Skip to main content
NCBI home page
NIHPA Author Manuscripts logoLink to NIHPA Author Manuscripts
. Author manuscript; available in PMC: 2020 Dec 1.
Published in final edited form as: Fertil Steril. 2019 Dec;112(6):1180–1189. doi: 10.1016/j.fertnstert.2019.07.1324

Cross-talk Between MiR-29c and TGF-β3 is Mediated by an Epigenetic Mechanism in Leiomyoma

Tsai-Der Chuang 1, Omid Khorram 1
PMCID: PMC6924575  NIHMSID: NIHMS1536024  PMID: 31843095

Abstract

Objective

To determine the expression of miR-29c and its target gene transforming growth factor-β3 (TGF-β3) in leiomyoma and the mechanisms of their reciprocal regulation.

DESIGN

Experimental study

SETTING

Academic research laboratory

PATIENT(S)

Women undergoing hysterectomy for leiomyoma.

Intervention

Over and under expression of miR-29c; blockade of DNA methyltransferase 1 (DNMT1).

MAIN OUTCOME MEASURE(S)

miR-29c, and its target gene TGF-β3 in leiomyoma and the effects of TGF-β3 and blockade of DNMT1 on miR-29c expression.

Results

Leiomyoma expressed significantly lower levels of miR-29c but higher expression of TGF-β3 compared with matched myometrium. The expression of TGF-β3 and miR-29c were independent of race/ethnicity. Using 3’UTR luciferase reporter assay we confirmed that TGF-β3 is a direct target of miR-29c in leiomyoma smooth muscle cells (LSMC). Gain-of-function of miR-29c in LSMC inhibited the expression of TGF-β3 at protein and mRNA levels, whereas loss-of-function of miR-29c had the opposite effect. Treatment of LSMC with TGF-β3 inhibited the expression of miR-29c while it stimulated DNMT1 expression. Knockdown of DNMT1 through transfection with siRNA significantly decreased the expression of TGF-β3, and induced miR-29c expression. Knockdown of DNMT1 also attenuated the inhibitory effect of TGF-β3 on miR-29c expression. Furthermore, we demonstrated that TGF-β3 increased the methylation level of miR-29c promoter in LSMC.

Conclusion

There is an inverse relationship in the expression of TGF-β3 and miR-29c in leiomyoma. TGF-β3 is a direct target of miR-29c and inhibits the expression of miR-29c through an epigenetic mechanism. The cross-talk between miR-29c and TGF-β3 provides a feed forward mechanism of fibrosis in leiomyoma.

Keywords: Leiomyoma, miR-29c, TGF-β3, fibrosis, epigenetics

Capsule

There is a cross talk between TGF-β3 and miR-29c in leiomyoma wherein TGF-β3 inhibits the expression of miR-29c through an epigenetic mechanism involving DNMT1.

Introduction

Leiomyomas are benign fibrotic tumors afflicting a significant number of women being the most common indication for all hysterectomies performed (1). These tumors whose growth is dependent on ovarian steroids are characterized by excess accumulation of extracellular matrix (ECM), inflammation, and increased angiogenesis (2, 3). Additionally, in leiomyomas altered expression of a number of non-protein coding genes, including microRNA (miRNAs), which target the expression of protein coding genes, has been documented (4, 5). The pathogenic mechanisms for leiomyoma initiation and progression have been under intense investigation (6–8). Our laboratory has focused on the mechanism underlying ECM accumulation and we have identified miR-29c, a member of small non-coding RNAs as being pivotal to the fibrosis associated with these tumors (9). The levels of miR-29c are suppressed in fibroids compared with myometrium and are under ovarian steroid control, transcription factors SP1 (specificity protein 1), NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) and epigenetic regulation (9). miR-29c has a number of targets including collagens (9–12), elastin (ELN) (10, 11, 13) and matrix metalloproteinases (MMPs) (11, 14) many of which have been demonstrated to play a role in fibroid pathogenesis (2), and previously published reports demonstrated an increased expression of miR-29c targets in leiomyomas including collagen I (COL-1), collagen III (COL-3), MMPs and epigenetic enzymes DNMTs (DNA methyltransferases) (9, 15, 16). Other groups have reported other members of the miR-29 family namely miR-29a and miR-29b to also be lower in fibroids compared with myometrium (16, 17). Furthermore, overexpression of miR-29b in an animal model for fibroid resulted in shrinkage of tumors (17). Collectively, these studies point to the highly significant role of miR-29 family in fibroid pathogenesis.

