MiRNA-Based Regulation of Hemostatic Factors through Hepatic Nuclear Factor-4 Alpha

MiRNAs have been reported as CIS-acting elements of several hemostatic factors, however, their mechanism as TRANS-acting elements mediated by a transcription factor is little known and could have important effects. HNF4α has a direct and important role in the regulation of multiple hepatic coagulation genes. Previous in vitro studies have demonstrated that miR-24-3p and miR-34a-5p regulate HNF4A expression. Here we aimed to investigate the molecular mechanisms of miR-24 and miR-34a on coagulation through HNF4A. Transfections with miR-24 and miR-34a in HepG2 cells decreased not only HNF4A but also F10, F12, SERPINC1, PROS1, PROC, and PROZ transcripts levels. Positive and significant correlations were observed between levels of HNF4A and several hemostatic factors (F5, F8, F9, F11, F12, SERPINC1, PROC, and PROS1) in human liver samples (N = 104). However, miR-24 and miR-34a levels of the low (10th) and high (90th) percentiles of those liver samples were inversely correlated with HNF4A and almost all hemostatic factors expression levels. These outcomes suggest that miR-24 and miR-34a might be two indirect elements of regulation of several hemostatic factors. Additionally, variations in miRNA expression profiles could justify, at least in part, changes in HNF4A expression levels and its downstream targets of coagulation.


Introduction
High levels of coagulation factors may disturb the fragile balance of the hemostatic system leading to thrombotic events. However, coagulation factors have a substantial interindividual variability in healthy human plasma [1,2] so that the threshold for the individual thrombotic risk will come defined by the joint action of genetic, environmental, and acquired factors [1,[3][4][5]. Among the genetic elements that drive the synthesis of coagulation factors a hereditary component has been described for several of them although the heritable basis for high or low levels of factors remains unknown [1]. Interestingly, common regulatory genes coordinate simultaneously the expression of several clotting factors which would allow to categorize individuals in those with high or low levels of coagulation factors [2,4].
Several inductors and repressors, that weave a complex regulatory net, participate in the expression of HNF4A gene [25,26]. In addition, HNF4A is post-transcriptionally regulated by miRNA [27,28], small non-coding RNAs implicated in protein regulation in a large number of physiological and pathological processes [29]. Takagi et al. were the first describing in vitro the regulation of HNF4α by miR-24 and miR-34a [30].
The more intuitive and better described mechanism of action of miRNAs is as CIS-suppressive-regulatory elements. Thus, up to seven coagulation factor genes have been described to be targets of miRNAs [31]. However, the hypothesis that miRNAs repress common transcription factors then working as TRANS-regulatory elements for some genes remains to be explored. Here, we aimed to thoroughly gain a deeper insight into the physiological modulator role of miRNA in the expression of downstream coagulation targets of HNF4α.

Materials and Methods
Cell culture and tissue samples HepG2 cells (American Type Culture Collection, Manassas, VA) were cultured in modified Eagle's medium (MEM) (ThermoFisher Scientific, Madrid, Spain) at 37°C under 5% CO 2 . Medium was supplemented with 0.1 mM non-essential amino acids and with 10% fetal calf serum (ThermoFisher Scientific, Madrid, Spain). 104 liver samples from white donors were provided by the Research Center of Experimental Pathology Department of La Fe Hospital and CIBERehd (Valencia, Spain) and by St. Jude Children's Research Hospital Liver Resource (Liver Tissue Procurement and Distribution System (NIH Contract #N01-DK-9-2310) and the Cooperative Human Tissue Network) [32]. None of the donors were from a vulnerable population and all donors provided written informed consent that was freely given. Human liver studies were approved by Ethics Committee for Clinical Investigation from Morales Meseguer Hospital in Murcia, Spain (#ESTU-19/12) and performed in accord with the declaration of Helsinki.

Transfection of miRNAs
Briefly, HepG2 cells were seeded twenty four hours before transfection in complete MEM medium supplemented with 10% fetal bovine serum at 37°C in a humidified incubator with 5% CO 2 without antibiotics and transfected with 100 nM of chemically modified double-stranded RNAs that mimic endogenous miRNAs or SCR (Life Technologies, Madrid, Spain) using Pep-Mute transfection reagent from SignaGen laboratories (Rockville, MD). After 48 h, cells were collected for subsequent mRNA and protein analyses.

