Long noncoding RNA MALAT1 promotes cardiomyocyte apoptosis after myocardial infarction via targeting miR-144-3p

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  Figure 1. The expression of MALAT1 and miR-144-3p mRNA levels in AMI patients and mice LAD models (A) Total RNAs were extracted from the peripheral venous blood from each subject in Normal, Angina and AMI group. The relative expression levels of MALAT1 in AMI patients (n=40), normal control (n=20) and angina patient (n=20) were determined by qRT-PCR. (B) The relationship of MALAT1 and miR-144-3p expression was analyzed. (C,D) Male mice were subjected to LAD coronary artery occlusion followed by reperfusion (LAD/reperfusion). The expressions of MALAT1 and miR-144 mRNA in heart tissue were determined by real-time PCR. The results were presented from three independent experiments with the similar results. *P<0.05.

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  Figure 2. MALAT1 regulates cardiomyocytes apoptosis in HL-1 cells Cells were subjected to 10 h of hypoxia with 95% N2, 5% CO2, and subsequent 2 h of reoxygenation with 95% O2, 5% CO2; (A) Total RNAs were extracted from control (Ctrl) and H/R group were reverse transcribed into cDNA. Then qRT-PCR was performed to examine the relative expression of MALAT1. (B) HL-1 cells were transfected with pcDNA-MALAT1 or shMALAT1, and then the expression levels of MALAT1 were examined by qRT-PCR. (C) Western blot assay was performed to examine the cleaved caspase 3 and cleaved PARP in in control, H/R cell, H/R cell transfected with MALAT1-pcDNA and shMALAT1 and the expression levels of c-caspase3 and c-PARP were quantified. (D) Annexin V and PI double staining was conducted in control, H/R cell, H/R cell transfected with MALAT1-pcDNA and shMALAT1 and then flow cytometry assay was performed to determine the cell apoptosis. The experiments were repeated for at least three times. n=5, *P<0.05; **P<0.01.

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  Figure 3. Inhibition of MALAT1 reduced myocardium apoptosis in LAD mice Mice injected with shMALAT1 followed with I/R injury. (A) TUNEL staining assay was performed to detect the apoptosis in cardiac infarction sites. (B) The TUNEL positive cells were counted and analyzed. (C) Western blot assay was used to detect the c-casepase3 and c-PARP in the cardiac infarction sites and the expression levels were quantified. All the experiments were repeated for at least three times and got the similar results. n=5, *P<0.05; **P<0.01.

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  Figure 4. MALAT1 targets and regulates miR-144-3p expression (A) The putative target sequence for miR-144-3p on the 3′-UTR of MALAT1 was predicted by TargetScan prediction software. (B) The relative luciferase activity of WT or MUT MALAT1 3′-UTR reporter plasmids was detected by luciferase reporter assay after the cells transfected with miR-144-3p. (C) HL-1 cells were transfected with miR-144-3p mimic or miR-144-3p inhibitor and then the expression of miR-144 was detected by qRT-PCR. (D) The expression of MALAT1 was detected by qRT-PCR in the cells transfected with miR-144-3p mimic or miR-144-3p inhibitor. All the results were presented the data from three independent experiments with the similar results. n=5, *P<0.05.

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  Figure 5. The effect of MALAT1 on cardiomyocytes apoptosis was mediated by miR-144 (A) Mice injected with shMALAT1 followed with I/R injury. MiR-144 mRNA levels in the indicated each group was examined by qRT-PCR. (B) The cleaved caspase3 and PARP in each group were examined by Western blot assay in HL-1 cells. (C) Annexin V and PI double staining were performed then the cell apoptosis rate in each group were determined and analyzed by flow cytometry in HL-1 cells. (D) HL-1 cells were transfected miR-144-3p mimic, MALATA-pcDNA or miR-1444 together with MALAT1, and then Western blot was performed to check the protein expression of cleaved caspase3 and PARP. All the results presented the data from three independent experiments with the similar results. n=5, *P<0.05; **P<0.01.