TGF-β3 (transforming growth factor-β3) is a pro-fibrotic cytokine which interacts with type 1 and 2 receptors propagating its effect through activation of signaling pathways such as MAPK/ERK (mitogen activated protein kinase/extracellular-signal-regulated kinase) (18), Smad2/3 (SMAD family member 2/3) (19) and PI3K/Akt (phosphatidylinositol 3-kinases/protein kinase B) (20), the end result of which is increased synthesis of collagen I, CTGF (connective tissue growth factor), fibronectin (FN1) and versican V0 (VCAN V0) all of which are critical to leiomyoma progression (18–22). Furthermore, TGF-β3 inhibits the expression of MMPs thus resulting in ECM accumulation (22). Treatment of leiomyoma smooth muscle cell (LSMC) with TGF-β3 but not TGF-β1 (transforming growth factor-β1) stimulated DNA synthesis (23). Others have shown an increase in cell proliferation in response to TGF-β1 (24). A number of factors have been shown to reduce the levels of TGF-β3 and fibrotic genes regulated by TGF-β3 in in vitro studies, including tranilast (15), vitamin D3 (18) and selective progesterone receptor modulators (25–27), whereas activation of β-catenin (Catenin beta-1) which is critical in fibroid pathogenesis induces TGF-β3 (28). The ovarian steroid dependence of TGF-β family and their influence on smooth muscle cell proliferation underscore the significance of TGF-β and its receptors in fibroid pathophysiology. Since TGF-β3 is a predicted target of miR-29c (Targetscan, http://www.targetscan.org) and both miR-29c and TGF-β3 are critical in regulation of fibrosis which is the hallmark of fibroids, we hypothesized that miR-29c might exert its effects on the ECM through modulation of TGF-β3 levels, and TGFβ3 in turn through a feedback loop could influence miR-29c transcription. This hypothesis was tested in an in vitro primary cell culture obtained from fibroids removed at the time of hysterectomy from women on no hormonal medications three months prior to surgery.

Materials and Methods

Tissue Collection and Primary Cell Isolation

Leiomyomas and paired myometrium were obtained from patients (N=42) not on hormonal treatments for at least 3 months prior to surgery at Harbor-UCLA Medical Center with prior approval obtained from LA BioMed at Harbor-UCLA Medical Center Institutional Review Board (#036247). Informed consent was obtained from all the patients participating in the study before surgery. The paired tissues were obtained from white Hispanics (n=14), African Americans (n=14) and Caucasians (n=14) aged 35–56 years (mean 45 ± 5.1 years). Among the Caucasians group 6 pairs were kindly provided by Dr. Al-Hendy (University of Chicago). The tissues were either snap frozen and stored in liquid nitrogen for further analysis, or used for isolation of LSMC as previously described (29). Briefly, LSMC were cultured in DMEM supplemented with 10% fetal bovine serum until reaching confluence with a change of media every 2–3 days. Cells at passages p1 to p3 were used for all experiments. Cell culture experiments were performed at least three times using LSMC obtained from different patients. All supplies for isolation and cell culture were purchased from Sigma-Aldrich (St. Louis, MO), Invitrogen (Carlsbad, CA) and Fisher Scientific (Atlanta, GA).

Gain-or Loss-of-function of miR-29c

LSMC were seeded in 6-well plates and at sub-confluence transfected with 50 nM of pre-miR-29c, anti-miR-29c, pre-miR negative (NC) or anti-miR negative control (aNC) (Applied Biosystems, Foster city, CA) for 72 to 96 hours using PureFection transfection reagent (System Biosciences, Inc., Mountain View, CA) according to the manufacturer’s protocol.

siRNA transfection

LSMC were cultured as above and at sub-confluence transfected with 50 nM of siRNA negative control (siNC) or siRNA against DNMT1 (siDNMT1; Santa Cruz Biotechnology, Dallas, Texas) for 72–96 hours using PureFection transfection reagent (System Biosciences, Inc., Mountain View, CA) according to the manufacturer’s protocol.

