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

doi:10.3877/cma.j.issn.2095-1221.2023.06.004

Abstract

Abstract:

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.

Key words: Gastric cancer, MiR-340-5p, cAMP specific 3', 5'-cyclic phosphodiesterase 4D, cisplatin, Cell proliferation, Migration, Invasion

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

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URL: https://zhxbygxbzz.cma-cmc.com.cn/EN/10.3877/cma.j.issn.2095-1221.2023.06.004

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Figures/Tables 18

References 23

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基因	序列（5'→3'）	预期扩增长度（bp））
miR-340-5p	上游TAAAGCAATGAGACTGATTAAA	271
	下游CGGAATTCGTGGCGACCGTGATACC
U6	上游CTCGCTTCGGCAGCACA	116
	下游AACGCTTCACGAATTTGCGT
PDE4D	上游ACCATTACCATGCTGATGTGGCCT	243
	下游ACACAGCCTCCAAAGCAGGTG
β-actin	上游CTGACTTCAACAGCGACACC	89
	下游TGCTGTAGCCAAATTCGTTG

基因	序列（5'→3'）	预期扩增长度（bp））
miR-340-5p	上游TAAAGCAATGAGACTGATTAAA	271
	下游CGGAATTCGTGGCGACCGTGATACC
U6	上游CTCGCTTCGGCAGCACA	116
	下游AACGCTTCACGAATTTGCGT
PDE4D	上游ACCATTACCATGCTGATGTGGCCT	243
	下游ACACAGCCTCCAAAGCAGGTG
β-actin	上游CTGACTTCAACAGCGACACC	89
	下游TGCTGTAGCCAAATTCGTTG

分组	样本量	细胞活力（%）				IC50（μg/mL）
分组	样本量	1 μg/mL DDP	3 μg/mL DDP	5 μg/mL DDP	7 μg/mL DDP	IC50（μg/mL）
AGS	6	72.23 ± 4.12	57.65 ± 3.37	39.36 ± 2.08	14.26 ± 1.52	1.46 ± 0.11
AGS/DDP	6	89.33 ± 5.67	71.66 ± 4.28	51.39 ± 3.15	38.56 ± 2.95	5.89 ± 0.27
t值		5.980	6.307	7.824	17.926	37.610
P值		< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

分组	样本量	细胞活力（%）				IC50（μg/mL）
分组	样本量	1 μg/mL DDP	3 μg/mL DDP	5 μg/mL DDP	7 μg/mL DDP	IC50（μg/mL）
AGS	6	72.23 ± 4.12	57.65 ± 3.37	39.36 ± 2.08	14.26 ± 1.52	1.46 ± 0.11
AGS/DDP	6	89.33 ± 5.67	71.66 ± 4.28	51.39 ± 3.15	38.56 ± 2.95	5.89 ± 0.27
t值		5.980	6.307	7.824	17.926	37.610
P值		< 0.001	< 0.001	< 0.001	< 0.001	< 0.001

分组	样本量	miR-340-5p/U6	PDE4D mRNA	PDE4D/β-actin
AGS	6	1.00 ± 0.00	1.00 ± 0.00	0.25 ± 0.01
AGS/DDP	6	0.34 ± 0.17	3.56 ± 0.39	1.83 ± 0.11
t'值		9.350	16.168	36.409
P值		< 0.001	< 0.001	< 0.001

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