Long non-coding RNA MALAT1 enhances the apoptosis of cardiomyocytes through autophagy inhibition by regulating TSC2-mTOR signaling

Abstract

Background: Our previous study showed that knockdown of long noncoding RNA (lncRNA) metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) attenuated myocardial apoptosis in mouse acute myocardial infarction (AMI). This study aims to explore whether MALAT1 enhanced cardiomyocyte apoptosis via autophagy regulation and the underlying mechanisms of MALAT1 regulating autophagy.

Methods: Cardiomyocytes were isolated from neonatal mice and then stimulated with hypoxia/reoxygenation (H/R) injury to mimic AMI. The autophagy level was assessed using GFP-LC3 immunofluorescence and western blot analysis of autophagy-related proteins. RNA pull-down and RNA immunoprecipitation (RIP) was performed to analyze the binding of MALAT1 and EZH2. Chromatin immunoprecipitation (ChIP) assay was performed to analyze the binding of TSC2 promoter and EZH2. The cell apoptosis was evaluated using TUNEL staining and western blot analysis of apoptosis-related proteins.

Results: H/R injury increased MALAT1 expression in cardiomyocytes. Furthermore, MALAT1 overexpression inhibited, whereas MALAT1 knockdown enhanced the autophagy of cardiomyocytes. Moreover, MALAT1 overexpression recruited EZH2 to TSC2 promoter regions to elevate H3K27me3 and epigenetically inhibited TSC2 transcription. Importantly, TSC2 overexpression suppressed mTOR signaling and then activated the autophagy. Further results showed that MALAT1 inhibited proliferation and enhanced apoptosis of cardiomyocytes through inhibiting TSC2 and autophagy.

Conclusion: These findings demonstrate that the increased MALAT1 expression induced by H/R injury enhances cardiomyocyte apoptosis through autophagy inhibition by regulating TSC2-mTOR signaling.

Keywords: Autophagy; Cardiomyocyte apoptosis; EZH2; MALAT1; TSC2-mTOR.

Fig. 1

MALAT1 overexpression inhibited, whereas MALAT1 knockdown enhanced the autophagy of cardiomyocytes stimulated with H/R injury. Cardiomyocytes were isolated from neonatal mice and then stimulated with H/R injury. qRT-PCR analysis of relative MALAT1 level (a) and HIF-1α mRNA level (b) in cardiomyocytes. c Western blot analysis of HIF-1α protein level in cardiomyocytes. d LDH release in cardiomyocytes. e The autophagosome puncta of GFP-LC3 by immunofluorescence in cardiomyocytes. Scale bar: 20 μm. f Western blot was performed to examine the protein levels of LC3-I, LC3-II, and Beclin-1 in cardiomyocytes. g Western blot was performed to examine the protein levels of LC3-I, LC3-II, and Beclin-1 in cardiomyocytes which were transfected with pcDNA3.1-MALAT1 (MALAT1), empty pcDNA3.1 (Vector), si-MALAT1, or scramble siRNA (si-Ctrl), followed by stimulation with H/R injury. Their quantitative analysis was normalized to β-actin. a–f **p < 0.01 vs. Control group. g **p < 0.01 vs. Vector group, ^##p < 0.01 vs. si-Ctrl group

Fig. 2

MALAT1 overexpression recruited EZH2 to elevate H3K27me3 and epigenetically inhibited TSC2 expression. Cardiomyocytes were transfected with pcDNA3.1-MALAT1 (MALAT1), empty pcDNA3.1 (Vector), si-MALAT1, or scramble siRNA (si-Ctrl), followed by stimulation with H/R injury. a Western blot was performed to measure the protein levels of TSC2, p-mTOR, and H3K27me3. *p < 0.05, **p < 0.01 vs. Vector group, ^##p < 0.01 vs. si-Ctrl group. b RNA pull-down and c RIP was performed to analyze the binding of MALAT1 and EZH2. **p < 0.01 vs. IgG group. d Western blot was performed to detect the protein levels of TSC2 and H3K27me3 in cardiomyocytes transfected with pcDNA3.1-EZH2 (EZH2) or empty pcDNA3.1 (Vector). **p < 0.01 vs. Vector group. e, f Cardiomyocytes were co-transfected with pcDNA3.1-MALAT1 (MALAT1) or empty pcDNA3.1 (Vector), with si-EZH2 or si-Ctrl. e Western blot was performed to detect the protein levels of TSC2 and H3K27me3. *p < 0.05, **p < 0.01 vs. Vector + si-Ctrl group, ^##p < 0.01 vs. si-Ctrl + MALAT1 group. f CHIP experiments displayed that TSC2 promoter was enriched with H3K27me3. ^**p < 0.01 vs. IgG group

Fig. 3

MALAT1 inhibited autophagy and enhanced apoptosis of cardiomyocytes through inhibiting TSC2. Cardiomyocytes were co-transfected with pcDNA3.1-MALAT1 (MALAT1) or empty pcDNA3.1 (Vector), and pcDNA3.1-TSC2 (TSC2) or Vector, followed by stimulation with H/R injury. a The autophagosome puncta of GFP-LC3 by immunofluorescence in cardiomyocytes. Scale bar: 20 μm. b Western blot was performed to examine the protein levels of p-mTOR, LC3-I, LC3-II, and Beclin-1 in cardiomyocytes. c The cell proliferation was evaluated using MTT assay. d The cell apoptosis was evaluated using TUNEL staining. The nick-ends were labeled in green indicating the apoptotic cells and the cell nucleus was labeled in blue by DAPI. Scale bar: 20 μm. e Western blot was performed to detect protein expression of Caspase-3, Bax, and Bcl-2 in cardiomyocytes. *p < 0.05, **p < 0.01 vs. Vector group; ^#p < 0.05, ^##p < 0.01 vs. MALAT1 group

Fig. 4

MALAT1 inhibited proliferation and enhanced apoptosis of cardiomyocytes through inhibiting autophagy. Cardiomyocytes were transfected with pcDNA3.1-MALAT1 (MALAT1) or empty pcDNA3.1 (Vector), followed by treatment with autophagy activator rapamycin before stimulation with H/R injury. a The cell proliferation was evaluated using MTT assay. b The cell apoptosis was evaluated using TUNEL staining. The nick-ends were labeled in green indicating the apoptotic cells and the cell nucleus was labeled in blue by DAPI. Scale bar: 20 μm. c Western blot was performed to detect protein levels of Caspase-3, Bax, and Bcl-2 in cardiomyocytes. *p < 0.05, **p < 0.01 vs. Vector group; ^#p < 0.05, ^##p < 0.01 vs. MALAT1 group