RNA Isolation and qRT-PCR Analysis

Total RNA was extracted from LSMC using Trizol (Thermo Fisher Scientific, Waltham, MA) and their quantity and quality was determined (ND-1000 Spectrophotometer, NanoDrop Technologies, Wilmington, DE) as previously described (30–32). Subsequently, RNA sample of 1 μg each was reverse transcribed using random primers for TGF-β3. MiR-29c primer design and PCR conditions have been described previously (33). Quantitative RT-PCR was carried out using SYBR gene expression master mixes (Applied Biosystems, Carlsbad, CA). Reactions were incubated for 10 min at 95°C followed by 40 cycles for 15 seconds at 95°C and 1 min at 60°C. Levels of mRNA and miRNA were quantified using the Invitrogen StepOne System and normalized to FBXW2 (34) and RNU6B, respectively. All reactions were run in triplicate and relative expression was determined using the comparative cycle threshold method (2–ΔΔCT), as recommended by the supplier (Applied Biosystems). Abundance values were expressed as fold changes compared to the corresponding control group. The primer sequences used were as follows: TGF-β3 (sense, 5’-CGGGCTTTGGACACCAATTA-3’; antisense, 5’-GGGCGCACACAGCAGTTC-3’); and FBXW2 (sense, 5’-CCTCGTCTCTAAACAGTGGAATAA-3’; antisense, 5’-GCGTCCTGAACAGAATCATCTA-3’). miR-29c (sense, 5’-GCAGTAGCACCATTTGAAATC-3’; antisense, 5’-GGTCCAGTTTTTTTTTTTTTTTAACC-3’); and RNU6B (sense, 5’-ATTGGAACGATACAGAGAAGATTAG-3’; antisense, 5’-AATATGGAACGCTTCACGAAT-3’).

Immunoblotting

Total protein isolated from paired tissue samples and LSMC following treatment conditions was subjected to immunoblotting as previously described (10, 35). Briefly, samples were suspended in RIPA buffer containing 1 mM EDTA and EGTA (Boston BioProducts, Ashland, MA) supplemented with 1 mM PMSF and a complete protease inhibitor mixture (Roche Diagnostics, Indianapolis, IN), sonicated, and centrifuged at 4°C for 10 min at 14,000 rpm. The concentration of protein was determined using the BCA™ Protein Assay Kit (Thermo Scientific Pierce, Rockford, IL). Equal aliquots (Fifty micrograms) of total protein for each sample were denatured with SDS-PAGE sample buffer, and separated by electrophoresis on an SDS polyacrylamide gel. After transferring the samples to a nitrocellulose membrane, the membrane was blocked with TBS-Tween + 5% milk, and probed with the following primary antibodies: TGF-β3 and DNMT1 (Santa Cruz Biotechnology). The membranes were washed with TBS containing 0.1%Tween-20 wash buffer after each antibody incubation cycle. SuperSignal West Pico Chemiluminescent Substrate™ (Thermo Scientific Pierce, Rockford, IL) was used for detection, and photographic emulsion was used to identify the protein bands, which were subsequently quantified by densitometry. The membranes were also stripped and probed with glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz Biotechnology) serving as the loading control. The densities of the specific protein bands were quantified with a scanning densitometer (Bio-Rad GS-800, Hercules, CA), and the results were expressed as means ± SEM normalized to GAPDH.

Luciferase Reporter Assays

LSMC were seeded in six-well plates and at 70–80% sub-confluence were transfected with 50 nM pre-miR-29c oligonucleotides or a negative control (NC) using the PureFection transfection reagent as previously described (36). At the same time, the cells were co-transfected with a luciferase reporter plasmid (1 μg/well) containing a 3’ untranslated region (UTR) sequence of TGF-β3 (GeneCopoeia, Rockville, MD). After 48 hrs of transfection Firefly and Renilla luciferase activities were measured using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). Firefly luciferase activity was normalized to Renilla luciferase activity. The level of induction was reported as mean ± SEM of three experiments performed in triplicates and compared with a ratio in cells transfected with negative control independently set as 1.

