miR‐19b controls cardiac fibroblast proliferation and migration

Abstract Cardiac fibrosis is a fundamental constituent of a variety of cardiac dysfunction, making it a leading cause of death worldwide. However, no effective treatment for cardiac fibrosis is available. Therefore, novel therapeutics for cardiac fibrosis are highly needed. Recently, miR‐19b has been found to be able to protect hydrogen peroxide (H2O2)‐induced apoptosis and improve cell survival in H9C2 cardiomyocytes, while down‐regulation of miR‐19b had opposite effects, indicating that increasing miR‐19b may be a new therapeutic strategy for attenuating cellular apoptosis during myocardial ischaemia–reperfusion injury. However, considering the fact that microRNAs might exert a cell‐specific role, it is highly interesting to determine the role of miR‐19b in cardiac fibroblasts. Here, we found that miR‐19b was able to promote cardiac fibroblast proliferation and migration. However, miR‐19b mimics and inhibitors did not modulate the expression level of collagen I. Pten was identified as a target gene of miR‐19b, which was responsible for the effect of miR‐19b in controlling cardiac fibroblast proliferation and migration. Our data suggest that the role of miR‐19b is cell specific, and systemic miR‐19b targeting in cardiac remodelling might be problematic. Therefore, it is highly needed and also urgent to investigate the role of miR‐19b in cardiac remodelling in vivo.


Introduction
Cardiac fibrosis represents a fundamental constituent of many cardiac dysfunction, including dilated, ischaemic, and hypertrophic cardiomyopathies, myocardial infarction and heart failure [1,2]. In addition, cardiac fibrosis can also be a primary disease independent of preexisting cardiomyocyte injury [3][4][5][6]. Cardiac fibrosis has been constituted as a major cause of death worldwide due to the fact that it can increase cardiac stiffness, impair cardiomyocyte contractility and disrupt electrical coupling [7]. However, currently there is no effective treatment for cardiac fibrosis in general [1,4]. Therefore, novel therapeutics for cardiac fibrosis are highly needed [3].
The miR-17/92 cluster is a best-explored miRNA cluster and its dysregulation has been related to many diseases including cardiovas-cular diseases [22][23][24][25]. Among the miR-17/92 cluster, miR-19b is a key component [22,25,26]. Recently, it has been reported that miR-19b was able to protect hydrogen peroxide (H 2 O 2 )-induced apoptosis and improve cell survival in H9C2 cardiomyocytes, while down-regulation of miR-19b had inverse effects, indicating that increasing miR-19b may be a new therapeutic strategy for attenuating cellular apoptosis during myocardial ischaemia-reperfusion injury [27]. Considering the fact that miRNAs might exert a cell-specific role [19], it is highly interesting to determine the role of miR-19b in cardiac fibroblasts.
Cardiac fibroblasts are a major type of cells responsible for cardiac fibrosis [20]. The proliferation, migration and activation of cardiac fibroblasts play an important role in the genesis of cardiac fibrosis [6,20,28,29]. Activated cardiac fibroblasts are featured with increased synthesis of protein, including collagen I, the predominant extracellular matrix protein [20]. In this study, we found that miR-19b was able to promote cardiac fibroblast proliferation and migration. Phosphatase and tensin homologue (Pten) was identified as a target gene of miR-19b. Our data highly suggested that it was urgent to determine the role of miR-19b in cardiac remodelling in vivo. Moreover, as the role of miR-19b is cell specific, systemic miR-19b targeting in cardiac remodelling might be problematic.

Materials and methods
Cardiac fibroblast isolation and culture Neonatal rat cardiac fibroblasts (NRCFs) were isolated from the heart of 0-3 day old Sprague Dawley SD rats. Ventricle tissues were minced and digested in 0.06% collagenase (Gibco, San Diego, CA, USA) and 0.04% pancreatin (Sigma-Aldrich, St. Louis, MO, USA) containing solution at 37°C. All cells were pre-plated for 1 hr so that NRCFs could rapidly adhere to the plate. After that, cardiomyocytes were removed and NRCFs were cultured in Dulbecco's modified Eagle's medium (DMEM; Corning, NY, USA) containing 10% Fetal Bovine Serum (BI, Beit-Haemek, Israel), 100 mg/ml streptomycin and 100 U/ml penicillin. All cells were maintained in a 37°C incubator containing 5% CO 2 . These NRCFs were defined as passage 0 (P0). NRCFs were passaged once every 4 days, and NRCFs at passage 2 (P2) were used in the following studies.

