Impact of LncRNA TUG1 on high glucose-induced cardiomyocyte apoptosis by regulating the miR-181b-5p/PDCD4 axis

doi:10.11958/20230523

Abstract

Abstract:

Objective To investigate the impact of long non-coding RNA (LncRNA) taurine up-regulated gene 1 (TUG1) on high glucose-induced cardiomyocyte apoptosis by regulating miR-181b-5p/programmed cell death protein 4 (PDCD4) axis. Methods Diabetic cardiomyopathy (DCM) cell model was established in vitro with high glucose (HG,25 mmol/L glucose). AC16 cells were divided into the NG (5.5 mmol/L glucose) group, the HG group, the HG+sh-NC group, the HG+sh-TUG1 group, the HG+miR-NC group, the HG+miR-181b-5p group, the HG+sh-TUG1+anti-miR-NC group, the HG+sh-TUG1+anti-miR-181b-5p group, the HG+miR-181b-5p+pcDNA group and HG+miR-181b-5p+pc-PDCD4 group. The Cell Counting Kit-8 (CCK-8) method was applied to detect cell viability. Lactate dehydrogenase (LDH) assay was applied to detect LDH release. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect expression levels of TUG1, miR-181b-5p and PDCD4 mRNA. Flow cytometry was applied to detect apoptosis. Western blot assay was applied to detect levels of B-cell lymphoma 2-associated X (Bax), activated caspase 3 (cleaved caspase 3) and PDCD4 proteins. Caspase-Glo3 assay was applied to assess caspase 3 activity. Dual-luciferase reporter assay was applied to verify the targeting relationship between TUG1 or PDCD4 and miR-181b-5p. Results Compared with the NG group, the cell activity decreased in the HG group, and LDH release, apoptosis rate, Bax, cleaved caspase 3 expression and caspase 3 activity increased (P<0.05), which could be antagonized by TUG1 knockdown or miR-181b-5p overexpression (P<0.05). Inhibition of miR-181b-5p was able to alleviate the impact of TUG1 silencing on cardiomyocyte viability and apoptosis under high glucose treatment (P<0.05). The overexpression of PDCD4 attenuated the promotion effect of miR-181b-5p up-regulation on the viability of cardiomyocytes treated with high glucose and the inhibitory effect on apoptosis. TUG1 was able to increase the expression of PDCD4 through adsorption of miR-181b-5p (P<0.05). Conclusion TUG1 promotes high glucose-induced cardiomyocyte apoptosis by down-regulating miR-181b-5p and up-regulating PDCD4.

Key words: taurine up-regulated gene 1, miR-181b-5p, programmed cell death protein 4, high glucose, cardiomyocytes, apoptosis

CLC Number:

R542.2

Figures/Tables 17

References 20

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基因名称	引物序列（5′→3′）	产物大小/bp
TUG1	上游：TCAGCCCACCGTAACAAT	184
	下游：CAAACTTCTCGGCGTCAT	184
PDCD4	上游：AAAACTCATCCCGGGACTCT	162
	下游：CTGCACCACCTTTCTTTGGT	162
miR-181b-5p	上游：CCAGCTGGGCTCACTGAACAATGA	202
miR-181b-5p	下游：CAACTGGTGTCGTGGAGTCGGC	202
GAPDH	上游：CCAGGTGGTCTCCTCTGA	197
	下游：GCTGTAGCCAAATCGTTGT	197
U6 snRNA	上游：GCTCGCTTCGGCAGCACA	128
	下游：GAGGTATTCGCACCAGAGGA	128

基因名称	引物序列（5′→3′）	产物大小/bp
TUG1	上游：TCAGCCCACCGTAACAAT	184
	下游：CAAACTTCTCGGCGTCAT	184
PDCD4	上游：AAAACTCATCCCGGGACTCT	162
	下游：CTGCACCACCTTTCTTTGGT	162
miR-181b-5p	上游：CCAGCTGGGCTCACTGAACAATGA	202
miR-181b-5p	下游：CAACTGGTGTCGTGGAGTCGGC	202
GAPDH	上游：CCAGGTGGTCTCCTCTGA	197
	下游：GCTGTAGCCAAATCGTTGT	197
U6 snRNA	上游：GCTCGCTTCGGCAGCACA	128
	下游：GAGGTATTCGCACCAGAGGA	128

组别		细胞活力/%	LDH释放总量/%	TUG1	miR-181b- 5p
NG组		100.00±10.03	100.00±1.47	1.00±0.08	1.00±0.11
HG组	12 h	81.23±9.24^a	126.34±3.95^a	1.35±0.11^a	0.76±0.09^a
	24 h	60.45±5.16^a	176.51±5.87^a	2.16±0.18^a	0.51±0.06^a
	48 h	38.72±2.35^a	228.63±7.68^a	3.31±0.32^a	0.37±0.03^a
F		76.862^＊	697.645^＊	164.702^＊	75.206^＊

组别		细胞活力/%	LDH释放总量/%	TUG1	miR-181b- 5p
NG组		100.00±10.03	100.00±1.47	1.00±0.08	1.00±0.11
HG组	12 h	81.23±9.24^a	126.34±3.95^a	1.35±0.11^a	0.76±0.09^a
	24 h	60.45±5.16^a	176.51±5.87^a	2.16±0.18^a	0.51±0.06^a
	48 h	38.72±2.35^a	228.63±7.68^a	3.31±0.32^a	0.37±0.03^a
F		76.862^＊	697.645^＊	164.702^＊	75.206^＊

组别	TUG1	miR-181b-5p	细胞活力/%	LDH释放总量/%
NG组	1.00±0.05	1.00±0.05	100.00±9.42	10.03±0.05
HG组	3.46±0.37^a	0.31±0.02^a	42.31±2.36^a	59.43±3.62^a
HG+sh-NC组	3.42±0.35	0.33±0.03	42.28±2.34	58.27±3.71
HG+sh-TUG1组	1.23±0.11^bc	0.74±0.04^bc	81.25±5.47^bc	22.34±1.52^bc
HG+miR-NC组	3.44±0.36	0.32±0.02	43.12±2.41	60.13±3.84
HG+miR-181b-5p组	1.38±0.14^bd	2.89±0.31^bd	80.42±4.65^bd	27.52±1.68^bd
F	128.928^＊	353.223^＊	148.302^＊	382.182^＊