Methylation-specific PCR

LSMC at sub-confluence were treated with TGF-β3 (5 ng/ml) for 48 hours and genomic DNA was extracted and used for bisulfite conversion (EZ DNA Methylation Kit, Zymo Research Corporation, Irvine, CA) as previously described (37). Since methylated cytosine residues remained unchanged after bisulfite conversion this approach allows distinguishing DNA sequences which are methylated or un-methylated in specific genomic regions using sequence-specific PCR primers (38). The pair of primers used to detect the methylated (Me) sequence of miR-29c promoter were 5’-GATGGGATTAAATTTTGGAATATTC-3’ and 5’-AAATCCTAAAACCCGTCGAA-3’ and for the un-methylated (Un) sequence of miR-29c promoter were 5’-GGGATTAAATTTTGGAATATTTGG-3’ and 5’-ATCAAATCCTAAAACCCATCAAA-3’. DNA amplification was carried out following manufacturer’s protocol (HotStarTaq Plus PCR reagent, Qiagen, Valencia, CA). The PCR condition was 5 min at 95°C, followed by 40 cycles of amplification at 94°C for 30 second s, 30 seconds at 55°C, and 1 min at 72°C. PCR amplified products were electrophoresed on 2% agarose gel and visualized under ultraviolet illumination.

Statistical analysis

Power calculation indicated that the sample size estimates for studies have been based on a power of 80% to detect 30% changes between the two groups (Myometrium vs Leiomyoma) (assuming an expected standard deviation of 20% of mean values). This analysis results in a requirement for 10 patients in each group. Throughout the text, all data are presented as mean ± SEM and analyzed by PRISM software (Graph-Pad, San Diego, CA). Dataset normality was determined by the Kolmogorov-Smirnov test. Comparisons involving two groups were analyzed using paired or un-paired Student’s t-tests as appropriate. One-way ANOVA was used for comparisons involving multiple groups. Statistical significance was established at P<0.05.

Results

Using paired leiomyoma and matched myometrium (N=42) the co-expression of miR-29c and TGF-β3 in the same specimens was determined. The analysis indicated that miR-29c (Fig. 1A) expression is reduced while TGF-β3 expression at both mRNA (Fig. 1A) and protein (Fig. 1B and 1C) levels is elevated in leiomyomas as compared to paired myometrium. The analysis further indicated no significant racial/ethnic differences in expression of miR-29c and TGF-β3 (Fig. 1D and 1E). Collectively, this data confirmed previous results from our laboratory and others with regard to miR-29c and TGF-β3 expression in leiomyomas demonstrating an inverse pattern of expression (9, 21, 23).

Figure 1.

Figure 1.

Open in a new tab

(A) The relative expression of miR-29c and TGF-β3 in paired (N=42) myometrium (MYO) and leiomyoma (LYO). *: P<0.05. (B) Western blot analysis of TGF-β3 in paired (N=16) myometrium (M) and leiomyoma (L) with dot plot (C) showing their relative band densities in myometrium (MYO) and leiomyoma (LYO). *: P<0.05. Relative (mean ± S.E.M.) expression of miR-29c (D) and TGF-β3 (E) in LYO and matched MYO from untreated group based on ethnicity in Caucasians (n=14), African Americans (N=14) and Hispanics (N=14). The relative expression was determined by setting the averaged expression value of each gene in myometrium from Caucasians as 1. *: P<0.05.

