL DDP, and the changes of AGS/DDP cell proliferation, migration and invasion were observed. Independent sample t-test was used for comparison between two groups, and univariate analysis of variance and SNK-q test were used for comparison between multiple groups.

Results

Compared with AGS cells, the cell viability of AGS/DDP cells was increased when treated with 1, 3, 5, and 7 μg/mL DDP, and the IC50 value was significantly increased [ (5.89 ± 0.27) μg/ mL vs (1.46 ± 0.11) μg/mL, P < 0.05]; compared with AGS cells, the expression of miR-340-5p in AGS/ DDP cells was decreased (0.34 ± 0.17 vs 1.00 ± 0.00, P < 0.05), while the level of PDE4D protein was significantly increased (1.83 ± 0.11 vs 0.25 ± 0.01, P < 0.05) ; PDE4D was the miR- 340- 5p target gene; overexpression of miR-340-5p or knockdown of PDE4D inhibited proliferation, migration [ (54.17 ± 4.12) vs (26.00 ± 2.28), (55.00 ± 4.43) vs (24.50 ± 2.07) ] and invasion [ (37.17 ± 3.19) vs (19.33 ± 1.63), (38.33 ± 3.14) vs (16.33 ± 1.63) ] of AGS/DDP cells (P < 0.05) ; upregulation of PDE4D expression reversed the inhibitory effect of overexpression of miR-340-5p on proliferation, migration, and invasion of AGS/DDP cells (P < 0.05) .

Conclusion

MiR-340-5p inhibits the proliferation, migration and invasion of AGS/DDP cells by targeting down-regulation of PDE4D expression to reduce their resistance to DDP.

表1 qRT-PCR引物序列
表2 DDP对AGS、AGS/DDP细胞活力的影响(±s
表3 miR-340-5p和PDE4D在胃癌细胞AGS和DDP耐药细胞AGS/DDP中表达(±s
图1 PDE4D在AGS和AGS/DDP细胞中表达水平
图2 预测的miR-340-5p与PDE4D的结合位点
表4 Western blot检测上调miR-340-5p后PDE4D蛋白表达水平变化(±s
图3 上调miR-340-5p表达后AGS和AGS/DDP细胞中PDE4D蛋白表达情况
表6 qRT-PCR检测转染miR-340-5p mimic后AGS/DDP细胞中miR-340-5p和PDE4D mRNA表达变化(±s
表7 上调miR-340-5p表达联合DDP对AGS/DDP细胞增殖的影响(±s
表8 上调miR-340-5p表达联合DDP对AGS/DDP细胞迁移与侵袭的影响(个,±s
图4 光学显微镜下观察上调miR-340-5p表达联合DDP对AGS/DDP细胞迁移和侵袭的影响(结晶紫染色,×200)注:与DDP和转染miR-NC联合DDP干预的细胞比较,上调miR-340-5p表达联合DDP干预的AGS/DDP细胞迁移与侵袭的细胞数降低
表9 下调PDE4D表达联合DDP对AGS/DDP细胞增殖的影响(±s
表10 下调PDE4D表达联合DDP对AGS/DDP细胞迁移与侵袭的影响(个,±s
图5 Western blot检测转染siRNA PDE4D后AGS/DDP细胞中PDE4D表达情况
图6 光学显微镜下观察下调PDE4D表达联合DDP对AGS/DDP细胞迁移与侵袭的影响(结晶紫染色,×200)注:与DDP和转染siRNA-NC联合DDP干预的细胞比较,下调PDE4D表达联合DDP干预的AGS/DDP细胞迁移和侵袭能力降低
表11 上调PDE4D表达逆转过表达miR-340-5p对AGS/DDP细胞增殖的影响
表12 上调PDE4D表达逆转过表达miR-340-5p对AGS/DDP细胞迁移与侵袭的影响(个,±s
图7 光学显微镜下观察上调PDE4D表达逆转过表达miR-340-5p对AGS/DDP细胞迁移与侵袭的影响(结晶紫染色,×200)注:与DDP和miR-NC+DDP干预比较,过表达miR-340-5p联合DDP干预的AGS/DDP细胞迁移与侵袭的细胞数降低;上调miR-340-5p和PDE4D表达联合DDP干预的AGS/DDP细胞迁移和侵袭能力高于过表达miR-340-5p联合DDP干预和转染miR-340-5p mimics+pcDNA-NC联合DDP干预的细胞
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