MiR‐590‐3p regulates proliferation, migration and collagen synthesis of cardiac fibroblast by targeting ZEB1

Abstract Previous studies have implicated the attractive and promising role of miR‐590‐3p to restore the cardiac function following myocardial infarction (MI). However, the molecular mechanisms for how miR‐590‐3p involves in cardiac fibrosis remain largely unexplored. Using human cardiac fibroblasts (HCFs) as the cellular model, luciferase report assay, mutation, EdU assay and transwell migration assay were applied to investigate the biological effects of miR‐590‐3p on the proliferation, differentiation, migration and collagen synthesis of cardiac fibroblasts. We found that miR‐590‐3p significantly suppressed cell proliferation and migration of HCFs. The mRNA and protein expression levels of α‐SMA, Col1A1 and Col3A were significantly decreased by miR‐590‐3p. Moreover, miR‐590‐3p directly targeted at the 3’UTR of ZEB1 to repress the translation of ZEB1. Interfering with the expression of ZEB1 significantly decreased the cell proliferation, migration activity, mRNA and protein expressions of α‐SMA, Col1A1 and Col3A. Furthermore, the expressions of miR‐590‐3p and ZEB1 were identified in infarct area of MI model in pigs. Collectively, miR‐590‐3p suppresses the cell proliferation, differentiation, migration and collagen synthesis of cardiac fibroblasts by targeting ZEB1. These works will provide useful biological information for future studies on potential roles of miR‐590‐3p as the therapeutic target to recover cardiac function following MI.

to myofibroblasts to produce fibrillar collagens (types I and III) 2,7 to support the structural and shape of myocardial cells. 8 Although a growing body of works has been carried out on cardiac fibrosis, 9 the underlying mechanisms of cardiac fibrosis after MI remain unclear.
Currently, microRNAs (miRNAs), comprising short endogenous non-coding RNAs of approximately 22 nucleotides in length, have been highly suggested to involve in the processes of cardiac fibrosis following MI. [10][11][12] Previous studies have demonstrated that, compared to normal humans, the expression levels of miR-26a-1, miR-146a and miR-199a-1 are significantly higher in the plasma of acute MI patients, 13 and these miRNAs are suggested to be the potential biomarkers for acute MI diagnosis. 13 miR-486-5p has been reported to suppress cardiomyocyte apoptosis and improve cardiac function though activation of PI3K/Akt pathway in rats. 14 Compared to the wild-type mice, double deletion of miR-133a-1 and miR-133a-2 mice show a 2.5-fold increase in cardiomyocyte proliferation 15 in hearts with an elevated expression of α-smooth muscle actin (α-SMA), 15 which is critical for fibroblasts differentiation to myofibroblasts. 16 miR-185-5p dramatically expresses lower in hearts from mice following MI than that from normal mice, and miR-185-5p has been confirmed to inhibit cell proliferations, migrations and tube formations at cellular level. 17  Previous studies have demonstrated miR-590-3p gets involved in cardiac fibrosis. [18][19][20] The administration of synthetic miR-590-3p lipid formulations immediately after MI in mice results in marked reduction of infarct size and persistent recovery of cardiac function. 18 Besides, we previously predicted that zinc finger E-box binding homeobox 1 (ZEB1) gene, which is a transcription factor and is known for the proliferation and invasion of various cells 21,22 including human cardiomyocytes, 10 is the potential target of miR-590-3p.
After MI, the expression of ZEB1 is significantly induced in mice. 23 ZEB1 has been reported to bind at the promoter of CXCR4 gene, which seems to restore cardiac fibrosis, 24 to inhibit the expression of CXCR4 in human cardiac cells to aggravate MI. 25 Moreover, overexpression of ZEB1 up-regulates the expressions of collagen crosslinking enzymes as well as the expressions of Col1A1 and Col3A1 to mediate collagen stabilization and deposition of ECM. 26 Therefore, we hypothesize that miR-590-3p gets involved in the processes of cardiac fibrosis via regulating the biological function of cardiac fibroblasts by targeting ZEB1.
In this study, using human cardiac fibroblasts (HCFs) as the cellular model, the biological functions of miR-590-3p on the proliferation, differentiation, migration and collagen synthesis of cardiac fibroblasts were first explored. The molecular regulation between miR-590-3p and ZEB1 were further identified, and then the biological functions of miR-590-3p-mediated-ZEB1 were characterized. These works will provide useful biological information for future studies on potential roles of miR-590-3p as the therapeutic target to recover cardiac function following MI.