Since TGF-β3 is a predicted target of miR-29c based on Targetscan, we assessed such regulatory function of miR-29c on TGF-β3 expression in isolated LSMC. As shown in Fig. 2A and 2B, luciferase reporter assay revealed that miR-29c directly interacts with the 3’ UTR of TGF-β3 thereby regulating its expression in LSMC. Transfection of LSMC with pre-miR-29c downregulated the expression of TGF-β3, whereas knockdown of miR-29c with anti-miR-29c, which mimics the expression pattern of miR-29c in leiomyoma, increased the expression of TGF-β3 at mRNA (Fig. 2C) and protein (Fig. 2D and 2E) levels. We also demonstrated that treatment of LSMC with TGF-β3 (5 ng/ml) resulted in inhibition of miR-29c expression (Fig 3A). Since miR-29c expression in LSMC is under epigenetic control (15, 37, 39), we determined whether TGF-β3 mediated inhibition of miR-29c expression is mediated through an epigenetic mechanism. As such we determined the effect of TGF-β3 on epigenetic enzymes namely, DNMT1 and DNMT3A which catalyze DNA methylation and EZH2 which is involved in histone methylation. The analysis indicated that treatment of LSMC with TGF-β3 had no significant effect on DNMT3A and EZH2 expression (data not shown); however, TGF-β3 induced the expression of DNMT1 protein levels (Fig. 3B and 3C). To further establish the role of DNMT1 in mediating the effects of TGF-β3 on miR-29c expression, LSMC were treated with DNMT1 siRNA which effectively inhibited DNMT1 (Fig. 3D and 3E). Inhibition of DNMT1 in LSMC resulted in suppression of TGF-β3 at both mRNA and protein levels (Fig. 3D, 3E and 3F), while it increased the expression of miR-29c (Fig. 3G). Furthermore, treatment of LSMC with DNMT1 siRNA attenuated the inhibitory effect of TGF-β3 on miR-29c expression indicating that the effect of TGF-β3 on miR-29c is mediated by DNMT1 (Fig. 3G). To provide additional support that TGF-β3-induced inhibition of miR-29c is epigenetically mediated, LSMC were treated with TGF-β3 and the methylation status of miR-29c promoter was determined by methylation specific PCR. As shown in Fig. 4A and 4B, TGF-β3 increased the methylation level of miR-29c promoter, thus highlighting the importance of an epigenetic mechanism for TGF-β3-induced inhibition of miR-29c.

Figure 2.

Figure 2.

Open in a new tab

(A) Complementary sequences between miR-29c and 3’UTR of TGF-β3. (B) Shows the relative luciferase activity in isolated LSMC co-transfected with Renilla and firefly luciferase reporter carrying a 3’UTR fragment of TGFβ3, pre-miR-29c or control oligonucleotides (NC) for 48 hrs. The relative luciferase activity is presented as the ratio of Firefly: Renilla as compared to NC which is independently set as 1. (C) qRT-PCR analysis of TGF-β3 mRNA expression in LSMC following transfection with pre-miR-29c or anti-miR-29c oligonucleotides for 72 hrs. (D) Representative western blot analysis of TGF-β3 following transfection of LSMC with pre-miR-29c or anti-miR-29c oligonucleotides for 96 hrs. Bar graph (E) shows the relative band densities of TGF-β3. The results are presented as mean ± SEM of at least three independent experiments with P values (*P<0.05) indicated by corresponding lines.

Figure 3:

Figure 3:

Open in a new tab

(A) The effect of TGF-β3 (5 ng/ml) following 48 hours of culture on the expression of miR-29c in LSMC. The results are presented as mean ± SEM of three independent culture experiments. *: P<0.05. (B-C) Western blot analysis of DNMT1 in LSMC following 24 hours of TGF-β3 (5 ng/ml) treatment and the relative band densities is shown in (C). (D-F) The effect of DNMT1 knockdown through transfection of LSMC with siRNA against DNMT1 for 72 hrs on protein levels of DNMT1 and TGF-β3 determined by western blot (D) along with the relative band densities analysis (E). The mRNA level of TGF-β3 determined by qRT-PCR is shown in (F). The results shown in (B) and (D) are representative of three sets of independent experiments. (G) Shows the effect of transfection with siRNA against DNMT1 for 96 hrs along with TGF-β3 (5 ng/ml) treatment in the last 48 hrs on miR-29c levels in isolated LSMC (n=4). *: P<0.05.

