Arq Bras Cardiol abc Arquivos Brasileiros de Cardiologia Arq. Bras. Cardiol. 0066-782X 1678-4170 Sociedade Brasileira de Cardiologia - SBC y3XWfqbLCN8HzHtqqgRZYzR S0066-782X2025001100306 00306 10.36660/abc.20240703 Artigo Original Knockdown do RNA Longo Não Codificante ZEB1-AS1 Acelera a Hipertrofia Cardíaca pela Via miR-186-5p/HDAC2 0009-0004-8118-8347 Cao Bingfeng Concepção e desenho da pesquisa e Redação do manuscrito Obtenção de dados 1 0009-0007-6074-1619 Liu Qingxia Análise e interpretação dos dados 2 0009-0002-3623-6982 Zhang Xiaoyuan Análise estatística 1 0009-0004-9989-7969 Wang Yufeng Concepção e desenho da pesquisa e Redação do manuscrito Revisão crítica do manuscrito quanto ao conteúdo 1 Department of Cardiology Weihai Central Hospital Shandong China Department of Cardiology – Weihai Central Hospital, Shandong – China Department of Outpatient Weihai Central Hospital Shandong China Department of Outpatient – Weihai Central Hospital, Shandong – China Yufeng Wang Department of Cardiology, Weihai Central Hospital, No. 3 Mishan East Road West, Weihai, 264400, Shandong - China E-mail: cbf_1978@126.com, bgf70@163.com

Natália Olivetti

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O papel do RNA longo não codificante ZEB1-AS1 na hipertrofia cardíaca (HC) permanece incerto.

 Objetivos

Investigar a função do ZEB1-AS1 no desenvolvimento e na progressão da HC, bem como elucidar seu mecanismo molecular subjacente.

 Métodos

Os níveis de expressão de RNA foram quantificados por PCR quantitativa em tempo real. A área de superfície das células AC16 foi avaliada por coloração por imunofluorescência. A expressão proteica foi analisada por Western blotting. As interações entre RNAs foram examinadas por ensaios com repórter de luciferase e por imunoprecipitação de RNA. A significância estatística foi definida como p < 0,05.

 Resultados

A expressão de ZEB1-AS1 esteve aumentada em tecidos miocárdicos e em células AC16 estimuladas com isoproterenol (ISO). O knockdown de ZEB1-AS1 atenuou as respostas hipertróficas induzidas por ISO. Mecanisticamente, ZEB1-AS1 modulou a expressão de desacetilase de histonas 2 (HDAC2) atuando como “esponja molecular” para miR-186-5p. De forma consistente, o knockdown de ZEB1-AS1 reduziu a HDAC2 e diminuiu a expressão de marcadores hipertróficos, incluindo o peptídeo natriurético tipo B, o peptídeo natriurético atrial e a cadeia pesada de miosina β, restringindo assim a progressão da HC.

 Conclusões

ZEB1-AS1 encontra-se superexpresso em tecidos miocárdicos e em células AC16 estimuladas com ISO. Nossos achados indicam que o eixo ZEB1-AS1/miR-186-5p/HDAC2 contribui para a HC, oferecendo uma base mecanística e um potencial alvo terapêutico para intervenção clínica.

<italic>Knockdown</italic> do RNA Longo Não Codificante ZEB1-AS1 Acelera a Hipertrofia Cardíaca pela Via miR-186-5p/HDAC2 Knockdown do RNA Longo Não Codificante ZEB1-AS1 Acelera a Hipertrofia Cardíaca Pela Via miR-186-5p/HDAC2. ANP: peptídeo natriurético atrial; BNP: peptídeo natriurético tipo B; β-MHC: cadeia pesada de miosina β; HDAC2: desacetilase de histonas 2; ISSO: isoproterenol; LncRNA: RNA longo não codificante.

Palavras-chave Cardiomegalia RNA Longo não Codificante Histona Desacetilase 2 Fontes de financiamento:O presente estudo não teve fontes de financiamento externas. Introdução

A hipertrofia cardíaca (HC) caracteriza-se pelo aumento do tamanho dos cardiomiócitos, e não pelo aumento de seu número. Trata-se, principalmente, de uma resposta compensatória ao estresse biomecânico, destinada a preservar o débito cardíaco e manter a homeostase fisiológica.1,2 A HC é amplamente classificada como patológica ou fisiológica. A HC patológica associa-se comumente à apoptose de cardiomiócitos, fibrose miocárdica, arquitetura celular desorganizada, alterações epigenéticas e reativação aberrante de programas gênicos fetais, incluindo os marcadores peptídeo natriurético tipo B (BNP), peptídeo natriurético atrial (ANP) e cadeia pesada de miosina β (β-MHC).3-5 A persistência da HC patológica pode levar a desfechos clínicos graves, como arritmia, remodelamento cardíaco maladaptativo, insuficiência cardíaca (IC), infarto do miocárdio e morte súbita cardíaca.6,7 Apesar dos avanços terapêuticos, os desfechos — particularmente na IC relacionada à HC — permanecem subótimos, com cerca de 50% de sobrevida em 5 anos após o diagnóstico.8 Elucidar os mecanismos moleculares que impulsionam o desenvolvimento e a progressão da HC é, portanto, uma prioridade de pesquisa.

Os RNAs não codificantes (ncRNAs) — incluindo RNAs circulares (circRNAs), RNAs longos não codificantes (lncRNAs) e micro RNAs (miRNAs) — têm atraído considerável atenção por seus papéis em diversos processos regulatórios, principalmente por meio da modulação da expressão de RNAs codificadores de proteína.9-13 Os lncRNAs, definidos como transcritos não codificadores com ≥ 200 nucleotídeos e sem capacidade de codificação proteica, emergiram como reguladores-chave em um amplo espectro de condições patológicas.14,15 Numerosos estudos mostram que lncRNAs contribuem para a patogênese de doenças como nefropatia diabética,16 hipertensão pulmonar,17 neoplasias malignas18 e doenças cardiovasculares.19-21 Evidências crescentes também implicam lncRNAs no desenvolvimento e na progressão da HC. Por exemplo, o silenciamento do lncRNA TUG1 atenua a HC ao modular a via miR-497/MEF2C,22 o ncRNA PVT1 promove a HC patológica atuando como esponja molecular para miR-196b23 e o lncRNA NEAT1 acelera a HC por meio do eixo miR-19a-3p/SMYD2.24 O RNA longo não codificante ZEB1-AS1 (lncRNA ZEB1-AS1), um lncRNA recentemente identificado e inicialmente estudado em câncer, tem sido implicado em múltiplos processos patológicos;25 contudo, seu papel na HC permanece amplamente indefinido.

Os miRNAs constituem uma classe de ncRNAs curtos e altamente conservados (tipicamente > 22 nucleotídeos [nt]) que reprimem a expressão de mRNAs-alvo.26 O miR-186-5p é um miRNA bem caracterizado envolvido na regulação de várias doenças humanas, incluindo condições cardiovasculares.27-29 Entretanto, seu papel específico na HC ainda não é bem compreendido. A desacetilase de histonas 2 (HDAC2), membro da família de desacetilases de histonas, tem sido implicada em diversos estados patológicos, incluindo HC.30-32 Resta determinar se o eixo regulatório miR-186-5p/HDAC2 participa da progressão da HC mediada por ZEB1-AS1.

Este estudo teve como objetivo investigar o papel do ZEB1-AS1 no desenvolvimento e na progressão da HC e delinear seu mecanismo subjacente, a fim de identificar um potencial alvo terapêutico.

 Métodos Espécimes de tecido cardíaco humano

Coletamos amostras de tecido cardíaco de 20 pacientes com hipertrofia ventricular esquerda (HVE) — secções de coração obtidas durante transplante cardíaco — e de 10 corações de doadores sem histórico de doença cardíaca, em nosso hospital, entre março de 2018 e setembro de 2023. A Tabela 1 resume as características clínicas de pacientes e doadores.

