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3653-3660-Lncrna MIAT Enhances Cardiac Hypertrophy Partly Through Eur opean Rev iew for Med ical and Pharmacol ogical Sci ences 2016; 20: 3653-3660 LncRNA MIAT enhances cardiac hypertrophy partly through sponging miR-150 X.-H. ZHU 1, Y.-X. YUAN 2, S.-L. RAO 3, P. WANG 4 1VIP Ward, Zhumadian Central Hospital, Zhumadian, Henan, China 2Department of Cardiology, Zoucheng People’s Hospital, Zoucheng, Shandong, China 3Department of Emergency, Binzhou People’s Hospital, Binzhou, Shandong, China Shandong, China 4The 2 nd Department of Cardiology, Feicheng mining industry Central Hospital, Feicheng, Shandong, China Xiuhua Zhu and Yuxiang Yuan contribtued equally to this study Abstract. – OBJECTIVE: In this work, we characterized by an enlargement of cardiomy - aimed to study whether myocardial infarction ocytes and the addition of contractile proteins 1,2 . associated transcript (MIAT) exerts a regulative Sustained cardiac hypertrophy and maladaptive effect on cardiac hypertrophy via acting as a cardiac remodeling significantly increased the risk miR-150 sponge. 3 MATERIALS AND METHODS: Cardiac hyper - of heart failure and cardiac death . The causes of trophy was induced using Angiotensin II (Ang cardiac hypertrophy are quite complex ; previous II). MIAT and miR-150 expression were quanti - studies 4,5 suggested that diverse factors , including fied using qRT-PCR. Rat heart-derived H9c2 physiological, mechanical, hormonal, and genetic cells were used as the in vitro model. Cardiac hypertrophic features were assessed by quanti - influence are all involved in the regulation . fying cardiac hypertrophic genes and measure - Long non-coding RNAs (lncRNAs) refers the ment of cell surface, protein synthesis and total non-protein-coding RNA transcripts longer than protein content. 200 nucleotides 6,7 . Recent studies suggest that RESULTS: MIAT is significantly increased in several dysregulated lncRNAs are associated Ang II induced cardiac hypertrophy in a mouse with the pathological development of cardiac hy - model and in H9c2 cells. MIAT siRNA substantial - ly alleviated the Ang II induced upregulation of pertrophy through transcriptional and post-tran - ANP, BNP and β-MHC in H9c2 cells and markedly scriptional regulation. At transcriptional level, attenuated the Ang II induced increase of the cell for example, lncRNA H19 is upregulated in surface area and the protein synthesis. MIAT pathological cardiac hypertrophy 8. Both H19 and overexpression in H9c2 cells significantly re - its encoded miR-675 can inhibit the hypertrophic duced the miR-150 expression. MIAT inhibition al - growth of cardiomyocytes, the effect of which is so partly restored the miR-150 levels under Ang II partly mediated by suppressing the expression of treatment. MiR-150 overexpression could attenu - 8 ate the Ang II induced upregulation of hyper - miR-675 target gene CaMKIIdelta . At the post- trophic marker genes and suppress the Ang II in - transcriptional level, lncRNA can function as duced hypertrophic phenotypes of the cells. miRNA sponge and modulate the development CONCLUSIONS: MIAT is significantly in - of cardiac hypertrophy. For example, lncRNA creased in Ang II induced cardiac hypertrophy and contributes to the pathological develop - CHRF can act as an endogenous sponge of miR- ment. MIAT can suppress miR-150 expression in 489 and increase the expression of miR-489 tar - cardiomyocytes and miR-150 is a downstream get gene myeloid differentiation primary re - effector of MIAT in the development of cardiac sponse gene 88 (Myd88), thereby promoting car - hypertrophy. diac hypertrophy 9. lncRNA-ROR can also pro - Key Words: mote cardiac hypertrophy via interacting with MIAT, miR-150, Cardiac hypertrophy . miR-133 10 . Myocardial infarction–associated transcript (MIAT), is an lncRNA predominantly expressed in heart and fetal brain tissue. Constant Introduction researches showed that dysregulated MIAT is in - volved in myocardial infarction 11,12 . However, Cardiac hypertrophy is an adaptive response of whether this lncRNA has a regulative effect on the heart to the increased cardiac load, which is cardiac hypertrophy is not clear. Corresponding Author: Peng Wang, MD; e-mail: [email protected] 3653 X.-H. Zhu, Y.-X. Yuan, S.-L. Rao, P. Wang Functionally, MIAT upregulation can lead to with 100 nM MIAT siRNA, 100 nM miR-150 microvascular dysfunction via enhancing en - mimics or pcDNA3.1-MIAT using Lipofecta - dothelial cell proliferation and migration 13 . In mine 2000 (Invitrogen) according to manufactur - both microvascular endothelial cells and lens ep - er’s instruction. ithelial cells, MIAT can act as a sponge of miR- 150 13,14 . miR-150 is an important miRNA with QRT-PCR Analysis inhibitive effect on pressure overload-induced Total RNA from the tissue and cell samples cardiac hypertrophy 15 and high glucose-induced were extracted using Trizol reagent (Invitrogen) cardiomyocyte hypertrophy 16 . Therefore, we hy - according to manufacturer’s instruction. The first pothesized that MIAT might exert a regulative strand cDNA were synthesized by reverse tran - effect on cardiac hypertrophy via acting as a scription using a First Strand Synthesis kit (Invit - miR-150 sponge. rogen, Carlsbad, CA, USA). QRT-PCR was per - formed to detect the expression of MIAT, ANP, BNP, β-MHC using gene specific primers and Materials and Methods SYBR ® Green qPCR MasterMix (Applied Biosystems, Foster City, CA, USA) in ABI 7500 Animals and Protocols Real-Time PCR Systems. The primers were: Animal based study was approved by the Re - search Commit tee of Zoucheng People’s Hospi - MIAT , 5’-TTTACTTTAACAGACCAGAA-3’ tal. All animal based studies followed the Guide (forward), for the Care and Use of Laboratory Animals (US 5’-CTCCTTTGTTGAATCCAT-30 and National Institutes of Health). Eight-week- (reverse); old male, healthy and specific pathogen-free ANP , 5’-ATCTGATGGATTTCAAGAACC-3’ C57BL/6J mice weighing 19-21 g was purchased (forward), from the Experimental Animal Center of the 5’-CTCTGAGACGGGTTGACTTC-3’ Shandong University. The mice were random - (reverse); ized into three groups (n=10/group): a blank BNP , 5’-ACAATCCACGATGCAGAAGCT- 3’ group without any treatment; a sham group only (forward), received PBS infusion; an Angiotensin II (Ang 5’-GGGCCTTGGTCCTTTGAGA-3’ II) (Sigma-Aldrich, St . Louis, MO, USA)-in - (reverse); fused group. The animals in Ang II group re - β-MHC , 5’-CCTCGCAATATCAAGGGAAA-3 ’ ceived Ang II dissolved in PBS with 10 µmol/L (forward), acetic acid at a dose of 2.5 µg/kg/min using a 5’-TACAGGTGCATCAGCTCCAG-3 subcutaneously implanted minipump (model (reverse). 2002, Alza, Moun tain View, CA, USA) for 15 days. The mice were housed in SPF conditions MiR-150 expression was determined by qRT- with a 12-h light/12-h dark cycle and with free PCR using the mirVana qRT-PCR miRNA De - access to drinking water and food. tection Kit and Primer Sets from Ambion as well as Taqman ® miRNA assays (Applied Biosys - Cell Culture and Treatment tems). The relative expression of the RNAs was Rat heart-derived H9c2 cells were cultured us - calculated using the 2 -∆∆ CT method. ing Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine Immunofluorescent Staining and serum (FBS) at 37 °C in a 5% CO 2 incubator. For Measurement of Cell Surface Area Ang II treatment, the cells were incubated with 1 Cells on coverslips were fixed with PBS con - µmol/L Ang II or PBS for 48 hours. MIAT siR - taining 4% paraformaldehyde, permeabilized in NA, miR-150 mimics and the scramble negative PBS containing 0.2% Triton X-100, and then controls were purchased from Ribobio blocked with PBS containing 3% BSA. After that (Guangzhou, China). The Full length of MIAT the cells were incubated with primary anti- α- cDNA was amplified and inserted into the sites actinin at a dilution of 1:100 in 1% goat serum between BamHI and XhoI of pcDNA3.1 plasmid overnight at 4°C, and then incubated with Alexa (Invitrogen, Carlsbad, CA, USA) and the recon - Fluor ® 594 goat anti-mouse IgG for 1 h at structed MIAT expression plasmids is named as 37°C. The coverslips were mounted onto glass pcDNA3.1-MIAT. H9c2 cells were transfected slides with SlowFade Gold antifade reagent with 3654 LncRNA MIAT enhances cardiac hypertrophy partly through sponging miR-150 DAPI. The cell surface area using a quantitative Results digital image analysis system (Image Pro-Plus version 7.0; Media Cybernetics, Rockville, MD, MIAT is Significantly Increased in Ang II USA) with a digital camera (Olympus IX-81, Induced Cardiac Hypertrophy Olympus, Tokyo, Japan) . 100 cells were random - Previous studies suggest that MIAT is an ly selected in three wells of different treatment. lncRNA that is significantly increased in patients with myocardial infarction and with dilated car - Measurement of [3H]leucine diomyopathy 12,18 . In this study, we firstly ex - Incorporation and Protein Content Assay plored the expression of MIAT in Ang II induced [3H]leucine incorporation was measured ac - cardiac hypertrophy in a mouse model. The re - cording to the method introduced in one previous sults showed that the hypertrophic cardiac tissues study 17 . Briefly, Cells were cultured in 24-well of the mice received 15 d administration of Ang plates in serum-free medium for 24 h. After indi - II had significantly increased MIAT expression cated treatment, the cells were pulsed with 1 (Figure 1A). In H9c2 cells, we also confirmed µCi/mL of [3H]leucine (Amersham Biosciences, that Ang II treatment induced a significant in - Piscataway, NJ, USA) for 4 hours before harvest. crease of MIAT expression (Figure 1B). There - After three times washing with PBS, the cells fore, we decided to further explore the biological were treated with 5% trichloroacetic acid for 30 function of MIAT using H9c2 cells as the in-vit - min. Finally, cells were solubilized in 500 µL of ro model. 1 mol/L NaOH and neutralized using 0.5 mol/L HCl. Then, an aliquot was taken to determine the Inhibition of MIAT Attenuates Ang II level of incorporated radioactivity using the Induced Cardiac Hypertrophy Beckman LS 3801 liquid scintillation counter Cardiac hypertrophy is associated with signifi - (Beckman, Fullerton, CA, USA).
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