Figure 4.

Figure 4.

Open in a new tab

(A) Graphical depiction of the miR-29b-2/29c with the location of “CpG” indicated the regions tested for DNA methylation status in miR-29c promoters. Methylation-specific PCR shows TGF-β3 (5 ng/ml) mediated increased miR-29c promoter methylation after 48 hours of incubation. The result is a representative of at least 3 sets of independent experiments using LSMC isolated from 3 patients. (B) The bands were quantified with NIH Image J software and the percentage of methylation was calculated as: Methylation / Methylation+Un-methylation (Me / Me+Un). The results are presented as mean ± standard error of the mean (SEM) of at least 3 independent experiments using LSMC isolated from 3 patients. *P <0.05. (C) Schematic diagram representing our working model in which leiomyoma are characterized by reduced miR-29c and elevated TGF-β3 expression. Lower expression of miR-29c leads to overexpression of its target gene, TGF-β3, which causes an increase in extracellular matrix accumulation, and by induction of DNMT1 causes increased methylation of miR-29c promoter resulting in its decreased expression.

Discussion

In the present study we provide evidence for a novel feed forward crosstalk between miR-29c and TGF-β3. We demonstrate an inverse pattern of expression between miR-29c and TGF-β3 in fibroids which is race independent. We confirm that TGF-β3 is a direct target of miR-29c in LMSC and knockdown of miR-29c results in overexpression of TGF-β3 whereas its overexpression has the opposite effect in LMSC. TGF-β3 in turn influences miR-29c expression through an epigenetic mechanism by increasing the expression of DNMT1 resulting in increased methylation of miR-29c promoter. Blockade of DNMT1 by siRNA transfection of LMSC blocked the inhibitory effect of TGFβ3 on miR-29c expression, thus establishing the importance of this epigenetic mechanism. A schematic demonstrating this cross talk between miR-29c and TGF-β3 is shown in Fig. 4C.

Leiomyomas are considered a fibrotic disorder characterized by increased cell proliferation, inflammation, angiogenesis and excess accumulation of ECM where elevated expression of TGF-β family plays a central role (2, 3, 6–8). Both driver mutations [MED12 (mediator complex subunit 12), HMGA2 (high mobility group AT-hook 2), FH (fumarate hydratase), COL4A5 (collagen type IV, alpha 5)] and chromosomal alteration in addition to widespread alteration in DNA methylation in multiple genes critical to fibroid growth have been reported (40, 41). The miR-29 family consists of miR-29a, miR-29b, and miR-29c which are encoded by two gene clusters, and are predicted to target many genes functionally associated with cellular transformation, inflammatory responses and ECM turnover (30, 42–47). It is clear that a low level of anti-fibrotic miR-29c and elevated level of pro-fibrotic TGF-β3 in leiomyoma provides an environment necessary to promote and maintain tumor fibrotic characteristic. MiR-29c directly interacted with the 3’UTR of TGF-β3 regulating TGF-β3 at both transcriptional and translational levels which we further confirmed using gain-or loss-of-function of miR-29c. Although our result is the first to confirm the regulatory function of miR-29c on TGF-β3 expression in leiomyoma, in a recent study using unilateral ureteral obstruction (UUO) model for renal fibrosis, intramuscular injection of exosome-encapsulated miR-29 was shown to target TGF-β3 (48), thus providing support for our observation. Furthermore, we found that TGF-β3 through an epigenetic mechanism involving DNMT1 expression inhibited miR-29c expression. The regulatory action of TGF-β3-induced DNMT1 occurred by increased methylation of miR-29c promoter, thus further support the importance of epigenetic regulatory mechanism in leiomyoma (49). A similar crosstalk involving miR-29b and TGF-β1 and TGF-β2 (transforming growth factor-β2) has been previously reported in trabecular meshwork cells (50), in renal tubular cells where miR-29b was downregulated by TGF-β1 via Smad3 (51, 52), and in cardiac fibroblast cells where TGF-β1 inhibited the expression of miR-29 family (46). However, to our knowledge our data is the first evidence demonstrating a feedback interaction between miR-29c and TGF-β3.