– Sequências de <italic>primers</italic> utilizadas no qRT-PCR
Gene Forward (5′ → 3′) Reverse (5′ → 3′)
ZEB1-AS1 5′-CCGTGGGCACTGCTGAAT-3′ 5′-CTGCTGGCAAGCGGAACT-3′
miR-186-5p 5′-CAAAGAAUUCUCCUUUUGGGCU-3′ 5′-CCCAAAAGGAGAAUUCUUUGUU-3′
HDAC2 5′-TGAGATTCCCAATGAGTTGCCA-3′ 5′-TACTGACATCTGGTCAGACA-3′
ANP 5′-CAACGCAGA-CCTGATGGATTT-3′ 5′-AGCCCCCGC-TTCTTCATTC-3′
BNP 5′-TGGAAACGTCCGGGTTACAG-3′ 5′-CTGATCCGGTCCATCTTCCT-3′
β-MHC 5′-CCGAGTCCCAGGTCAACAA-3′ 5′-CTTCACGGGCACCCTTGGA-3′
U6 5′-GCTTCGGCAGCACATATACTAAAAT-3′ 5′-CGCTTCACGAATTTGCGTGTCAT-3′
GAPDH 5′-CCATCAACGACCCCTTCATT-3′ 5′-GACCAGCTTCCCATTCTCAG-3′

ANP: peptídeo natriurético atrial; BNP: peptídeo natriurético tipo B; GAPDH: gliceraldeído-3-fosfato desidrogenase; HDAC2: histone deacetylase 2; miR-186-5p: microRNA-186-5p; qRT-PCR: reação em cadeia da polimerase quantitativa em tempo real; U6: RNA nuclear pequeno U6; ZEB1-AS1: zinc finger E-box binding homeobox 1 antisense RNA 1; β-MHC: cadeia pesada de miosina β.

Os critérios de inclusão para HVE foram espessura do septo interventricular em diástole e/ou espessura da parede posterior do VE ≥ 1,2 cm, com fração de ejeção do VE > 40% à ecocardiografia. Critérios de exclusão incluíram IC sistólica, neoplasia, infecção aguda, BNP > 400 ng/L, doença de Parkinson, fibrose pulmonar idiopática, choque, cirrose hepática ou tratamento recente com interferon ou fenobarbital.33 Os corações de doadores eram provenientes de indivíduos falecidos por causas não cardíacas; os tecidos foram coletados entre 0,5 e 6 h pós-morte. Todas as amostras de tecido cardíaco foram imediatamente congeladas por snap-freezing em nitrogênio líquido (Delun, Xangai, China) antes da extração de RNA.

O estudo foi aprovado pelo Comitê de Ética Institucional de nosso hospital, e todos os participantes forneceram consentimento informado. Os procedimentos de pesquisa aderiram aos princípios da revisão de 2024 da Declaração de Helsinque da Associação Médica Mundial.34

 Cultura celular e transfecção

A linhagem de cardiomiócitos humanos AC16 foi obtida da American Type Culture Collection (EUA). As células foram cultivadas em Dulbecco’s Modified Eagle Medium (DMEM; HyClone Laboratories, EUA) suplementado com 20% de soro fetal bovino (Gibco, EUA) e mantidas em incubadora umidificada a 37 °C com 5% de CO2. Para estabelecer um modelo in vitro de HC, células AC16 foram tratadas com 10 µmol/L de isoproterenol (ISO; Sigma-Aldrich, EUA), conforme protocolos publicados.35

Os construtos short hairpin RNA–negative control (sh-NC) e sh-ZEB1-AS1 foram obtidos da GeneChem (Xangai, China). Mimics de miR-186-5p, um inibidor de miR-186-5p e seus respectivos controles negativos foram adquiridos da RiboBio (Guangzhou, China). As transfecções celulares foram realizadas com Lipofectamine 3000 (Sigma-Aldrich, EUA), seguindo as instruções do fabricante.

 PCR quantitativa em tempo real

O RNA total foi extraído de amostras de tecido cardíaco humano e de células AC16 usando o reagente TRIzol (Sigma-Aldrich, EUA). A síntese de cDNA e a amplificação foram realizadas com SYBR Green Master Mix (Takara, Japão), conforme instruções do fabricante. GAPDH e U6 serviram como controles endógenos para normalização da expressão de lncRNA/mRNA e miRNA, respectivamente. A expressão relativa foi calculada pelo método 2Ct. A Tabela 2 apresenta as sequências de primers utilizadas no qRT-PCR.

– Características clínicas de doadores saudáveis e pacientes com HVE
Variável Saudáveis (n = 10) HVE (n = 20) Valor p
Idade (anos) 67 ± 8 68 ± 12 0,8141
Masculino/feminino (n/n) 4/6 11/9 0,4390
Tabagismo, n (%) 4 (40%) 11 (55%) 0,4390
DM, n (%) 3 (30%) 12 (60%) 0,1210
Hipertensão, n (%) 4 (40%) 18 (90%) 0,0040
SCA, n (%) 2 (20%) 14 (70%) 0,0100
FA, n (%) 0 (0%) 5 (25%) 0,0830
Glicose em jejum (mmol/L) 5,51 ± 0,54 6,23 ± 1,85 0,2417
PAS (mmHg) 115,25 ± 18,25 159,91 ± 28,24 0,0001
PAD (mmHg) 73,52 ± 12,25 95,41 ± 15,36 0,0005
HDL-C (mmol/L) 1,36 ± 0,11 1,15 ± 0,29 0,0365
LDL-C (mmol/L) 2,61 ± 0,56 3,05 ± 0,81 0,1354
BNP (ng/L) 129,61 ± 74,12 158,36 ± 95,21 0,4112
FE (%) 78,55 ± 5,81 58,25 ± 6,25 0,0001
Diâmetro do VE (cm) 4,25 ± 0,45 4,38 ± 0,66 0,5807
DDFVE (cm) 4,55 ± 0,50 4,99 ± 0,91 0,1673
EIVd (cm) 0,98 ± 0,08 1,24 ± 0,13 0,0001
EPPVEd (cm) 0,95 ± 0,12 1,29 ± 0,12 0,0001
ZEB1-AS1 relativo (média ± DP) 0,895 ± 0,394 2,194 ± 0,633 0,0001

BNP: peptídeo natriurético tipo B; DDFVE: diâmetro diastólico final do ventrículo esquerdo; DM: diabetes melito; EIVd: espessura do septo interventricular em diástole; EPPVEd: espessura da parede posterior do ventrículo esquerdo em diástole; FA: fibrilação atrial; FEVE: fração de ejeção do ventrículo esquerdo; HDL-C: colesterol de lipoproteína de alta densidade; HVE: hipertrofia do ventrículo esquerdo; LDL-C: colesterol de lipoproteína de baixa densidade; PAD: pressão arterial diastólica; PAS: pressão arterial sistólica; SCA: síndrome coronariana aguda.

 <italic>Western blotting</italic>

A extração de proteínas de células AC16 foi realizada conforme descrito anteriormente.36 Em resumo, as células foram lisadas em tampão radioimmunoprecipitation assay e as proteínas foram desnaturadas por fervura. Quantidades iguais de proteína foram separadas por eletroforese em gel de poliacrilamida com dodecil sulfato de sódio a 10% (SDS-PAGE) e transferidas para membranas de polifluoreto de vinilideno (PVDF; Millipore, EUA). As membranas foram bloqueadas com 1% de albumina sérica bovina (BSA) por 1 h à temperatura ambiente e lavadas com solução salina tamponada com fosfato (PBS). As blots foram incubadas a 4 °C, durante a noite, com anticorpos primários contra ANP (1:2000, Abcam, EUA), BNP (1:2000, CST, EUA), β-MHC (1:2000, CST, EUA) e GAPDH (1:2000, Abcam, EUA). Após lavagem, as membranas foram incubadas com anticorpo secundário (1:10.000, Jackson, EUA) por 2 h à temperatura ambiente. As bandas foram visualizadas por quimioluminescência aprimorada (Amersham, Reino Unido) e quantificadas no ImageJ (National Institutes of Health, EUA).

 Coloração por imunofluorescência

Células AC16 foram fixadas com paraformaldeído a 4% a 37 °C por 30 min, lavadas três vezes com PBS e permeabilizadas com Triton X-100 a 0,1% por 15 min. As células foram bloqueadas por 30 min em soro normal de cabra a 10% com 1% de BSA (Sigma-Aldrich) e incubadas, no escuro, durante a noite a 4 °C, com anticorpo primário anti-β-actina (Abcam, EUA) diluído no tampão de bloqueio. Após três lavagens em PBS, as células foram incubadas com o anticorpo secundário apropriado por 2 h a 37 °C, contracoradas com DAPI e imageadas em microscópio de fluorescência (Carl Zeiss). A área de superfície celular foi quantificada no ImageJ.

 Ensaio repórter de dupla luciferase

Sequências de ligação para miR-186-5p em ZEB1-AS1 e na 3′-RNT de HDAC2 foram clonadas em vetores pGL3-Basic (LMAI Bio, Xangai, China). Células AC16 foram cotransfectadas com mimic controle negativo ou mimic de miR-186-5p juntamente com construções repórter wild type (WT) ou mutantes (MUT). Após a transfecção, as células foram coletadas e a atividade de luciferase foi medida com o Dual-Luciferase Reporter Assay System (Promega, EUA).