Cell proliferation assay
Forty-eight hours after miR-19b mimics, miR-19b inhibitors, and their negative-controls transfection, cell proliferation was analysed by Cell Counting Kit (CCK-8, Dojindo, Japan) and 5-ethynyl-2 0 -deoxyuridine (EdU) Kit (RiboBio, Guangzhou, China). CCK-8 was added 1 hr before the terminal time of the experiment and was incubated in the 37°C incubator. The data were analysed by a Microplate Absorbance Reader (Biorad, Richmond, CA, USA) under the absorbance of 450 nm. During EdU assays, NRCFs were incubated with 10 lM EdU for 24 hrs before tested.

Cell migration assay
NRCFs at P2 were plated in the 12-well plate. After forming the monolayer, a wound was made by a line each well with a 200-ll pipette tip. Cell migration was determined by the width of the initial scratched area at 0 hr and the width of the terminal scratched area at 48 hrs.

Quantitative real-time reverse transcriptasepolymerase chain reactions (qRT-PCRs)
Total RNA was extracted from NRCFs using the Trizol Reagent (Invitrogen). For mRNA detection, 400 ng of total RNA was used to the reverse transcription reaction via Bio-Rad iScript TM cDNA Synthesis Kit (Bio-Rad). The RT product was subjected to 40 cycles of quantitative PCR with Takara SYBR Premix Ex Taq TM (Tli RNaseH Plus, TaKara, Dalian, Liaoning Province, China) in a CFX96TM Real-Time PCR Detection System (Bio-Rad). 18S was used to normalize Pten gene. The sequences of Pten primer: forward CAATGTTCAGTGGCGAACTT (5 0 -3 0 ) and reverse GGCAATGGCTGAGG GAACT (5 0 -3 0 ). The sequences of 18S primer: forward ATTCGAACGTCTGCCC TATCAA (5 0 -3 0 ) and reverse CGGGAGTGGG- The Bulge-Loop TM miRNA qRT-PCR Primer Set (Ribobio, Guangzhou, China) was used for reverse transcription reaction via the Bio-Rad iScript TM cDNA Synthesis Kit (Bio-Rad). The Takara SYBR Premix Ex Taq TM (Tli RNaseH Plus) was used to determine the expression level of miR-19b by qRT-PCRs in the CFX96 Real-time PCR Detection System. 5S was used to normalize the expression of miR-19b. Relative expression levels for each mRNA and miRNA expression were calculated by the 2 ÀMMCT method.

Statistical analysis
All data were presented as mean AE SEM, and an independent-sample t-test was used to calculate all data via SPSS version 19. P < 0.05 was settled as the limit of statistical significance.

miR-19b promotes cardiac fibroblast proliferation
To investigate the role of miR-19b in cardiac fibroblasts, cardiac fibroblasts were transfected with miR-19b mimics or miR-19b inhibitors to overexpress or knock-down miR-19b, respectively. Forty-eight hours after transfection, qRT-PCRs were used to determine the expression level of miR-19b. We confirmed that transfection with 50 nM miR-19b mimics increased miR-19b expression, whereas transfection with 100 nM miR-19b inhibitors decreased that (Fig. 1A). miR-19b mimics were found to be able to promote cardiac fibroblast proliferation as evidenced by both CCK-8 and EdU assays ( Fig. 1B and C), while miR-19b inhibitors had opposite effects ( Fig. 1B and C). Collectively, our data suggest that miR-19b was both sufficient and required for cardiac fibroblast proliferation.

miR-19b enhances cardiac fibroblast migration
The regulatory effect of miR-19b on migration was determined based on unhealing distance. The smaller the unhealing distance, the greater   the migration capacity. It was found that miR-19b mimics significantly decreased the unhealing distance of cardiac fibroblasts, while miR-19b inhibitors increased that ( Fig. 2A), indicating that miR-19b was a positive regulator of cardiac fibroblast migration.
As collagen synthesis is also a major function of cardiac fibroblasts [21,30], we determined the effect of miR-19b mimics and inhibitors in regulating collagen I expression. However, miR-19b mimics and inhibitors did not modulate the expression level of collagen I (Fig. 2B).