Our data indicated that TGF-β3 induced DNMT1 expression in LSMC which in turn resulted in methylation of miR-29c promoter and lowering of miR-29c. Interestingly, although DNMT1 is not a direct target of miR-29 recent studies have demonstrated that miR-29b by targeting the expression of transcription factor SP1 regulates DNMT1 expression in acute myeloid leukemia cell lines (53, 54). In addition, our laboratory and others have reported that leiomyoma express significantly higher DNMT1 as compared to matched myometrium (15, 55, 56). In line with our findings showing a significant decrease in the expression of TGF-β3 after DNMT1 knockdown in LSMC, zebrafish with a mutation in DNMT1 were reported to have 50% decrease in expression of TGF-β3 in the lens (57). Several studies in other tissues and cells have demonstrated an effect of the TGF-β1 on DNA methylation, including global changes in DNA methylation of genes critical to epithelial to mesenchymal transition in ovarian cancer cell line through induction of DNMT1 and DNMT3A (DNA methyltransferase 3A) and DNMT3B (DNA methyltransferase 3B) (58). In cardiac fibroblast TGF-β1 reduced the expression of DNMT1 and DNMT3A and induced COL1A1 expression (59). Collectively, these studies indicate the tissue/cell specific effect of TGFβ isoforms on DNA methylation.

Our data presented here further support the regulatory function of miRNAs and epigenetic modification of genes that drive tumorigenesis and tissue fibrosis (60–63), and leiomyoma growth and progression (56, 64–67). miRNAs have been implicated in the pathogenesis of a number of gynecologic and obstetrics conditions including endometriosis (68), polycystic ovary syndrome (69), fetal growth restriction (70) and pre-eclampsia (71), and as such have been proposed to serve as biomarkers or targets of therapeutics. Our profiling data indicating the inverse pattern of expression between miR-29c and TGF-β3 expression with anti-fibrotic and pro-fibrotic functions respectively, implicate their importance in leiomyoma pathogenesis. Additionally, the regulatory interaction between miR-29c and TGF-β3 provides a feed forward mechanism which could explain leiomyoma growth through ECM accumulation and an ideal therapeutic target to interrupt fibroid growth. This aim could be achieved by overexpression of miR-29c or inhibition of TGF-β3 to disrupt the fibrotic crosstalk between TGF-β3 and miR-29c. The strength of our study is the in vitro demonstration of the link between TGF-β3 known to play a key role in fibrosis associated with leiomyoma and the anti-fibrotic miRNA miR-29c and the limitation of the study is that it is an in vitro study and unknown if it applies to the in vivo setting.

In summary, our data established a novel interaction between miR-29c and TGF-β3 in leiomyoma cells. In this scheme (Fig. 4C) TGF-β3 is a direct target of miR-29c. Elevated levels of TGF-β3 in fibroids induce the expression of DNMT1 which in turn increases the methylation of miR-29c promoter and its reduced expression. This feed forward axis would result in increased accumulation of the ECM and progression of fibroid growth.

Acknowledgements

We thank Dr. Al-Hendy for kindly providing 6 Caucasian specimens. This study was supported by NIH (HD088868).