| Ethics approval
All experiments in the present study were performed in accordance with the guidelines of the Animal Care and Use Committee of Guangdong Provincial Key Laboratory for Laboratory Animals and Guangdong Laboratory Animals Monitoring Institute.

| Creation of MI model in pigs
Six young male Juema 27 minipigs weighting 20-25 kg were used to create MI model according to previous studies. 11,28 Briefly, these pigs were raised in Guangdong Provincial Key Laboratory for Laboratory Animals, and this laboratory has been identified and recognized by Association for Assessment and Accreditation of Laboratory Animal Care International. These anesthetized pigs were randomly divided into sham operation control group (n = 3) and MI group (n = 3). After supine bound, these pigs were transected 7-10 cm in the left third intercostal space to expose the heart. Three MI pigs were created by permanent ligation of the trunk near one third of the apex after the first branch. The thoracic cavity was opened, and sutures were placed in the approximate position without ligation for the other three pigs for sham operation as the control group. BeneViewT5 and EDAN H100 were used to monitor the basic vital signs of animals. The success of ligation was judged and elevated by ST segment of electrocardiogram.

| Quantitative real-time polymerase chain reaction (qRT-PCR)
The total RNA was extracted from HCFs by using Trizol reagent (Invitrogen) according to the manufacturers protocol. The quantity of RNA was assessed spectrophotometrically using a Nanodrop One (NanoDrop Technologies, Thermo). Then, 0.5 g of total RNA was reverse transcribed into cDNA using Reverse TransScript Kit (Toyobo, Takara).  Table 1.

| Western blotting
Total protein was isolated from the HCFs using the total protein kit (APPLYGEN). Then, the protein was determined using the BCA Protein Assay Kit (Thermo). The primary antibodies were α-SMA

| Statistical analysis
All experiments were repeated at least three times independently.
All data were shown as mean ± standard deviation (SD) of repeated experiments. Student's t test (two-tailed) was used to analyse the significance of mean differences in data by R software. *Indicates P < .05; **indicates P < .05; #indicates P > .05.

| miR-590-3p inhibits differentiation and collagen synthesis of cardiac fibroblasts
To further explore the function of miR-590-3p on differentiation and collagen synthesis of HCFs, the expression levels of markers of differentiation and collagen synthesis were shown in Figure 2.
Compared to control group, the mRNA and protein expression levels of α-SMA were significantly decreased (Figure 2A,B) by miR-590-3p mimic but were significantly increased by miR-590-3p inhibitor ( Figure 2A,B). Furthermore, miR-590-3p mimic was observed to significantly decrease the mRNA and protein levels of Col1A1 and Col3A1 ( Figure 2C,D), and miR-590-3p inhibitor was observed to significantly increase the mRNA and protein levels of Col1A1 and Col3A1 ( Figure 2C,D).

| miR-590-3p suppresses the proliferation and migration by targeting ZEB1
To further explore whether miR-590-3p inhibits the proliferation and migration of cardiac fibroblast by targeting ZEB1 in HCFs, the expressions of ZEB1 was interfered with specific siRNAs (Figures 4 and 5).
Three ZEB1-specific siRNAs (si-ZEB1-1, si-ZEB1-2, and si-ZEB1-3) and a negative control (si-ZEB1-NC) were transfected into HCFs. As shown in Figure 4A, si-ZEB1-1 exhibited the highest inhibition efficiency and thus was selected for knockdown of ZEB1 in HCFs. Compared to NC, siZEB1 significantly decreased the cell proliferation ( Figure 4B) and migration activity ( Figure 4C) of HCFs, and this observation was in line with that of miR-590-mimic ( Figure 4B,C). Compared to NC, miR-590-3p inhibitor significantly increased the cell proliferation ( Figure 4B) and migration activity ( Figure 4C) of HCFs, but miR-590-3p inhibitor + siZEB1 could reverse the biological functions of miR-590-3p inhibitor on the proliferation and migration of cardiac fibroblast ( Figure 4B,C).