 Imunoprecipitação de RNA

A imunoprecipitação de RNA (RIP) foi realizada com o RNA-Binding Protein Immunoprecipitation Kit (Millipore, EUA) para avaliar as interações entre ZEB1-AS1, miR-186-5p e HDAC2, conforme instruções do fabricante. Em resumo, as células foram lisadas em tampão de lise RIP e incubadas com esferas magnéticas conjugadas a anti-Argonaute-2 (Ago2; Abcam, EUA) ou IgG de coelho controle (Abcam, EUA). Após três lavagens com PBS, o RNA coprecipitado com Ago2 ou IgG foi purificado e analisado por qRT-PCR.

 Análise dos dados

As análises foram realizadas no GraphPad Prism versão 7.0.0 para Windows, GraphPad Software, Boston, Massachusetts, EUA; www.graphpad.com. Os dados são apresentados como média ± desvio-padrão (DP). Comparações entre dois grupos utilizaram teste t de Student bicaudal e não pareado; diferenças entre múltiplos grupos foram avaliadas por ANOVA de um fator com pós-teste de Bonferroni. As associações entre a expressão de ZEB1-AS1 e variáveis clínicas foram avaliadas com o teste do qui-quadrado. Normalidade e homocedasticidade foram verificadas pelos testes de Shapiro–Wilk e de Levene, respectivamente; p > 0,05 indicou que os pressupostos foram atendidos. A significância estatística foi definida como p < 0,05.

 Resultados ZEB1-AS1 em pacientes com HVE e em HC induzida por ISO em células AC16

Quantificamos inicialmente ZEB1-AS1 em tecidos cardíacos de 20 pacientes com hipertrofia ventricular esquerda (HVE) e de 10 corações de doadores normais por qRT-PCR. ZEB1-AS1 esteve significativamente superexpresso nos tecidos com HVE em comparação aos controles (Figura 1A).

– Superexpressão de ZEB1-AS1 em tecidos com HVE e em células AC16 estimuladas com ISO. A) Expressão de ZEB1-AS1 em corações com HVE (n = 20) versus doadores normais (n = 10) por qRT-PCR; B) Área de superfície de células AC16 após tratamento com PBS ou ISO, avaliada por coloração por imunofluorescência; C) Níveis de mRNA de BNP, ANP e β-MHC em células AC16 após PBS ou ISO por qRT-PCR; D) Níveis de proteína de BNP, ANP e β-MHC em células AC16 após PBS ou ISO por Western blot; E) Expressão de ZEB1-AS1 em células AC16 após PBS ou ISO por qRT-PCR. Todos os experimentos foram realizados em triplicata. *p < 0,05, *p < 0,01. ANP: peptídeo natriurético atrial; BNP: peptídeo natriurético tipo B; β-MHC: cadeia pesada de miosina β; HC: hipertrofia cardíaca; HVE: hipertrofia ventricular esquerda; ISO: isoproterenol; PBS: solução salina tamponada com fosfato; qRT-PCR: reação em cadeia da polimerase quantitativa em tempo real.

Para modelar a HC in vitro, células AC16 foram tratadas com ISO (10 µM). O ISO aumentou a área de superfície celular às 24 h, atingindo o máximo em 48 h em relação aos controles não tratados (Figura 1B). Em paralelo, o ISO promoveu aumentos dependentes do tempo nos níveis de mRNA e proteína de marcadores hipertróficos — BNP, ANP e β-MHC — quando comparados aos controles (Figuras 1C e 1D), confirmando o estabelecimento bem-sucedido do modelo de HC.

Notavelmente, a expressão de ZEB1-AS1 também aumentou de forma significativa nas células AC16 tratadas com ISO em relação aos controles (Figura 1E), sugerindo um papel de ZEB1-AS1 na progressão da HC.

 A deficiência de ZEB1-AS1 atenua a HC induzida por ISO em células AC16

Para avaliar o papel de ZEB1-AS1 na hipertrofia induzida por ISO, células AC16 foram transfectadas com sh-ZEB1-AS1 ou sh-NC. A expressão de ZEB1-AS1 foi significativamente reduzida nas células sh-ZEB1-AS1 em comparação com sh-NC, confirmando knockdown eficiente (Figura 2A). O tratamento com ISO aumentou acentuadamente a área de superfície das AC16, consistente com alterações hipertróficas, ao passo que esse aumento foi significativamente atenuado nas células transfectadas com sh-ZEB1-AS1 (Figura 2B). De modo semelhante, o ISO superexpressou os níveis de mRNA e proteína dos marcadores hipertróficos (BNP, ANP e β-MHC); essa indução foi substancialmente atenuada pelo knockdown de ZEB1-AS1 (Figuras 2C, D). Esses dados indicam que a redução de ZEB1-AS1 regula negativamente a HC induzida por ISO.

– A deficiência de ZEB1-AS1 atenua a HC induzida por ISO em células AC16. A) Expressão de ZEB1-AS1 em células AC16 transfectadas com sh-NC ou sh-ZEB1-AS1 por qRT-PCR; B) área de superfície das AC16 após estimulação com ISO com sh-NC ou sh-ZEB1-AS1, avaliada por coloração por imunofluorescência; C) níveis de mRNA de BNP, ANP e β-MHC em AC16 estimuladas com ISO com sh-NC ou sh-ZEB1-AS1 por qRT-PCR; D) níveis de proteína de BNP, ANP e β-MHC em AC16 estimuladas com ISO com sh-NC ou sh-ZEB1-AS1 por Western blot. Todos os experimentos foram realizados em triplicata. *p < 0,05, *p < 0,01. ANP: peptídeo natriurético atrial; BNP: peptídeo natriurético tipo B; β-MHC: cadeia pesada de miosina β; HC: hipertrofia cardíaca; ISO: isoproterenol; qRT-PCR: reação em cadeia da polimerase quantitativa em tempo real; sh-NC: short hairpin RNA–negative control; sh-ZEB1-AS1: shRNA direcionado a ZEB1-AS1.

ZEB1-AS1 regula a expressão de HDAC2 por sequestro de miR-186-5p

Utilizamos starBase 2.0 para predizer miRNAs que interagem com ZEB1-AS1 e identificamos o miR-186-5p como candidato (Figura 3A). Um ensaio repórter de dupla luciferase mostrou que a cotransfecção do mimic de miR-186-5p com o construto wild type de ZEB1-AS1 (ZEB1-AS1-WT) reduziu significativamente a atividade de luciferase em células AC16, ao passo que o construto mutante (ZEB1-AS1-MUT) não teve efeito (Figura 3B), confirmando ligação específica. Usando TargetScan, identificamos em seguida a HDAC2 como alvo a jusante putativo de miR-186-5p (Figura 3C). Essa interação foi validada por ensaio de luciferase: o mimic de miR-186-5p diminuiu a atividade do repórter HDAC2-WT, mas não do HDAC2-MUT (Figura 3D). O RIP demonstrou ainda enriquecimento de ZEB1-AS1, miR-186-5p e HDAC2 em imunoprecipitados com Ago2 a partir de lisados de AC16 (Figura 3E), indicando coassociação no complexo de silenciamento induzido por RNA.

– ZEB1-AS1 regula HDAC2 ao sequestrar miR-186-5p. A) Sítio previsto de ligação de miR-186-5p em ZEB1-AS1 (WT) e construto mutante (MUT) correspondente. B) Ensaio de dupla luciferase mostrando redução da atividade do repórter para ZEB1-AS1-WT, mas não para ZEB1-AS1-MUT, na presença do mimic de miR-186-5p. C) Sítio previsto de ligação de miR-186-5p na 3′-RNT de HDAC2 (WT) e construto MUT. D) Ensaio de dupla luciferase mostrando diminuição da atividade para HDAC2-WT, mas não para HDAC2-MUT, com mimic de miR-186-5p. E) RIP demonstrando enriquecimento de ZEB1-AS1, miR-186-5p e HDAC2 em imunoprecipitados com Ago2. F-G) Níveis de miR-186-5p e de HDAC2 em corações com HVE (n = 20) versus doadores normais (n = 10) por qRT-PCR. Todos os experimentos foram realizados em triplicata. *p < 0,05, *p < 0,01. Ago2: Argonauta-2; CMV: citomegalovírus; HDAC2: desacetilase de histonas 2; LVH: hipertrofia ventricular esquerda; miR-NC: mimic de controle negativo; MUT: mutante; qRT-PCR: reação em cadeia da polimerase quantitativa em tempo real; RIP: imunoprecipitação de RNA; RNT: região não traduzida; WT: wild type.

Em tecidos miocárdicos de 20 pacientes com HVE versus 10 corações de doadores normais, a qRT-PCR revelou menor miR-186-5p e maior expressão de HDAC2 (Figuras 3F,G). Em conjunto, esses dados sustentam um eixo ZEB1-AS1/miR-186-5p/HDAC2 na HC, pelo qual ZEB1-AS1 modula HDAC2 ao “esponjar” miR-186-5p.

