lncRNA GPRC5D-AS1对地塞米松诱导小鼠成肌细胞肌萎缩的抵抗和再生作用及其机制

doi:10.13481/j.1671-587X.20230608

Abstract

Abstract:

Objective To discuss the resistance and regeneration effects of long non-coding RNA （lncRNA） GPRC5D-AS1 on the muscle atrophy of myocytes in the mice induced by dexamethasone， and to clarify its mechanism. Methods The C2C12 cells at logarithmic growth phase were selected. The survival rates of the cells after treated with 0， 25， 50， 75， 100， 200 and 400 mg·L^－1 dexamethasone for 24， 48 and 72 h were detected， which was used to select the optimal dose and time to induce the muscle atrophy model.The expression level of lncRNA GPRC5D-AS1 in the C2C12 cells was detected by real-time fluroscence quantitative PCR（RT-qPCR） method to validate the cell model. The C2C12 cells were divided into normal group， control group，lncRNA GPRC5D-AS1-NC group，and lncRNA GPRC5D-AS1-OE group. The expression levels of lncRNA GPRC5D-AS1 in the cells in various groups were detected by RT-qPCR method. The levels of reactive oxygen species （ROS）， glutathione peroxidase 4 （GPX4）， ferrous ion （Fe²⁺） and malondialdehyde （MDA） in the cells in various groups were detected by flow cytometry and enzyme-linked immunosorbent assay （ELISA） method； the ultrastructural changes of mitochondria in the cells in various groups were observed under transmission electron microscope； the expression of myogenic differentiation protein （MyoD） in the cells in various groups was detected by immunofluorescence； the expression levels of ferroptosis-related proteins in the cells in various groups were detected by Western blotting method. Results The best modeling effect was achieved with 100 mg·L^－1 dexamethasone at 48 h. Compared with normal C2C12 cells， the expression level of lncRNA GPRC5D-AS1 in the C2C12 cells after treated with 100 mg·L^－1 dexamethasone for 48 h was decreased （P<0.05）， confirming that lncRNA GPRC5D-AS1 had a regulatory role in the muscle atrophy model. The RT-qPCR results showed that compared with normal group， the expression level of lncRNA GRPC5D-AS1 in the cells in model group was decreased （P<0.05）； compared with model group and lncRNA GRPC5D-AS1-NC group， the expression level of lncRNA GRPC5D-AS1 in the cells in lncRNA GRPC5D-AS1-OE group was increased （P<0.05）. The flow cytometry results showd that compared with normal group， the ROS level in the cells in model group was increased （P<0.05）； compared with model group and lncRNA GRPC5D-AS1-NC group， the ROS level in the cells in lncRNA GRPC5D-AS1-OE group was decreased （P<0.05）. The ELISA results showed that compared with normal group， the levels of Fe²⁺ and MDA in the cells in model group were increased （P<0.05）， and the level of GPX4 was decreased （P<0.05）； compared with model group and lncRNA GRPC5D-AS1-NC group， the levels of Fe²⁺ and MDA in the cells in lncRNA GRPC5D-AS1-OE group were decreased （P<0.05）， and the level of GPX4 was increased （P<0.05）. The transmission electron microscope results showed that the cells in normal group were round and normal in size with rich cilia on the membrane， and the mitochondria had a normal morphology； the cells in model group had less mitochondria and granulation in the cytoplasm， with blurred and swollen mitochondrial structures and typical ferroptosis-related manifestations； the number of mitochondria in the cells in lncRNA GRPC5D-AS1-OE group was increased，and the granulation in cytoplasm of the cells was decreased， and showed clearer mitochondrial structures. The immunofluorescence detection results showed that compared with normal group，the expression of MyoD in the cells in model group was decreased and the cells exhibited muscle atrophy； compared with model group and lncRNA GRPC5D-AS1-NC group， the expression of MyoD in the cells in lncRNA GRPC5D-AS1-OE group was increased， and the cells exhibited less muscle atrophy and more regeneration of myocytes. The Western blotting results revealed that compared with the normal group， the expression level of long-chain acyl-coenzyme A synthase （ACSL）4 protein in the cell in model group was increased （P<0.05）， and the expression levels of solute carrier family 7 member 11 （SLC7A11） and GPX4 proteins were decreased （P<0.05）； compared with model group and lncRNA GRPC5D-AS1-NC group， the expression level of ACSL4 protein in the cells in lncRNA GRPC5D-AS1-OE group was decreased （P<0.05）， and the expression levels of SLC7A11 and GPX4 proteins were increased （P<0.05）. Conclusion LncRNA GPRC5D-AS1 can protect the ferroptosis in the myocytes induced by dexamethasone and promote the repairment and regeneration of the cells，and its mechanism is related to regulating the SLC7A11/GPX4/ACSL4 signaling pathway.

Key words: Muscular dystrophy, Long non-coding RNA, GPRC5D-AS1, Ferroptosis, Dexamethasone, Myocytes

CLC Number:

R685

Ruipeng ZHANG,Jie LI. Resistance and regeneration effects of lncRNA GPRC5D-AS1 on muscle atrophy of myocytes in mice induced by dexamethasone and its mechanism[J].Journal of Jilin University(Medicine Edition), 2023, 49(6): 1457-1465.

Figures/Tables 9

Tab.1

Tab. 2

Survival rates of C2C12 cells after treated with different doses of dexamethasone"

Group	A value	Survival rate(η/%)
Dexamethosone[ρ_B/(mg·L^－1)]
0	1.000 $±$ 0.049	100.0
25	0.807 $±$ 0.013	80.7
50	0.771 $±$ 0.021	77.1
75	0.731 $±$ 0.018	73.1
100	0.623 $±$ 0.039	62.3
200	0.219 $±$ 0.023	21.9
400	0.136 $±$ 0.031	13.6

Tab. 2

Tab. 3

Survival rates of C2C12 cells after treated for different time"

Group	A value	Survival rate(η/%)
Induction time(t/h)
0	1.000 $±$ 0.071	100.0
24	0.742 $±$ 0.018	74.2
48	0.637 $±$ 0.042	63.7
72	0.281 $±$ 0.064	28.1

Tab. 3

Fig. 1

Fig. 2

Tab. 4

Levels of GPX4、Fe2+ and MDA in cells in various groups [n=3，x±s，ρB/（ng·L－1）］"

Group	GPX4	Fe²⁺	MDA
Normal	303.749 $±$ 27.244	0.572 $±$ 0.006	21.057 $±$ 0.829
Model	158.745 $±$ 11.129^*	0.837 $±$ 0.034^*	37.135 $±$ 0.749^*
LncRNA GPRC5D-AS1-NC	147.371 $±$ 22.815	0.863 $±$ 0.029	36.493 $±$ 0.681
LncRNA GPRC5D-AS1-OE	233.042 $±$ 34.649^△#	0.748 $±$ 0.019^△#	26.565 $±$ 1.124^△#

Tab. 4

Fig. 3

Fig. 4

Fig. 5

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Gene	Primer sequence
GAPDH	F：5'-ATTCAACGGCACAGTCAAGG-3'
GAPDH	R：5'-GCAGAAGGGGCGGAGATGA-3'
lncRNA GRPC5D- AS1	F：5'-TGGGACAGAACTGGAGTCAGA-3'
lncRNA GRPC5D- AS1	R：5'-CCAGCCTGGTTGAATGAAACG-3'

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Resistance and regeneration effects of lncRNA GPRC5D-AS1 on muscle atrophy of myocytes in mice induced by dexamethasone and its mechanism

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