| Expression of miR-590-3p and ZEB1 in infarct area of MI model based on miniature pigs
To further explore the biological functions of miR-590-3p and ZEB1 in cardiac fibroblasts after MI, the MI model was created and built in infarct area was significantly lower in MI pigs ( Figure 6A), but the mRNA ( Figure 6B) and protein ( Figure 6C) expression levels of ZEB1 in infarct area were significantly higher in MI pigs. These observations were in line with the supposed role of miR-590-3p and ZEB1 in cardiac fibroblast.

| D ISCUSS I ON
Previous studies have shown that MI causes cardiac fibrosis, cardiac remodelling and heart failure 2,3 due to inadequate regeneration of cardiomyocytes. The cardiac fibroblasts produce excessive extracellular matrix deposition 2 to support myocardial structure 4 and regulate cardiomyocyte proliferation and growth by directly connecting to cardiomyocytes via connexins. 29 Although previous studies have suggested the potential roles of miR-590-3p as the therapeutic target to recover cardiac function following MI, the biological role of miR-590-3p in cardiac fibrosis following MI remain vitally unexplored. In the present study, miR-590-3p was observed to regulate the proliferation, differentiation, migration and collagen synthesis of cardiac fibroblasts. As shown in Figures 1 and 2, miR-590-3p was observed to significantly decrease cell proliferation ( Figure 1C) and migration activity of HCFs (Figure1D).
Moreover, miR-590-3p was found to significantly decrease the mRNA and protein expression levels of α-SMA (Figure 2A,B), which was considered to be the marker for fibroblasts differentiation to myofibroblasts. 16 Also, miR-590-3p was observed to significantly ( Figure 2C,D). These results suggest that miR-590-3p suppresses the proliferation, differentiation, migration and collagen synthesis of cardiac fibroblasts.
In cancer cells, miR-590-3p has been often reported to suppress the cell proliferation and migration. [30][31][32] For example, in head and neck cancer cells, miR-590-3p is reported to suppress the expression of Cyclin B and Cdk1, which are key regulators of cell cycle progression, and then reduce cell proliferation and migration. 31 In hepatocellular carcinoma cells, miR-590-3p suppresses cell growth in vitro and in vivo by targeting TEAD1. 33 In intrahepatic cholangiocarcinoma cells, miR-590-3p dramatically suppresses cell migration and invasion. 32 In breast cancer cells, miR-590-3p suppresses cell survival and triggers cell apoptosis via targeting sirtuin-1 and deacetylation of p53. 30 In breast cancer cells, miR-590-3p has been suggested to decrease cell proliferation and increase cell apoptosis. 34 These findings coupling with the observations in this study support that miR-590-3p suppresses the proliferation, differentiation, migration and collagen synthesis of cardiac fibroblasts. Moreover, miR-590-3p has been shown to be able to attenuate the cardio sphere-derived cells differentiation, which is one type of stem cells in cardiac cell lineages. 35 The administration of synthetic miR-590-3p lipid formulations immediately after MI in mice results in marked reduction of infarct size and persistent recovery of cardiac function. 18 After MI in mice, miR-590-3p is reported to stimulate marked cardiac regeneration and almost complete recovery of cardiac functional parameters. 36 These findings recommend the attractive and promising role of miR-590-3p to restore the cardiac function. These observations suggest the possibility that miR-590-3p suppresses the proliferation, differentiation, migration and collagen synthesis of cardiac fibroblasts and thus promotes the cardiomyocyte proliferation following MI.
In this study, ZEB1 was predicted as the potential target of miR-590-3p by three algorithms: TaregetScan These results demonstrate that ZEB1 promotes the proliferation, differentiation, migration and collagen synthesis of cardiac fibroblasts.
Collectively, it is possible that miR-590-3p suppresses proliferation, differentiation, migration and collagen synthesis of cardiac fibroblasts by targeting ZEB1 and thus regulates the proliferation and differentiation cardiomyocytes during the processes of cardiac fibrosis following MI.

CO N FLI C T O F I NTE R E S T
The authors declare no competing interests.

AUTH O R CO NTR I B UTI O N S
XLY, HZ, and XLW conceived and designed this work; XLY, JCP, BYG and HBG acquired the biological samples and analysed the data; F I G U R E 6 Expression of miR-590-3p and ZEB1 in MI model of pigs. A, Expression of miR-590-3p at infarct area in control and MI pigs. The mRNA (B) and protein (C) expression levels of ZEB1 at infarct area in control and MI pigs. Data are shown as mean ± SD. *Indicates P < .05. **Indicates P < .01