 ZEB1-AS1 promove a HC regulando a via miR-186-5p/HDAC2

Para testar se ZEB1-AS1 impulsiona a HC via o eixo miR-186-5p/HDAC2, realizamos experimentos de rescue. O mRNA de HDAC2 foi medido em células AC16 estimuladas com ISO e transfectadas com sh-NC, sh-ZEB1-AS1 ou cotransfectadas com sh-ZEB1-AS1 mais um inibidor de miR-186-5p. O knockdown de ZEB1-AS1 reduziu significativamente o mRNA de HDAC2, e essa redução foi revertida pela inibição de miR-186-5p (Figura 4A). Do mesmo modo, o aumento induzido por ISO na área de superfície celular foi atenuado por sh-ZEB1-AS1, enquanto a inibição concomitante de miR-186-5p aboliu esse efeito (Figura 4B). Em paralelo, o knockdown de ZEB1-AS1 diminuiu a expressão de mRNA e proteína dos marcadores hipertróficos induzida por ISO (BNP, ANP e β-MHC), e esses efeitos supressores foram revertidos pela inibição de miR-186-5p (Figuras 4C e 4D). Coletivamente, os resultados indicam que ZEB1-AS1 promove a HC ao regular a via de sinalização miR-186-5p/HDAC2.

– ZEB1-AS1 promove a HC ao regular a via miR-186-5p/HDAC2. A) mRNA de HDAC2 em células AC16 estimuladas com ISO e transfectadas com os construtos indicados, medido por qRT-PCR; B) área de superfície das AC16 após estimulação com ISO e transfecção com os construtos indicados, avaliada por coloração por imunofluorescência; C) níveis de mRNA de BNP, ANP e β-MHC em AC16 estimuladas com ISO após transfecção, medidos por qRT-PCR; D) níveis de proteína de BNP, ANP e β-MHC em AC16 estimuladas com ISO após transfecção, medidos por Western blot. Todos os experimentos foram realizados em triplicata. *p < 0,05, *p < 0,01. ANP: peptídeo natriurético atrial; BNP: peptídeo natriurético tipo B; β-MHC: cadeia pesada de miosina β; HC: hipertrofia cardíaca; HDAC2: histone deacetylase 2; ISO: isoproterenol; qRT-PCR: reação em cadeia da polimerase quantitativa em tempo real; sh-NC: short hairpin RNA–negative control; sh-ZEB1-AS1: zinc finger E-box binding homeobox 1 antisense RNA 1 direcionado ao short hairpin RNA.

Discussão

Neste estudo, mostramos que ZEB1-AS1 está significativamente superexpresso na HC e que seu silenciamento atenua as respostas hipertróficas induzidas por ISO em células AC16. Mecanisticamente, ensaios de dupla luciferase e RIP confirmaram que miR-186-5p é alvo direto de ZEB1-AS1 e que HDAC2 é alvo de miR-186-5p, estabelecendo um eixo regulatório que contribui para a progressão da HC.

Os lncRNAs foram outrora considerados subprodutos transcripcionais não funcionais,37 mas evidências acumuladas indicam que regulam diversos processos biológicos por meio de interações com miRNAs, fatores de transcrição, DNA genômico e proteínas ligantes de RNA. Os lncRNAs têm sido cada vez mais reconhecidos no desenvolvimento cardíaco e na patogênese das doenças cardiovasculares. Por exemplo, o lncRNA MIR217HG exacerba a HC induzida por sobrecarga de pressão via a via miR-138/THBS1,38 ADAMTS9-AS1/miR-185-5p/KAT7 atua como uma rede supressora de ceRNA na cardiomiopatia hipertrófica obstrutiva39 e o knockdown de DANCR mitiga a IC ao atenuar HC e fibrose por meio do eixo miR-758-3p/PRG4/Smad.35 ZEB1-AS1, inicialmente caracterizado em câncer, também tem sido implicado em diversas neoplasias,25,40,41 mas seu papel na HC permanecia incerto. Aqui, identificamos ZEB1-AS1 como um lncRNA pró-hipertrófico: ele se encontra aumentado em tecidos com HVE e em células AC16 tratadas com ISO, e seu knockdown reduz a hipertrofia induzida por ISO, indicando um papel patogênico no desenvolvimento e na progressão da HC.

Os lncRNAs frequentemente atuam em redes de ceRNA para regular a expressão gênica no pós-transcricional, sequestrando miRNAs e, assim, modulando sua disponibilidade para mRNAs-alvo.42,43 A importância dos circuitos de ceRNA na HC foi corroborada por diversos estudos.44-46 De modo consistente, mostramos que ZEB1-AS1 funciona como sponge para miR-186-5p — um miRNA relatado como reduzido no tecido cardíaco em cardiomiopatia diabética47 — aliviando a repressão de HDAC2 e promovendo a hipertrofia induzida por ISO. Ao nosso conhecimento, este é o primeiro relato que implica miR-186-5p na HC, ressaltando seu potencial como ponto de entrada terapêutico.

As HDACs são contribuintes-chave para a patologia cardiovascular.30-32,48-50 Trabalhos prévios mostraram que a inibição de HDAC atenua a HC induzida por sobrecarga de pressão ou por infusão de ISO,51,52 em linha com nosso modelo in vitro baseado em ISO. Entre as HDACs, a HDAC2 tem recebido atenção particular; a fosforilação na serina 394 é um evento regulatório crítico na progressão da HC.53 Por exemplo, o efeito anti-hipertrófico de plantamajoside na HC induzida por ISO associa-se estreitamente à HDAC2,32 e EYA4-au1 promove hipertrofia de cardiomiócitos ao recrutar Med11 para superexpressar EYA4, levando à redução de p27Kip1 e à ativação do eixo CK2α/HDAC2.54 Apesar do papel estabelecido da HDAC2, sua regulação a montante via redes lncRNA–miRNA–mRNA tem sido menos clara. Nossos dados indicam que HDAC2 é alvo direto de miR-186-5p e que ZEB1-AS1 promove HC modulando o eixo miR-186-5p/HDAC2.

Limitações do estudo

Este estudo apresenta algumas limitações. Como lncRNAs frequentemente interagem com múltiplos miRNAs e miRNAs individuais tipicamente regulam numerosos alvos, ZEB1-AS1 pode influenciar vias adicionais relevantes para a HC que não foram examinadas aqui. Esses mecanismos alternativos merecem investigação futura. Além disso, embora o intervalo pós-morte para tecidos de autópsia tenha sido breve, a degradação de RNA permanece uma potencial fonte de viés e pode afetar análises transcriptômicas. Avaliação rigorosa da integridade do RNA é, portanto, essencial para assegurar validade e reprodutibilidade, e será foco de trabalhos futuros.

 Conclusão

ZEB1-AS1 está marcadamente superexpresso em tecidos com HVE e em células AC16 estimuladas com ISO. Funcionalmente, ZEB1-AS1 promove a HC atuando como ceRNA para miR-186-5p a fim de modular a expressão de HDAC2. Esses achados definem um eixo ZEB1-AS1/miR-186-5p/HDAC2 na patogênese da HC e fornecem base mecanística para explorar estratégias terapêuticas direcionadas.

