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, 42 (5), 397-405

Silence of LncRNA GAS5 Protects Cardiomyocytes H9c2 Against Hypoxic Injury via Sponging miR- 142- 5p

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Silence of LncRNA GAS5 Protects Cardiomyocytes H9c2 Against Hypoxic Injury via Sponging miR- 142- 5p

Jian Du et al. Mol Cells.

Abstract

The regulatory role of long noncoding RNA (lncRNA) growth arrest-specific transcript 5 (GAS5) in both cancerous and noncancerous cells have been widely reported. This study aimed to evaluate the role of lncRNA GAS5 in heart failure caused by myocardial infarction. We reported that silence of lncRNA GAS5 attenuated hypoxia-triggered cell death, as cell viability was increased and apoptosis rate was decreased. This phenomenon was coupled with the down-regulated expression of p53, Bax and cleaved caspase-3, as well as the up-regulated expression of CyclinD1, CDK4 and Bcl-2. At the meantime, the expression of four heart failure-related miRNAs was altered when lncRNA GAS5 was silenced (miR-21 and miR-142-5p were up-regulated; miR-30b and miR-93 were down-regulated). RNA immunoprecipitation assay results showed that lncRNA GAS5 worked as a molecular sponge for miR-142-5p. More interestingly, the protective actions of lncRNA GAS5 silence on hypoxia-stimulated cells were attenuated by miR-142-5p suppression. Besides, TP53INP1 was a target gene for miR-142-5p. Silence of lncRNA GAS5 promoted the activation of PI3K/AKT and MEK/ERK signaling pathways in a miR-142-5p-dependent manner. Collectively, this study demonstrated that silence of lncRNA GAS5 protected H9c2 cells against hypoxia-induced injury possibly via sponging miR-142-5p, functionally releasing TP53INP1 mRNA transcripts that are normally targeted by miR-142-5p.

Keywords: H9c2 cell; heart failure; hypoxia; lncRNA GAS5; miR-142-5p.

Figures

Fig. 1
Fig. 1. Effect of hypoxia on H9c2 cell growth
(A) Viability of H9c2 cells was detected by CCK-8 assay, after exposure of hypoxia for 0–24 h. The cells cultured in normoxic condition were used as control. Then, H9c2 cells were exposed to hypoxia for 6 h, after which (B–C) protein changes of p53, CyclinD1, and CDK4, (D) apoptosis rate, and (E–F) protein changes of apoptosis-related factors were assessed by flow cytometer and Western blot. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the normoxic control group.
Fig. 2
Fig. 2. Effect of lncRNA GAS5 silence on hypoxia-injured H9c2 cells
(A) Expression changes of lncRNA GAS5 in H9c2 cells were detected by RT-qPCR, after transfection with sh-NC or sh-GAS5. Untransfected cells served as control. Then, the transfected cells were exposed to hypoxia for 6 h, after which (B) viability, (C–D) protein expression changes of p53, CyclinD1, and CDK4, (E) apoptosis rate, and (F–G) protein expression changes of apoptosis-related factors were assessed by CCK-8 assay, flow cytometer and Western blot. Cells without any treatment were used as control. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the indicated group.
Fig. 3
Fig. 3. Effect of lncRNA GAS5 silence on miRNAs expression
(A) Expression changes of heart failure-related miRNAs (miR-378, miR-21, miR-30b, miR-93, and miR-142-5p) in H9c2 cells were detected by RT-qPCR, after transfection with sh-NC or sh-GAS5. Untransfected cells served as control. (B) Schematic representation of the miR-142-5p site in lncRNA GAS5. (C) A construct containing lncRNA GAS5 transcripts combined with MS2 elements. (D) MS2-RIP followed by qPCR to assay miR-142-5p endogenously associated with lncRNA GAS5. (E) AGO-RIP followed by qPCR to assay lncRNA GAS5 endogenously associated with miR-142-5p. (F) H9c2 cells transfected with sh-GAS5 or sh-NC were treated by hypoxia, and then the expression changes of miR-142-5p were measured by RT-qPCR. Cells without any treatment were used as control. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the indicated group.
Fig. 4
Fig. 4. Effect of miR-142-5p suppression together with lncRNA GAS5 silence on hypoxia-injured H9c2 cells
(A) Expression changes of miR-142-5p in H9c2 cells were detected by RT-qPCR, after transfection with miR-142-5p inhibitor or its negative control (NC). H9c2 cells were transfected with sh-GAS5 alone or in combination with miR-142-5p inhibitor. Then, the cells were exposed to hypoxic condition for 6 h. (B) Cell viability, (C–D) protein expression changes of p53, CyclinD1, and CDK4, (E) apoptosis rate, and (F–G) protein expression changes of apoptosis-related factors were assessed by CCK-8 assay, flow cytometer and Western blot. Cells without any treatment were used as control. *p < 0.05, **p < 0.01, ***p < 0.001 compared to the indicated group.
Fig. 5
Fig. 5. Effect of miR-142-5p suppression together with lncRNA GAS5 silence on the activation of PI3K/AKT and MEK/ERK signaling pathways
H9c2 cells were transfected with sh-GAS5 alone or in combination with miR-142-5p inhibitor. Then, the cells were exposed to hypoxic condition for 6 h. Cells without any treatment were used as control. The expression changes of core factors in (A) PI3K/AKT and (B) MEK/ERK signaling pathways were measured by Western blot. ns, no significant, *p < 0.05, **p < 0.01, ***p < 0.001 compared to the indicated group.
Fig. 6
Fig. 6. Regulatory relationship between miR-142-5p and TP53INP1
(A) Schematic representation of the TP53INP1 site in miR-142-5p. Dual-luciferase reporter assay was carried out to validate whether TP53INP1 was a target gene for miR-142-5p. (B) mRNA and (C–D) protein levels of TP53INP1 were measured by RT-qPCR and Western blot respectively, after H9c2 cells were transfected with miR-142-5p mimic, miR-142-5p inhibitor or scrambled negative controls (miR-NC and NC). Untransfected cells served as control. *p < 0.05, **p < 0.01 compared to the indicated group.

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