MALAT1 regulates hypertrophy of cardiomyocytes by modulating the miR-181a/HMGB2 pathway

Abstract

Noncoding RNAs are important for regulation of cardiac hypertrophy. The function of MALAT1 (a long noncoding mRNA), miR-181a, and HMGB2; their contribution to cardiac hypertrophy; and the regulatory relationship between them during this process remain unknown. In the present study, we treated primary cardiomyocytes with angiotensin II (Ang II) to mimic cardiac hypertrophy. MALAT1 expression was significantly downregulated in Ang II-treated cardiomyocytes compared with control cardiomyocytes. Ang II-induced cardiac hypertrophy was suppressed by overexpression of MALAT1 and promoted by genetic knockdown of MALAT1. A dual-luciferase reporter assay demonstrated that MALAT1 acted as a sponge for miR-181a and inhibited its expression during cardiac hypertrophy. Cardiac hypertrophy was suppressed by overexpression of a miR-181a inhibitor and enhanced by overexpression of a miR-181a mimic. HMGB2 was downregulated during cardiac hypertrophy and was identified as a target of miR-181a by bioinformatics analysis and a dual-luciferase reporter assay. miR-181a overexpression decreased the mRNA and protein levels of HMGB2. Rescue experiments indicated that MALAT1 overexpression reversed the effect of miR-181a on HMGB2 expression. In summary, the results of the present study show that MALAT1 acts as a sponge for miR-181a and thereby regulates expression of HMGB2 and development of cardiac hypertrophy. The novel MALAT1/miR-181a/HMGB2 axis might play a crucial role in cardiac hypertrophy and serve as a new therapeutic target.

Figure 1.

The establishment of cardiac hypertrophy model. A) Cultured primary cardiomyocytes were treated with Ang II or control, then the cells were subjected to immunocytochemistry with actin antibody. The images captured by LSM 780 confocal microscope and were analyzed and the relative cell surface area was measured to indicate hypertrophy response (B). The cells were subjected to RT-qPCR to detect the mRNA level (C) and the protein levels of ANP, BNP and β-MHC. *p<0.05; **p<0.01; ***p<0.001; repeated three times; scale bar: 20 μm.

Figure 2.

MALAT1 is important for cardiac hypertrophy. A) Cells were treated as in figure 1. The cells were detected with RT-qPCR for the level of MALAT1. B) The levels of MALAT1 were verified. Cell were transfected with MALAT1 overexpression plasmids (C-E) or genetic knockdown siRNA fragments (D-F), or the controls, then the representative images were shown and the relative cell surface area was determined. *p<0.05; **p<0.01; ***p<0.001; ns, no significance; repeated three times; scale bar: 20 μm.

Figure 3.

The effect of miR-181a in cardiac hypertrophy. A) Cells were treated as in figure 1. The miR-181a level was detected by RTqPCR. (B) The efficiency of miR-181a inhibitor or mimic was determined by RT-qPCR in H9c2 cells. Cells were transfected with miR- 181a inhibitor (C-E) or mimic (D-F), then the representative images were shown and the relative cell surface area was measured. *p<0.05; **p<0.01; ns, no significance; repeated three times.

Figure 4.

MALAT1 targets miR-181a in cardiac hypertrophy. A) The bioinformatics analysis between the 3’ UTR of MALAT1 against miR-181a and the construction of MALAT1 MUT was shown. B) H9c2 cells were transfected with MALAT1 WT or MUT luciferase reporter plasmids, with or without miR-181a mimic. Then the relative luciferase activity was determined. C) The levels of MALT1 in primary cardiomyocytes were determined upon the transfection of miR-181a inhibitor or mimic. D) Cardiomyocytes were transfected with the indicated plasmids or fragments, then were subjected with Ang II treatment. The relative cell surface area was determined. *p<0.05; **p<0.01; repeated three times.

Figure 5.

HMGB2 is a target of miR-181a in cardiac hypertrophy. A,B) The expression level of HMGB2 was show in cardiac hypertrophy model. C) The bioinformatics analysis between the 3’ UTR of HMGB2 and miR-181a was shown. D) The luciferase activity from HMGB2 WT or MUT transfected with or without miR-181a mimic was shown. E,F) The mRNA and protein levels of HMGB2 were shown. *p<0.05; **p<0.01; repeated three times.

Figure 6.

MALAT1/miR-181a axis regulate HMGB2 in cardiac hypertrophy. A) The luciferase reporter of HMGB2 WT were co-transfected with indicated fragments or MALT1 encoding plasmids; then the relative luciferase activity of HMGB2 was determined. The mRNA level (B) and the protein level (C) of HMGB2, and the relative cell surface area (D) were determined in cells same treated in (A). *p<0.05; **p<0.01; repeated three times.

Figure 7.

The schematic diagram of regulatory mechanism of MALAT1/miR-181a/HMGB2 in cardiac hypertrophy.