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Kaohsiung J Med Sci. 2024;40(1):46-62. doi: 10.1002/kjm2.12774. Jiao W Hao J Liu JM Gao WN Zhao JJ Li YJ Mesenchymal Stem Cells-Derived Extracellular Vesicle-Incorporated H19 Attenuates Cardiac Remodeling in Rats with Heart Failure Kaohsiung J Med Sci 2024 40 1 46 62 10.1002/kjm2.12774 45. Liu P, Dong X, Dong C, Hou G, Liu W, Jiang X, et al. LncRNA MHRT Prevents Angiotensin II-Induced Myocardial Oxidative Stress and NLRP3 Inflammasome Via Nrf2 Activation. Antioxidants. 2023;12(3):672. doi: 10.3390/antiox12030672. Liu P Dong X Dong C Hou G Liu W Jiang X et al LncRNA MHRT Prevents Angiotensin II-Induced Myocardial Oxidative Stress and NLRP3 Inflammasome Via Nrf2 Activation Antioxidants 2023 12 3 672 10.3390/antiox12030672 46. Zhu W, Peng F, Cui X, Li J, Sun C. LncRNA SOX2OT Facilitates LPS-Induced Inflammatory Injury by Regulating Intercellular Adhesion Molecule 1 (ICAM1) Via Sponging miR-215-5p. Clin Immunol. 2022;238:109006. doi: 10.1016/j.clim.2022.109006. Zhu W Peng F Cui X Li J Sun C LncRNA SOX2OT Facilitates LPS-Induced Inflammatory Injury by Regulating Intercellular Adhesion Molecule 1 (ICAM1) Via Sponging miR-215-5p Clin Immunol 2022 238 109006 10.1016/j.clim.2022.109006 47. Liu Y, Zheng W, Pan Y, Hu J. Low Expression of miR-186-5p Regulates Cell Apoptosis by Targeting Toll-Like Receptor 3 in High Glucose-Induced Cardiomyocytes. J Cell Biochem. 2019;120(6):9532-8. doi: 10.1002/jcb.28229. Liu Y Zheng W Pan Y Hu J Low Expression of miR-186-5p Regulates Cell Apoptosis by Targeting Toll-Like Receptor 3 in High Glucose-Induced Cardiomyocytes J Cell Biochem 2019 120 6 9532 9538 10.1002/jcb.28229 48. Wang L, Ahn YJ, Asmis R. Inhibition of Myeloid HDAC2 Upregulates Glutaredoxin 1 Expression, Improves Protein Thiol Redox State and Protects Against High-Calorie Diet-Induced Monocyte Dysfunction and Atherosclerosis. Atherosclerosis. 2021;328:23-32. doi: 10.1016/j.atherosclerosis.2021.05.002. Wang L Ahn YJ Asmis R Inhibition of Myeloid HDAC2 Upregulates Glutaredoxin 1 Expression, Improves Protein Thiol Redox State and Protects Against High-Calorie Diet-Induced Monocyte Dysfunction and Atherosclerosis Atherosclerosis 2021 328 23 32 10.1016/j.atherosclerosis.2021.05.002 49. Yang P, Liang K, Wang W, Zhou D, Chen Y, Jiang X, et al. LncRNA SOX2-OTinhibitionprotects Against Myocardialischemia/Reperfusion-Inducedinjury Via themicroRNA-186-5p (miR-186-5p)/Yin Yang 1 (YY1)Pathway. Bioengineered. 2022;13(1):280-90. doi: 10.1080/21655979.2021.2000229. Yang P Liang K Wang W Zhou D Chen Y Jiang X et al LncRNA SOX2-OTinhibitionprotects Against Myocardialischemia/Reperfusion-Inducedinjury Via themicroRNA-186-5p (miR-186-5p)/Yin Yang 1 (YY1)Pathway Bioengineered 2022 13 1 280 290 10.1080/21655979.2021.2000229 50. Feng Y, Yan B, Cheng H, Wu J, Chen Q, Duan Y, et al. Knockdown circ_0040414 Inhibits Inflammation, Apoptosis and Promotes the Proliferation of Cardiomyocytes Via miR-186-5p/PTEN/AKT Axis in Chronic Heart Failure. Cell Biol Int. 2021;45(11):2304-15. doi: 10.1002/cbin.11678. Feng Y Yan B Cheng H Wu J Chen Q Duan Y et al Knockdown circ_0040414 Inhibits Inflammation, Apoptosis and Promotes the Proliferation of Cardiomyocytes Via miR-186-5p/PTEN/AKT Axis in Chronic Heart Failure Cell Biol Int 2021 45 11 2304 2315 10.1002/cbin.11678 51. Cheng T, Liu C, Wang Y, Li G, Feng L, Zhang S, et al. A Novel Histone Deacetylase Inhibitor Se-SAHA Attenuates Isoproterenol-Induced Heart Failure Via Antioxidative Stress and Autophagy Inhibition. Toxicol Appl Pharmacol. 2024;487:116957. doi: 10.1016/j.taap.2024.116957. Cheng T Liu C Wang Y Li G Feng L Zhang S et al A Novel Histone Deacetylase Inhibitor Se-SAHA Attenuates Isoproterenol-Induced Heart Failure Via Antioxidative Stress and Autophagy Inhibition Toxicol Appl Pharmacol 2024 487 116957 10.1016/j.taap.2024.116957 52. Zhou H, Kee HJ, Wan L, Asfaha Y, Fischer F, Kassack MU, et al. YAK577 Attenuates Cardiac Remodeling and Fibrosis in Isoproterenol-Infused Heart Failure Mice by Downregulating MMP12. Korean Circ J. 2025;55(3):231-47. doi: 10.4070/kcj.2024.0093. Zhou H Kee HJ Wan L Asfaha Y Fischer F Kassack MU et al YAK577 Attenuates Cardiac Remodeling and Fibrosis in Isoproterenol-Infused Heart Failure Mice by Downregulating MMP12 Korean Circ J 2025 55 3 231 247 10.4070/kcj.2024.0093 53. Raghunathan S, Goyal RK, Patel BM. Selective Inhibition of HDAC2 by Magnesium Valproate Attenuates Cardiac Hypertrophy. Can J Physiol Pharmacol. 2017;95(3):260-7. doi: 10.1139/cjpp-2016-0542. Raghunathan S Goyal RK Patel BM Selective Inhibition of HDAC2 by Magnesium Valproate Attenuates Cardiac Hypertrophy Can J Physiol Pharmacol 2017 95 3 260 267 10.1139/cjpp-2016-0542 54. Zhao J, Gong F, Yang Q, Yang R, Yan Z, Xi Z, et al. Exercise in Ozone-Polluted Air Evokes Pathological Cardiac Hypertrophy Via Up-Regulation of Nuclear lncRNA EYA4-au1 and Recruiting Med11 to Activating EYA4/p27kip1/CK2a/HDAC2 Cascade. Ecotoxicol Environ Saf. 2024;287:117264. doi: 10.1016/j.ecoenv.2024.117264. Zhao J Gong F Yang Q Yang R Yan Z Xi Z et al Exercise in Ozone-Polluted Air Evokes Pathological Cardiac Hypertrophy Via Up-Regulation of Nuclear lncRNA EYA4-au1 and Recruiting Med11 to Activating EYA4/p27kip1/CK2a/HDAC2 Cascade Ecotoxicol Environ Saf 2024 287 117264 10.1016/j.ecoenv.2024.117264

Não há vinculação deste estudo a programas de pós-graduação.

Este estudo foi aprovado pelo Comitê de Ética do Weihai Central Hospital sob o número de protocolo WKY-2023-008. Todos os procedimentos envolvidos nesse estudo estão de acordo com a Declaração de Helsinki de 1975, atualizada em 2013. O consentimento informado foi obtido de todos os participantes incluídos no estudo.

Os autores não utilizaram ferramentas de inteligência artificial no desenvolvimento deste trabalho.

Todo o conjunto de dados que dá suporte aos resultados deste estudo está disponível mediante solicitação ao autor correspondente Bingfeng Cao.

O presente estudo não teve fontes de financiamento externas.

 10.36660/abc.20240703i Original Article Knockdown of the Long Noncoding RNA ZEB1-AS1 Accelerates Cardiac Hypertrophy via the miR-186-5p/HDAC2 Pathway 0009-0004-8118-8347 Cao Bingfeng Conception and design of the research and Writing of the manuscript Acquisition of data 1 0009-0007-6074-1619 Liu Qingxia Analysis and interpretation of the data 2 0009-0002-3623-6982 Zhang Xiaoyuan Statistical analysis 1 0009-0004-9989-7969 Wang Yufeng Conception and design of the research and Writing of the manuscript Critical revision of the manuscript for content 1 China Department of Cardiology – Weihai Central Hospital, Shandong – China China Department of Outpatient – Weihai Central Hospital, Shandong – China Yufeng Wang Department of Cardiology, Weihai Central Hospital, No. 3 Mishan East Road West, Weihai, 264400, Shandong - China E-mail: cbf_1978@126.com, bgf70@163.com

No potential conflict of interest relevant to this article was reported.

Natália Olivetti

 Abstract Background

The role of the long noncoding RNA ZEB1-AS1 in cardiac hypertrophy (CH) remains unclear.

 Objectives

To investigate the function of ZEB1-AS1 in the development and progression of CH as well as elucidate its underlying molecular mechanism.

 Methods

RNA expression levels were quantified by quantitative real-time PCR. Surface area of AC16 cells was assessed by immunofluorescence staining. Protein expression was evaluated by Western blotting. Interactions among RNAs were examined using luciferase reporter assays and RNA immunoprecipitation. Statistical significance was set at p < 0.05.

 Results

The expression of ZEB1-AS1 was upregulated in myocardial tissues and in isoproterenol (ISO)-stimulated AC16 cells. The knockdown of ZEB1-AS1 mitigated ISO-induced hypertrophic responses. Mechanistically, ZEB1-AS1 modulated histone deacetylase 2 (HDAC2) expression by acting as a molecular sponge for miR-186-5p. Consistently, the knockdown of ZEB1-AS1 reduced HDAC2 and decreased the expression of hypertrophic markers, including B-type natriuretic peptide, atrial natriuretic peptide, and β-myosin heavy chain, thereby restraining the progression of CH.

 Conclusions

ZEB1-AS1 is upregulated in myocardial tissues and ISO-stimulated AC16 cells. Our findings indicate the ZEB1-AS1/miR-186-5p/HDAC2 axis contributes to CH, providing a mechanistic basis and potential therapeutic target for clinical intervention.