RNA isolation and real-time RT-PCR
Total RNA was isolated using RNAzol1RT Reagent (Molecular Research Center, Inc. Cincinnati, OH). The RNA concentration and 260/280 ratio were determined by using NanoDrop spectrophotometer (Thermo Scientific, Wilmington, DE). Retrotranscription reactions were performed using 100 ng of total RNA for each sample according to the manufacturer instructions (Life Technologies, Madrid, Spain).
Quantitative real-time PCR, using TaqMan1 Gene Expression Assays (Applied Biosystems, Madrid, Spain) and gene-specific primers/probes (S1 Table), was performed on a Light-Cycler1 480 Real-Time PCR System (Roche Applied Science, Barcelona, Spain). F11 and F9 were previously quantified in our previous study [33] and their mRNA expression data were used for analyses in this study. Data were analyzed using the comparative threshold cycle method (2 -ΔCt method) with β-actin as an endogenous reference control for quantification and normalization. To quantify expression levels of miRNAs, commercial RT-PCR assays for miR-24, miR-34a, and U6 snRNA (endogenous control) from Life Technologies were used.

Statistical analysis
Statistical differences between groups were calculated by non-parametric Mann-Whitney U test using GraphPad Prism 6 software (GraphPad Software Inc., San Diego, CA). A p-value <0.05 was considered to be statistically significant. Correlations were observed with Pearson's correlation of coefficient and analyses were carried out using Statistical Package for Social Science (version 21.0; SPSS, Chicago, IL).

HNF4A correlates with coagulation factor expression levels in human liver
To verify whether HNF4A modulates the expression of coagulation factors in human liver, we quantified mRNA levels of HNF4A and 9 genes involved in coagulation (F5, F9, F10, F11, F12, SERPINC1, PROC, PROZ, and PROS1) in 104 human liver samples. We observed a widespread interindividual variation in expression levels of the analyzed hepatic transcripts (Fig 1).
As shown, the wider expression variability was found for F9 (14-400%) and SERPINC1 (6-640%) whereas PROC and PROZ had the lower range of variability (Fig 1). The distribution by percentiles according with the expression of the analyzed factors showed up to 3-fold differences among percentile 25 th and 75 th (Fig 1). Similarly, a wide variability was also observed for miR-24, as shown in Fig 1. Next, we analyzed the correlation between HNF4A expression and the same 9 coagulation factors in human livers. We found that those samples with higher coagulation factor expressions had also higher HNF4A expression (all p<0.0001; Fig 2).
These results suggest that differences in HNF4A expression might explain, at least in part, interindividual variations seen in the expression of coagulation factors in human liver.

In vitro study in HepG2 cells
To study the possible indirect effect of miRNAs on coagulation factor expression, we selected two miRNAs, miR-24 and miR-34a, previously pointed as direct inhibitors of HNF4α [27,30]. Both miRNAs bind to several sites in human HNF4A, as described in Fig 3. While miR-34a interacts in three different sites located within HNF4A 3'UTR, miR-24 mostly inhibits HNF4α expression by binding to sites located within the coding region [27] (Fig 3).
MiR-24 regulates coagulation factors by targeting HNF4A. The functional role of miR-24 in regulating HNF4α downstream targets was tested in HepG2 cells by transfecting with miRNA mimics. As expected, results from western blot analysis using whole cell lysates from HepG2 confirmed a decrease of 70% of HNF4α mediated by miR-24 (p = 0.01) ( Fig 4A) and a decrease of 25% in mRNA levels ( Fig 4B). To investigate whether the decrease of HNF4α was   accompanied by a decrease of coagulation factors, we determined mRNA levels by qRT-PCR analysis. HepG2 transfection with miR-24 caused a decrease in mRNA of all selected factors although such reduction was only statistically significant for F10, F12, PROS1 and SERPINC1 (Fig 4C).
MiR-34a regulates coagulation factors by targeting HNF4A. The functional role of miR-34a on HNF4α was tested in HepG2 cells. Similarly to that seen for miR-24, western blot analysis of lysates from HepG2 showed a significant decrease of HNF4α (Fig 4A) and HNF4A mRNA (Fig 4B), as previously described [27,30].
We next investigated the consequences of HNF4α inhibition by miR-34a on coagulation factors. For this, mRNA levels of selected factors were tested by qRT-PCR. As shown in Fig 4B, miR-34a induced a decrease in the expression of all tested factors.
Overall, these results confirmed the role of miR-24 and miR-34a in regulating HNF4α expression and showed a new trans-mechanism of regulation of several coagulation factors by miRNA through HNF4α.

