Abstract

Background

   MicroRNA-200b-3p (miR-200b-3p) plays a pivotal role in inflammatory
   responses and is implicated in various inflammatory disorders. In this
   study, we aim to explore the role of miR-200b-3p in the inflammatory
   response in heart failure (HF).

Methods

   Patients diagnosed with heart failure and age-matched healthy controls
   were studied. Peripheral blood samples from participants were collected
   for RNA-seq analysis to explore the expression profile of miR-200b-3p.
   The predictive value of miR-200b-3p and ZEB1 in the prognosis of heart
   failure was evaluated by analyzing the receiver operating
   characteristic (ROC) curve. Bioinformatics analysis and double
   luciferase reporter gene analysis were used to confirm the interaction
   between miR-200b-3p and ZEB1. Real-time quantitative polymerase chain
   reaction (QRT-PCR) was used to detect the expression levels of
   miR-200b-3p and ZEB1 in cardiopulmonary bypass. Additionally, the
   effects of miR-200b-3p on myocardial cell line (H9c2) injury were
   evaluated by enzyme-linked immunosorbent assay (ELISA).

Results

   In the extracardiac circulation of HF patients, miR-200b-3p expression
   was significantly reduced, while ZEB1 levels were notably elevated.
   Analysis of the ROC curve revealed that miR-200b-3p and ZEB1 have
   predictive value in the prognosis of HF patients. The double luciferase
   reporter experiment demonstrated that miR-200b-3p binds to ZEB1 and
   inhibits its expression. Overexpression of miR-200b-3p demonstrated a
   remarkable ability to alleviate inflammation and inhibit the damage to
   myocardial cells in vivo.

Conclusion

   MiR-200b-3p can target and inhibit ZEB1, reducing the inflammatory
   reaction of myocardial cells. The miR-200b-3p/ZEB1 network may be
   helpful in preventing and treating HF.

   Keywords: Heart failure, MiR-200b-3p, ZEB1, Inflammatory response, ROC
   curve

Introduction

   Heart failure (HF) is a prevalent global epidemic and a primary cause
   of sudden mortality. As the population ages, the incidence and
   prevalence of this condition continue to escalate [[33]1, [34]2]. HF is
   a complicated condition that may be driven on by many factors, the most
   common of which include hypertension, age, diabetes, coronary heart
   disease, and obesity [[35]3]. Over the last several decades, various
   treatment methods, such as pacing and defibrillation treatment, heart
   transplantation or mechanical assisted circulation support, have been
   widely used in HF [[36]3]. However, insufficient research progress has
   been made on the pathophysiology and molecular mechanisms of HF,
   leading to a significant decrease in the quality of life for HF
   patients [[37]4]. Consequently, gaining a comprehensive understanding
   of the intricate mechanisms underlying HF will help identify new
   targets and even expand the window for treating HF.

   Numerous studies have illustrated that microRNAs (miRNAs) participated
   in various cellular processes. Within the miR-200 family, miR-200b-3p
   holds significant importance [[38]5, [39]6]. Prior investigations have
   illustrated that miR-200b-3p participated in regulating the prognosis
   of HF [[40]7, [41]8]. Nevertheless, the precise function of miR-200b-3p
   in the onset and progression of HF remains limited, especially its
   signal transduction pathway and its interaction with some mRNA in the
   prognosis of HF.

   ZEB1, as a transcription regulatory factor, induces endometrial
   transformation and exerts a pivotal influence on both normal
   physiological and pathological processes [[42]9]. It was originally
   recognized as a DNA-binding protein that encompasses a homology domain
   along with two clusters of zinc fingers, which facilitate its
   interaction with two high-affinity binding sites located in the
   E-cadherin promoter region, thereby regulating the biological function
   of cells [[43]10]. Research has found that ZEB1 can regulate IL-6 and
   IL-8 protein production, promoting inflammatory response. Inflammation
   assumes a close relationship with acute and chronic HF, and multiple
   inflammatory mediators are crucial to the progression of HF and
   cardiovascular disease [[44]11]. Despite this recognition, the
   regulatory impact of ZEB1 on cellular immune inflammation in HF remains
   insufficiently explored. Consequently, our study aims to clarify the
   regulating impact of miR-200b-3p on ZEB1, and further analyzed the
   biological role of this miRNA in the prognosis of HF.

Materials and methods

Main reagent

   Myocardial cell line H9c2 (National Biomedical Experimental Cell
   Resource Bank, USA), DMEM medium (Gibco, USA), Fetal bovine serum (FBS,
   Hyclone, USA), X-tremeGENE9 (Roche, Switzerland), double luciferase
   reporter gene detection system kit (Sigma, Germany), overexpression of
   miR-200b-3p vector (Genechem, Shanghai), ELISA kit (Mlbio, Shanghai),
   Polyclonal antibodies (Abcam, USA), isoproterenol (Sigma, Germany).

Collection of sample tissues

   The subjects were 100 patients diagnosed with heart failure at
   Yongchuan Hospital of Chongqing Medical University. The inclusion
   criteria were as follows: (1) adults over 18 years old, (2) diagnosed
   with heart failure (clinically using the Framingham standard or
   echocardiography), and (3) received follow-up care in the heart clinic
   for at least three months. The exclusion criteria included severe
   respiratory diseases, chronic inflammation, organic heart disease, and
   hyperthyroidism. The normal control group consisted of 50 age-matched
   healthy subjects without cardiovascular disease or metabolic disorders.
   This study received approval from the Medical Ethics Committee of
   Yongchuan Hospital of Chongqing Medical University. The peripheral
   blood of the patients was collected and separated into serum and blood
   cells. The blood cells were stored at -80 °C after adding the
   appropriate RNAlater solution.

RNA-seq analysis

   RNA extraction from blood cells was performed using TRIzol reagent.
   Following quality inspection, a cDNA library was constructed. After
   passing the quality control, the Illumina HiSeq4000 platform was
   utilized for high-throughput sequencing. The sequencing process and
   result analysis were conducted by Beijing Nuohe Zhiyuan Company.
   Specifically, |log2(fold change, FC)| > 1 and P < 0.05 were used as
   cutoffs to screen differentially expressed genes. An online website was
   employed to create heat maps. The KOBAS software was utilized to
   conduct KEGG pathway enrichment analysis on the genes associated with
   each distinct set of differentially expressed transcripts, and the
   screening threshold for significant differences was set to P < 0.05.

Cell culture and experimental grouping

   H9c2 cardiomyocytes were cultured in DMEM medium containing 10% FBS and
   Penicillin-Streptomycin (80 U/ml) at 37 °C and 5% O2. For the
   experimental setup, four distinct groups were established: the control
   group, ISO group, ISO-miR-NC group, and ISO-miR-200b-3p group. The
   control group utilized culture medium as the control and added 50 µM
   ISO. Following the instructions of the X-tremeGENE9 kit, the expression
   vector was transfected into H9c2 myocardial cells. After 24 to 48 h of
   cell culture, well-growing cells were selected for subsequent
   experiments.

QRT-PCR experiment

   QRT-PCR was conducted following the protocols outlined in the SYBR
   fluorescence quantitative assay kit instructions. The QRT-PCR reaction
   was set for 40 cycles with an initial denaturation at 94 °C for 5 min,
   followed by denaturation at 94 °C for 20 s, annealing at 60 °C for
   1 min, and signal collection at 60 °C. To perform relative quantitative
   analysis, internal references such as GAPDH and U6 were utilized. The