Knockdown of the Long Noncoding RNA ZEB1-AS1 Accelerates Cardiac Hypertrophy via the miR-186-5p/HDAC2 Pathway Knockdown of the Long Noncoding RNA ZEB1-AS1 Accelerates Cardiac Hypertrophy via the miR-186-5p/HDAC2 Pathway. ANP: atrial natriuretic peptide; BNP: B-type natriuretic peptide; β-MHC: beta-myosin heavy chain; HDAC2: histone deacetylase 2; ISO: isoproterenol; LncRNA: long noncoding RNA.

Cardiomegaly Long Noncoding RNA Histone Deacetylase 2 Sources of funding:There were no external funding sources for this study. Introduction

Cardiac hypertrophy (CH) is characterized by enlargement of cardiomyocytes rather than an increase in their number. It primarily represents a compensatory response to biomechanical stress, intended to preserve cardiac output and maintain physiological homeostasis.1,2 CH is broadly classified as pathological or physiological. Pathological CH is commonly associated with cardiomyocyte apoptosis, myocardial fibrosis, disorganized cellular architecture, epigenetic alterations, and aberrant reactivation of fetal gene programs, including the markers B-type natriuretic peptide (BNP), atrial natriuretic peptide (ANP), and β-myosin heavy chain (β-MHC).3-5 Persistent pathological CH can lead to serious clinical outcomes, such as arrhythmia, maladaptive cardiac remodeling, heart failure (HF), myocardial infarction, and sudden cardiac death.6,7 Despite advances in therapy, outcomes — particularly for CH-related HF — remain suboptimal, with about 50% survival at 5 years after diagnosis.8 Elucidating the molecular mechanisms driving the development and progression of CH is therefore an urgent research priority.

Noncoding RNAs (ncRNAs) — including circular RNAs (circRNAs), long noncoding RNAs (lncRNAs), and microRNAs (miRNAs) — have attracted considerable attention for their roles in diverse regulatory processes, primarily through modulation of protein-coding RNA expression.9-13 LncRNAs, defined as noncoding transcripts ≥ 200 nucleotides that lack protein-coding capacity, have emerged as key regulators across a wide spectrum of pathological conditions.14,15 Numerous studies show lncRNAs contribute to the pathogenesis of diseases such as diabetic nephropathy,16 pulmonary hypertension,17 malignancies,18 and cardiovascular diseases.19-21 Growing evidence also implicates lncRNAs in the development and progression of CH. For example, silencing lncRNA TUG1 attenuates CH by modulating the miR-497/MEF2C pathway,22 ncRNA PVT1 promotes pathological CH by acting as a molecular sponge for miR-196b,23 and lncRNA NEAT1 accelerates CH via the miR-19a-3p/SMYD2 axis.24 The long noncoding RNA ZEB1-AS1 (lncRNA ZEB1-AS1), a recently identified lncRNA initially studied in cancer, has been implicated in multiple pathological processes;25 however, its role in CH remains largely undefined.

MiRNAs are a class of highly conserved, short ncRNAs (typically > 22 nucleotides [nt]) that repress target mRNA expression.26 miR-186-5p is a well-characterized miRNA involved in the regulation of several human diseases, including cardiovascular conditions.27-29 Nevertheless, its specific role in CH is not well understood. Histone deacetylase 2 (HDAC2), a member of the histone deacetylase family, has been implicated in various pathological states, including CH.30-32 Whether the miR-186-5p/HDAC2 regulatory axis participates in ZEB1-AS1–mediated CH progression remains to be determined.

This study aimed to investigate the role of ZEB1-AS1 in CH development and progression and delineates its underlying mechanism in order to identify a potential therapeutic target.

 Methods Human heart tissues specimens

We collected human cardiac tissue specimens from 20 patients with left ventricular hypertrophy (LVH) — heart sections obtained during heart transplantation — and from 10 normal donor hearts without a history of cardiac disease, at our hospital between March 2018 and September 2023. Table 1 summarizes clinical characteristics of patients and donors.

– Primer sequences used for qRT-PCR
Gene Forward (5′ → 3′) Reverse (5′ → 3′)
ZEB1-AS1 5′-CCGTGGGCACTGCTGAAT-3′ 5′-CTGCTGGCAAGCGGAACT-3′
miR-186-5p 5′-CAAAGAAUUCUCCUUUUGGGCU-3′ 5′-CCCAAAAGGAGAAUUCUUUGUU-3′
HDAC2 5′-TGAGATTCCCAATGAGTTGCCA-3′ 5′-TACTGACATCTGGTCAGACA-3′
ANP 5′-CAACGCAGA-CCTGATGGATTT-3′ 5′-AGCCCCCGC-TTCTTCATTC-3′
BNP 5′-TGGAAACGTCCGGGTTACAG-3′ 5′-CTGATCCGGTCCATCTTCCT-3′
β-MHC 5′-CCGAGTCCCAGGTCAACAA-3′ 5′-CTTCACGGGCACCCTTGGA-3′
U6 5′-GCTTCGGCAGCACATATACTAAAAT-3′ 5′-CGCTTCACGAATTTGCGTGTCAT-3′
GAPDH 5′-CCATCAACGACCCCTTCATT-3′ 5′-GACCAGCTTCCCATTCTCAG-3′

ANP: atrial natriuretic peptide; BNP: B-type natriuretic peptide; β-MHC: beta-myosin heavy chain; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HDAC2: histone deacetylase 2; miR-186-5p: microRNA-186-5p; qRT-PCR: quantitative real-time polymerase chain reaction; U6: U6 small nuclear RNA; ZEB1-AS1: zinc finger E-box binding homeobox 1 antisense RNA 1.

Inclusion criteria for LVH were interventricular septal thickness at end-diastole and/or LV posterior wall depth ≥ 1.2 cm, with a LV ejection fraction > 40% on echocardiography. Exclusion criteria included systolic HF, malignancy, acute infection, BNP > 400 ng/L, Parkinson’s disease, idiopathic pulmonary fibrosis, shock, liver cirrhosis, or recent treatment with interferon or phenobarbital.33 Donor hearts were from individuals who died of noncardiac causes; tissues were collected within 0.5–6 h postmortem. All cardiac tissue specimens were immediately snap-frozen in liquid nitrogen (Delun, Shanghai, China) before RNA extraction.

The study was approved by the Institutional Ethics Committee of our hospital, and informed consent was obtained from all participants. Research procedures adhered to the principles of the 2024 revision of the World Medical Association Declaration of Helsinki.34

 Cell culture and transfection

Human cardiomyocyte cell line AC16 was obtained from the American Type Culture Collection (USA). Cells were cultured in Dulbecco’s Modified Eagle Medium (HyClone Laboratories, USA) supplemented with 20% fetal bovine serum (Gibco, USA) and maintained in a humidified incubator at 37 °C with 5% CO2. In order to establish an in vitro model of CH, AC16 cells were treated with 10 µmol/L isoproterenol (ISO; Sigma-Aldrich, USA) according to published protocols.35

Short hairpin RNA–negative control (sh-NC) and sh-ZEB1-AS1 constructs were obtained from GeneChem (Shanghai, China). miR-186-5p mimics, a miR-186-5p inhibitor, and their corresponding negative controls were purchased from RiboBio (Guangzhou, China). Cell transfections were performed with Lipofectamine 3000 (Sigma-Aldrich, USA) following the manufacturer’s instructions.

 Quantitative real-time PCR

Total RNA was extracted from human heart tissue specimens and AC16 cells using TRIzol reagent (Sigma-Aldrich, USA). cDNA synthesis and amplification were performed with a SYBR Green Master Mix kit (Takara, Japan) following the manufacturer’s instructions. GAPDH and U6 served as endogenous controls for normalization of lncRNA/mRNA and miRNA expression, respectively. Relative expression was calculated using the 2Ct method. Table 2 lists primer sequences used for qRT-PCR.