Inverse correlation between HNF4A and both miR-24 and miR-34a expression levels in human livers
To verify the impact of miR-24 and miR-34a variations on HNF4Aand because substantial variability in the expression of HNF4A and its downstream coagulation genes targets remains unexplained, we analyzed ex vivo miRNA-mRNA correlations in human liver samples. We found a slight inverse correlation between HNF4A and both miR-24 and miR-34a hepatic transcript levels (r = -0.170; p = 0.08 and r = -0.228; p<0.05; respectively) ( Fig 5).
Interestingly, liver samples with extreme miRNA levels (percentiles 10 th and 90 th ) showed inverse levels of HNF4A and coagulation factors transcripts, in some cases, such correlations were statistically significant (Fig 6). As shown in Fig 6A, liver samples with lower expression of miR-24 had significantly higher expression of HNF4A, F9, F11, PROS1 and PROZ. In turn, liver samples with lower levels of miR-34a also had the following higher coagulation factors levels: HNF4α, F9, PROS1 and PROZ (Fig 6B).

Discussion
Previous data from our group showed a noticeable variability in transcript levels of F11 and F9 in human healthy livers [33] suggesting that a common genetic regulator is behind specific clustering patterns for human hepatic coagulation factors. However, molecular basis underlying the wide coagulation factors variability in normal population probably involve a combination of factors. Transcriptional factors are ideal candidates to investigate in this framework given their crucial role in controlling mRNAs processing. Moreover, the tandem of some transcription factors and miRNA is an essential network for several biological processes, and its identification has elucidated some of its functions in inflammation [34] and drug metabolism [35] but it still remains to be described in hemostasis. Therefore, we aimed to investigate the contribution of miRNAs as novel participants in the variable expression of coagulation factors as well as to investigate the role of HNF4α in this regulatory loop.
Our first interesting finding was positive correlations between HNF4A and the mRNAs of several coagulation genes. To our knowledge these are the first data describing transcriptional relationship between HNF4A and a large number of hepatic coagulation factors in human liver.
The bibliographic review ofstudies that experimentally validated HNF4A regulation by miRNA, conducted us to select miR-24 and miR-34a as indirect regulators candidates of several coagulation factors [27,30]. Our in vitro results showed that miR-24 and miR-34a had a significant impact on the expression of coagulation factors in HepG2 (Fig 4B). Similarly, both miRNAs inhibited HNF4α expression in human hepatocytes, suggesting a TRANS-regulation of several coagulation factors under miRNA control as a novel mechanism in hemostasis.
Additionally, we investigated whether the integrated regulatory association of miR-24, miR-34a, and HNF4A observed in vitro could also take place in human healthy livers. We first tested the relationship between HNF4A mRNA and both miRNA levels confirming that liver samples with higher HNF4A transcript are those with the lower miRNAs levels, and vice versa. The idea that miR-24 and miR-34a gene regulation is mediating as a significant mechanism contributing to variation in gene expression has been previously documented. Lamba et al [35] demonstrated that miR-34a expression was significantly and negatively correlated with expression levels of multiple hepatic transcription factors (including HNF4α) and that it was involved in a significant mediation of the association observed between CYP2C19 and HNF4α [35]. Then, our results while confirming the miR-34a/HNF4A interaction, for the first time also showed a key regulatory role of miR-34a in hepatic coagulation genes in humans.
In turn, we also confirmed here a regulatory connection between miR-24 and HNF4A, as Hatziapostolou et al did in samples from 12 healthy livers [34]. Our series of human liver samples extended to 104, and provide additional consequences for miR-24/HNF4A interaction, as it had repercussions on the levels of coagulation factors transcripts. Of note, liver samples with extreme expression of miR-24 levels (percentiles 10 th and 90 th ) were, in the opposite, those with utmost HNF4A and coagulation factors expression. Similar findings can be inferred from data reported by Hatziapostolou et al [34].Thus, these authors described that transient inhibition of HNF4A in an hepatocellular carcinoma model drives a feedback loop circuit through several inflammatory miRNAs, and among them miR-24 [34]. Moreover, our in vitro data supported a lower inhibitory effect for miR-24 in comparison with miR-34a, which might explain that only statistically significant values are found for liver samples from extreme percentiles (Fig 6). Alternatively, the sample size might be insufficient to reach statistical power (Fig 5).
In summary, our results suggest that miR-24 and miR-34a participate in the interindividual variability observed in expression of coagulation factors genes in humans. Therefore, in both circuits HNF4A would work as a key switchboard. It remains to be clarified to what extent genetic variations of miRNAs that change HNF4α expression and activity may affect the expression of hepatic coagulation factors, and to what extent this may affect the development of thrombotic or hemorrhagic disorders.
Supporting Information S1