Pten is identified as a target gene of miR-19b in cardiac fibroblasts
Phosphatase and tensin homologue (Pten) is a well-known target gene of miR-19b and has recently been found to be a target gene of miR-19b in H9C2 cardiomyocytes [27]. However, due to the cell-specific effects of miRNA [13,20,21,31], if Pten is a target gene of miR-19b in cardiac fibroblasts is unclear. To investigate the relationship between miR-19b and Pten in cardiac fibroblasts, we transfected cardiac fibroblasts with miR-19b mimics and inhibitors and determined the protein expression level of Pten using Western blotting. We found that miR-19b mimics down-regulated while miR-19b inhibitors upregulated Pten at the protein level (Fig. 3A). Two siRNAs for Pten were used in this study to exclude the off-target effects. Both Pten siRNAs used here could decrease Pten at least at mRNA level (Fig. 3B). Thus, in the following studies, Pten siRNAs were used to further investigate if Pten was responsible for the effect of miR-19b in cardiac fibroblast proliferation and migration. As determined by EdU staining, silencing Pten was able to increase cardiac fibroblasts proliferation and migration, while the inhibitory effect of miR-19b inhibitor in both cardiac fibroblast proliferation and migration could be blocked by Pten siRNAs (Fig. 3C and Fig. 4). These results indicate that Pten is a target gene of miR-19b responsible for the effects of miR-19b in cardiac fibroblasts.

Discussion
Cardiac fibrosis is the final most common pathway of lots of cardiac disorders and diseases [21]. Unfortunately, currently no specific treatment has been approved for cardiac fibrosis worldwide [21]. Thus, new therapeutics for cardiac fibrosis is highly desirable [20]. Here, we found that miR-19b was able to promote cardiac fibroblast proliferation and migration, and Pten was identified as a target gene of miR-19b responsible for the effects of miR-19b in cardiac fibroblasts. The miR-19/Pten axis might be a novel therapeutic target for cardiac fibrosis.
It should be noted that although we consider reactive fibrosis to be undesirable within viable myocardium, fibrosis sometimes is desirable [30]. For example, replacement fibrosis has been considered as an essential initial step for heart repair, and it is also important for compensating the limited regenerative capacity of the adult heart in damage [30]. As miR-19b could improve cell survival and protect H 2 O 2 -induced apoptosis in H9C2 cardiomyocytes [27], it is highly interesting and urgent to detect the effects of miR-19b in cardiac remodelling and heart failure in vivo.
Emergency evidence have proved that abnormal expressions of miRNAs were linked to cardiac fibrosis, including miR-1, miR-21, miR-22, miR-24, miR-26a, miR-101, miR-652, etc. [19,21,32]. In this study, we found that miR-19b could promote cardiac fibroblast proliferation and migration, while miR-19 has also been proved to be able to protect cardiomyocyte apoptosis and improve cell survival [27]. This is not a unique case as miRNA exerts biological effects in a cell-specific manner [27]. For example, miR-21 inhibition has been reported to be able to reduce cardiac fibrosis, while it (miR-21 inhibition) may also enhance cardiomyocyte hypertrophy [5,6,19,21,32]. Besides that, similar phenomenon has been observed with miR-24 and miR-15 [19]. Interestingly, miR-19b has been reported to blunt the activated hepatic stellate cell phenotype by morphological assessment and decreased a-SMS expression by targeting TGFbRII [33]. Moreover, miR-19b has been found to reduce hepatic stellate cell proliferation by targeting GRB2 in hepatic fibrosis models both in vivo and in vitro [34]. This indicates a tissue-specific role of miR-19b. Thus, considering the fact that off-target effects or opposing effects of miRNAs exist in different cells and tissues, in vivo studies are needed to test if enhancing or inhibiting a miRNA could be viewed as a useful therapy for cardiac fibrosis treatment. Also, it is extremely important that novel cardiovascular therapy delivery systems by targeting a specific cell population are supposed be developed to optimize miRNA therapy strategies in cardiac repair.
Several limitations of this study should be highlighted. First, this study investigated the effects of miR-19b in cardiac fibroblasts in baseline condition. It would be interesting to explore the effects of miR-19b in cardiac fibroblasts under stress such as TGF-b1 and angiotensin II stimulation. Second, the upstream molecules responsible for the inducible effects of miR-19b in cardiac fibroblasts are unclear. Third, though beyond the scope of this study, it is highly urgent and important to investigate if miR-19b inhibition in vivo is beneficial or harmful in cardiac remodelling and heart failure in the future.
In conclusion, this study indicates that miR-19b promotes cardiac fibroblast proliferation and migration by targeting Pten. The effects of pharmacological inhibition of miR-19b in cardiac remodelling and heart failure in vivo need to be determined in the future.