– Clinical characteristics of healthy donors and patients with LVH
Variable Healthy (n = 10) LVH (n = 20) p-value
Age (years) 67 ± 8 68 ± 12 0.8141
Male/female (n/n) 4/6 11/9 0.4390
Smoking, n (%) 4 (40%) 11 (55%) 0.4390
DM, n (%) 3 (30%) 12 (60%) 0.1210
Hypertension, n (%) 4 (40%) 18 (90%) 0.0040
ACS, n (%) 2 (20%) 14 (70%) 0.0100
AF, n (%) 0 (0%) 5 (25%) 0.0830
Fasting glucose (mmol/L) 5.51 ± 0.54 6.23 ± 1.85 0.2417
SBP (mmHg) 115.25 ± 18.25 159.91 ± 28.24 0.0001
DBP (mmHg) 73.52 ± 12.25 95.41 ± 15.36 0.0005
HDL-C (mmol/L) 1.36 ± 0.11 1.15 ± 0.29 0.0365
LDL-C (mmol/L) 2.61 ± 0.56 3.05 ± 0.81 0.1354
BNP (ng/L) 129.61 ± 74.12 158.36 ± 95.21 0.4112
EF (%) 78.55 ± 5.81 58.25 ± 6.25 0.0001
LV diameter (cm) 4.25 ± 0.45 4.38 ± 0.66 0.5807
LVIDd (cm) 4.55 ± 0.50 4.99 ± 0.91 0.1673
IVSd (cm) 0.98 ± 0.08 1.24 ± 0.13 0.0001
PWd (cm) 0.95 ± 0.12 1.29 ± 0.12 0.0001
Relative ZEB1-AS1 (mean ± SD) 0.895 ± 0.394 2.194 ± 0.633 0.0001

ACS: acute coronary syndrome; AF: atrial fibrillation; BNP: B-type natriuretic peptide; DBP: diastolic blood pressure; DM: diabetes mellitus; HDL-C: high-density lipoprotein cholesterol; IVSd: interventricular septum thickness at end-diastole; LDL-C: low-density lipoprotein cholesterol; LV: left ventricle; LVEF: LV ejection fraction; LVIDd: LV internal dimension at end-diastole; LVH: LV hypertrophy; PWd: LV posterior wall thickness at end-diastole; SBP: systolic blood pressure.

 Western blotting

Protein extraction from AC16 cells was performed as described previously.36 Briefly, cells were lysed in radioimmunoprecipitation assay buffer and proteins were denatured by boiling. Equal amounts of protein were resolved on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, USA). Membranes were blocked with 1% bovine serum albumin (BSA) for 1 h at room temperature and washed with phosphate-buffered saline (PBS). Blots were incubated overnight at 4 °C with primary antibodies against ANP (1:2000, Abcam, USA), BNP (1:2000, CST, USA), β-MHC (1:2000, CST, USA), and GAPDH (1:2000, Abcam, USA). After washing, membranes were incubated with secondary antibody (1:10,000, Jackson, USA) for 2 h at room temperature. Bands were visualized using enhanced chemiluminescence (Amersham, UK) and quantified in ImageJ (National Institutes of Health, USA).

 Immunofluorescent staining

AC16 cells were fixed with 4% paraformaldehyde at 37 °C for 30 min, washed three times with PBS, and permeabilized with 0.1% Triton X-100 for 15 min. Cells were blocked for 30 min in 10% normal goat serum with 1% BSA (Sigma-Aldrich). They were then incubated overnight at 4 °C in the dark with an anti–β-actin primary antibody (Abcam, USA) diluted in blocking buffer. After three PBS washes, cells were incubated with the appropriate secondary antibody for 2 h at 37 °C, counterstained with DAPI, and imaged on a fluorescence microscope (Carl Zeiss). Cell surface area was quantified using ImageJ.

 Dual-luciferase reporter assay

Binding sequences for miR-186-5p within ZEB1-AS1 and the HDAC2 3′-UTR were cloned into pGL3-Basic vectors (LMAI Bio, Shanghai, China). AC16 cells were co-transfected with either negative-control mimic or miR-186-5p mimic together with the wild-type (WT) or mutant (MUT) reporter constructs. After transfection, cells were harvested and luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega, USA).

 RNA immunoprecipitation

RNA immunoprecipitation (RIP) was performed with the RNA-Binding Protein Immunoprecipitation Kit (Millipore, USA) to assess interactions among ZEB1-AS1, miR-186-5p, and HDAC2, following the manufacturer’s instructions. Briefly, cells were lysed in RIP lysis buffer and incubated with magnetic beads conjugated to anti-Argonaute-2 (Ago2; Abcam, USA) or control rabbit IgG (Abcam, USA). After three PBS washes, RNA co-precipitated with Ago2 or IgG was purified and analyzed by qRT-PCR.

 Data analysis

Analyses were performed in GraphPad Prism version 7.0.0 for Windows, GraphPad Software, Boston, Massachusetts USA, www.graphpad.com. Data are presented as mean ± standard deviation (SD). Two-group comparisons used an unpaired two-tailed Student t test; multiple-group differences were assessed by one-way ANOVA with Bonferroni post hoc testing. Associations between ZEB1-AS1 expression and clinical variables were evaluated with the chi-square test. Normality and homoscedasticity were assessed by the Shapiro-Wilk and Levene tests, respectively; p > 0.05 indicates the assumptions were met. Statistical significance was set at p < 0.05.

 Results ZEB1-AS1 in patients with LV and in ISO-induced CH in AC16 cells

We first quantified ZEB1-AS1 in heart tissues from 20 patients with LVH and 10 normal donor hearts by qRT-PCR. ZEB1-AS1 was significantly upregulated in LVH tissues versus controls (Figure 1A).

To model CH in vitro, AC16 cells were treated with ISO (10 µM). ISO increased cell surface area at 24 h and reached a maximum at 48 h relative to untreated controls (Figure 1B). In parallel, ISO elicited time-dependent increases in the mRNA and protein levels of hypertrophic markers — namely BNP, ANP, and β-MHC — compared with controls (Figures 1C and 1D), confirming successful establishment of the CH model.

Notably, ZEB1-AS1 expression was also significantly increased in ISO-treated AC16 cells versus controls (Figure 1E), suggesting a role for ZEB1-AS1 in CH progression.

– Upregulation of ZEB1-AS1 in LVH tissues and ISO-stimulated AC16 cells. A) ZEB1-AS1 expression in LVH hearts (n = 20) versus normal donors (n = 10) by qRT-PCR. B) AC16 cell surface area after PBS or ISO treatment assessed by immunofluorescence. C) mRNA levels of BNP, ANP, and β-MHC in AC16 cells after PBS or ISO by qRT-PCR. D) Protein levels of BNP, ANP, and β-MHC in AC16 cells after PBS or ISO by Western blot. E) ZEB1-AS1 expression in AC16 cells after PBS or ISO by qRT-PCR. All experiment was performed in triplicate. *p < 0.05, **p < 0.01. ANP: atrial natriuretic peptide; BNP: B-type natriuretic peptide; β-MHC: beta-myosin heavy chain; CH: cardiac hypertrophy; ISO: isoproterenol; LVH: left ventricular hypertrophy; PBS: phosphate-buffered saline; qRT-PCR: quantitative real-time polymerase chain reaction.

ZEB1-AS1 deficiency mitigates ISO-induced CH in AC16 cells

To assess the role of ZEB1-AS1 in ISO-induced hypertrophy, AC16 cells were transfected with sh-ZEB1-AS1 or sh-NC. ZEB1-AS1 expression was significantly reduced in sh-ZEB1-AS1 cells versus sh-NC, confirming efficient knockdown (Figure 2A). ISO treatment markedly increased AC16 cell surface area, consistent with hypertrophic changes, whereas this enlargement was significantly attenuated in sh-ZEB1-AS1–transfected cells (Figure 2B). Likewise, ISO upregulated the mRNA and protein levels of hypertrophic markers (namely, BNP, ANP, and β-MHC); this induction was substantially blunted by ZEB1-AS1 knockdown (Figure 2C,D). These data indicate ZEB1-AS1 downregulation negatively regulates ISO-induced CH.

– ZEB1-AS1 deficiency mitigates ISO-induced CH in AC16 cells. A) ZEB1-AS1 expression in AC16 cells transfected with sh-NC or sh-ZEB1-AS1 by qRT-PCR; B) AC16 cell surface area after ISO stimulation with sh-NC or sh-ZEB1-AS1 assessed by immunofluorescence; C) mRNA levels of BNP, ANP, and β-MHC in ISO-stimulated AC16 cells with sh-NC or sh-ZEB1-AS1 by qRT-PCR; D) protein levels of BNP, ANP, and β-MHC in ISO-stimulated AC16 cells with sh-NC or sh-ZEB1-AS1 by Western blot. All experiments were performed in triplicate. *p < 0.05, **p < 0.01. ANP: atrial natriuretic peptide; BNP: B-type natriuretic peptide; β-MHC: beta-myosin heavy chain; CH: cardiac hypertrophy; ISO: isoproterenol; qRT-PCR: quantitative real-time polymerase chain reaction; sh-NC: short hairpin RNA–negative control; sh-ZEB1-AS1: ZEB1-AS1-targeting shRNA.

ZEB1-AS1 regulates HDAC2 expression by sequestering miR-186-5p

We used starBase 2.0 to predict miRNAs that interact with ZEB1-AS1 and identified miR-186-5p as a candidate (Figure 3A). A dual-luciferase reporter assay showed that co-transfection of miR-186-5p mimic with the ZEB1-AS1 wild-type (ZEB1-AS1-WT) construct significantly reduced luciferase activity in AC16 cells, whereas the mutant construct (ZEB1-AS1-MUT) had no effect (Figure 3B), confirming specific binding. Using TargetScan, we next identified HDAC2 as a putative downstream target of miR-186-5p (Figure 3C). This interaction was validated by luciferase assay: the miR-186-5p mimic decreased activity of the HDAC2-WT reporter but not the HDAC2-MUT reporter (Figure 3D). RIP further demonstrated enrichment of ZEB1-AS1, miR-186-5p, and HDAC2 in Ago2 immunoprecipitates from AC16 cell lysates (Figure 3E), indicating co-association within the RNA-induced silencing complex.

In myocardial tissues from 20 patients with LVH versus 10 normal donor hearts, qRT-PCR revealed lower miR-186-5p and higher HDAC2 expression (Figure 3F,G). Together, these data support a ZEB1-AS1/miR-186-5p/HDAC2 axis in CH, whereby ZEB1-AS1 modulates HDAC2 by sponging miR-186-5p.

– ZEB1-AS1 regulates HDAC2 by sequestering miR-186-5p. A) Predicted miR-186-5p binding site in ZEB1-AS1 (WT) and corresponding mutant (MUT) construct. B) Dual-luciferase assay showing reduced reporter activity for ZEB1-AS1-WT, but not ZEB1-AS1-MUT, in the presence of miR-186-5p mimic. C) Predicted miR-186-5p binding site in the HDAC2 3′-UTR (WT) and MUT construct. D) Dual-luciferase assay showing decreased activity for HDAC2-WT, but not HDAC2-MUT, with miR-186-5p mimic. E) RIP demonstrating enrichment of ZEB1-AS1, miR-186-5p, and HDAC2 in Ago2 immunoprecipitates. F-G) miR-186-5p and HDAC2 levels in LVH hearts (n = 20) versus normal donors (n = 10) by qRT-PCR. All experiments were performed in triplicate. *p < 0.05, **p < 0.01. Ago2: Argonaute-2; CMV: cytomegalovirus; HDAC2 histone deacetylase 2; LVH: left ventricular hypertrophy; miR-NC: negative-control mimic; MUT: mutant; qRT-PCR: quantitative real-time polymerase chain reaction; RIP: RNA immunoprecipitation; UTR: untranslated region; WT: wild type.

ZEB1-AS1 promotes CH by regulating the miR-186-5p/HDAC2 pathway

To test whether ZEB1-AS1 drives CH via the miR-186-5p/HDAC2 axis, we performed rescue experiments. HDAC2 mRNA was measured in ISO-stimulated AC16 cells transfected with sh-NC, sh-ZEB1-AS1, or co-transfected with sh-ZEB1-AS1 plus a miR-186-5p inhibitor. ZEB1-AS1 knockdown significantly reduced HDAC2 mRNA, and this reduction was reversed by miR-186-5p inhibition (Figure 4A). Likewise, the ISO-induced increase in cell surface area was attenuated by sh-ZEB1-AS1, whereas concurrent inhibition of miR-186-5p abrogated this effect (Figure 4B). In parallel, ZEB1-AS1 knockdown decreased the ISO-induced mRNA and protein expression of hypertrophic markers (namely, BNP, ANP, and β-MHC), and these suppressive effects were reversed by miR-186-5p inhibition (Figures 4C and 4D). Collectively, these results indicate ZEB1-AS1 promotes CH by regulating the miR-186-5p/HDAC2 signaling pathway.

– ZEB1-AS1 promotes CH by regulating the miR-186-5p/HDAC2 pathway. A) HDAC2 mRNA in ISO-stimulated AC16 cells transfected with the indicated constructs measured by qRT-PCR; B) AC16 cell surface area after ISO stimulation and transfection with the indicated constructs assessed by immunofluorescence; C) mRNA levels of BNP, ANP, and β-MHC in ISO-stimulated AC16 cells after transfection measured by qRT-PCR; D) protein levels of BNP, ANP, and β-MHC in ISO-stimulated AC16 cells after transfection measured by Western blot. All experiments were performed in triplicate. *p < 0.05, **p < 0.01. ANP: atrial natriuretic peptide; BNP: B-type natriuretic peptide; β-MHC: beta-myosin heavy chain; CH: cardiac hypertrophy; HDAC2: histone deacetylase 2; ISO: isoproterenol; qRT-PCR: quantitative real-time polymerase chain reaction; sh-NC: short hairpin RNA–negative control; sh-ZEB1-AS1: zinc finger E-box binding homeobox 1 antisense RNA 1-targeting short hairpin RNA; miR-186-5p: microRNA-186-5p.

Discussion

In this study, we show ZEB1-AS1 is significantly upregulated in CH and its silencing attenuates ISO-induced hypertrophic responses in AC16 cells. Mechanistically, dual-luciferase and RIP assays confirmed miR-186-5p is a direct target of ZEB1-AS1 and HDAC2 is targeted by miR-186-5p, establishing a regulatory axis that contributes to CH progression.

LncRNAs were once considered nonfunctional transcriptional byproducts,37 but accumulating evidence indicates they regulate diverse biological processes through interactions with miRNAs, transcription factors, genomic DNA, and RNA-binding proteins. LncRNAs are increasingly recognized in cardiac development and in the pathogenesis of cardiovascular disease. For example, lncRNA MIR217HG exacerbates pressure-overload–induced CH via the miR-138/THBS1 pathway,38 ADAMTS9-AS1/miR-185-5p/KAT7 acts as a suppressive ceRNA network in hypertrophic obstructive cardiomyopathy,39 and the knockdown of DANCR mitigates HF by attenuating CH and fibrosis through the miR-758-3p/PRG4/Smad axis.35 ZEB1-AS1, initially characterized in cancer, has also been implicated across malignancies,25,40,41 but its role in CH remained unclear. Here, we identify ZEB1-AS1 as a pro-hypertrophic lncRNA: it is increased in LVH tissues and in ISO-treated AC16 cells, and its knockdown blunts ISO-induced hypertrophy, indicating a pathogenic role in CH development and progression.

LncRNAs frequently act within ceRNA networks to regulate gene expression post-transcriptionally by sequestering miRNAs and thereby modulating their availability to target mRNAs.42,43 The importance of ceRNA circuitry in CH has been substantiated in a number of studies.44-46 Consistently, we show ZEB1-AS1 functions as a sponge for miR-186-5p — a miRNA reported to be downregulated in cardiac tissue in diabetic cardiomyopathy47 — thus relieving repression of HDAC2 and promoting ISO-induced hypertrophy. To our knowledge, this is the first report implicating miR-186-5p in CH, highlighting its potential as a therapeutic entry point.

HDACs are key contributors to cardiovascular pathology.30-32,48-50 Prior work have shown that HDAC inhibition attenuates CH induced by pressure overload or ISO infusion,51,52 in line with our ISO-based in vitro model. Among HDACs, HDAC2 has received particular attention; phosphorylation at serine 394 is a critical regulatory event in CH progression.53 For example, the anti-hypertrophic effect of plantamajoside in ISO-induced CH is closely associated with HDAC2,32 and EYA4-au1 promotes cardiomyocyte hypertrophy by recruiting Med11 to upregulate EYA4, leading to p27Kip1 downregulation and activation of the CK2α/HDAC2 axis.54 Despite HDAC2’s established role, its upstream regulation via lncRNA–miRNA–mRNA networks has been less clear. Our data indicate HDAC2 is a direct target of miR-186-5p and ZEB1-AS1 promotes CH by modulating the miR-186-5p/HDAC2 axis.

Limitations of study

This study has some limitations. Because lncRNAs often interact with multiple miRNAs and individual miRNAs typically regulate numerous targets, ZEB1-AS1 may influence additional pathways relevant to CH that were not examined here. These alternative mechanisms warrant further investigation. In addition, although the postmortem interval for autopsy-derived tissues was brief, RNA degradation remains a potential source of bias and could affect transcriptomic analyses. Rigorous assessment of RNA integrity is therefore essential to ensure validity and reproducibility, and will be a focus of future work.

 Conclusion

ZEB1-AS1 is markedly upregulated in LVH tissues and in ISO-stimulated AC16 cells. Functionally, ZEB1-AS1 promotes CH by acting as a ceRNA for miR-186-5p to modulate HDAC2 expression. These findings define a ZEB1-AS1/miR-186-5p/HDAC2 axis in CH pathogenesis and provide a mechanistic basis for exploring targeted therapeutic strategies.

This study is not associated with any thesis or dissertation work.

This study was approved by the Ethics Committee of the Weihai Central Hospital under the protocol number WKY-2023-008. All the procedures in this study were in accordance with the 1975 Helsinki Declaration, updated in 2013. Informed consent was obtained from all participants included in the study.

The authors did not use any artificial intelligence tools in the development of this work.

All datasets supporting the results of this study are available upon request from the corresponding author Bingfeng Cao.

There were no external funding sources for this study.