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Reference List

  • 1.Doherty L, Mutlu L, Sinclair D, Taylor H. Uterine fibroids: clinical manifestations and contemporary management. Reprod Sci 2014;21:1067–92. [DOI] [PubMed] [Google Scholar]
  • 2.Islam MS, Ciavattini A, Petraglia F, Castellucci M, Ciarmela P. Extracellular matrix in uterine leiomyoma pathogenesis: a potential target for future therapeutics. Hum Reprod Update 2018;24:59–85. [DOI] [PubMed] [Google Scholar]
  • 3.Chegini N Proinflammatory and profibrotic mediators: principal effectors of leiomyoma development as a fibrotic disorder. Semin Reprod Med 2010;28:180–203. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Karmon AE, Cardozo ER, Rueda BR, Styer AK. MicroRNAs in the development and pathobiology of uterine leiomyomata: does evidence support future strategies for clinical intervention? Hum Reprod Update 2014;20:670–87. [DOI] [PubMed] [Google Scholar]
  • 5.McWilliams MM, Chennathukuzhi VM. Recent Advances in Uterine Fibroid Etiology. Semin Reprod Med 2017;35:181–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 6.Islam MS, Protic O, Stortoni P, Grechi G, Lamanna P, Petraglia F et al. Complex networks of multiple factors in the pathogenesis of uterine leiomyoma. Fertil Steril 2013;100:178–93. [DOI] [PubMed] [Google Scholar]
  • 7.Segars JH, Parrott EC, Nagel JD, Guo XC, Gao X, Birnbaum LS et al. Proceedings from the Third National Institutes of Health International Congress on Advances in Uterine Leiomyoma Research: comprehensive review, conference summary and future recommendations. Hum Reprod Update 2014;20:309–33. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Taylor DK, Holthouser K, Segars JH, Leppert PC. Recent scientific advances in leiomyoma (uterine fibroids) research facilitates better understanding and management. F1000Res 2015;4:183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Chuang TD, Khorram O. Mechanisms underlying aberrant expression of miR-29c in uterine leiomyoma. Fertil Steril 2016;105:236–45.e1. [DOI] [PubMed] [Google Scholar]
  • 10.Chuang TD, Sakurai R, Gong M, Khorram O, Rehan VK. Role of miR-29 in mediating offspring lung phenotype in a rodent model of intrauterine growth restriction. Am J Physiol Regul Integr Comp Physiol 2018;315:R1017–r26. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Chuang TD, Pearce WJ, Khorram O. miR-29c induction contributes to downregulation of vascular extracellular matrix proteins by glucocorticoids. Am J Physiol Cell Physiol 2015;309:C117–25. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Maegdefessel L, Azuma J, Toh R, Merk DR, Deng A, Chin JT et al. Inhibition of microRNA-29b reduces murine abdominal aortic aneurysm development. J Clin Invest 2012;122:497–506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Zhang P, Huang A, Ferruzzi J, Mecham RP, Starcher BC, Tellides G et al. Inhibition of microRNA-29 enhances elastin levels in cells haploinsufficient for elastin and in bioengineered vessels--brief report. Arterioscler Thromb Vasc Biol 2012;32:756–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Wang H, Zhu Y, Zhao M, Wu C, Zhang P, Tang L et al. miRNA-29c Suppresses Lung Cancer Cell Adhesion to Extracellular Matrix and Metastasis by Targeting Integrin beta1 and Matrix Metalloproteinase2 (MMP2). PLoS One 2013;8:e70192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 15.Chuang TD, Khorram O. Tranilast Inhibits Genes Functionally Involved in Cell Proliferation, Fibrosis, and Epigenetic Regulation and Epigenetically Induces miR-29c Expression in Leiomyoma Cells. Reprod Sci 2017;24:1253–63. [DOI] [PubMed] [Google Scholar]
  • 16.Marsh EE, Steinberg ML, Parker JB, Wu J, Chakravarti D, Bulun SE. Decreased expression of microRNA-29 family in leiomyoma contributes to increased major fibrillar collagen production. Fertil Steril 2016;106:766–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Qiang W, Liu Z, Serna VA, Druschitz SA, Liu Y, Espona-Fiedler M et al. Down-regulation of miR- 29b is essential for pathogenesis of uterine leiomyoma. Endocrinology 2014;155:663–9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Halder SK, Goodwin JS, Al-Hendy A. 1,25-Dihydroxyvitamin D3 reduces TGF-beta3-induced fibrosis-related gene expression in human uterine leiomyoma cells. J Clin Endocrinol Metab 2011;96:E754–62. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Norian JM, Malik M, Parker CY, Joseph D, Leppert PC, Segars JH et al. Transforming growth factor beta3 regulates the versican variants in the extracellular matrix-rich uterine leiomyomas. Reprod Sci 2009;16:1153–64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Salama SA, Diaz-Arrastia CR, Kilic GS, Kamel MW. 2-Methoxyestradiol causes functional repression of transforming growth factor beta3 signaling by ameliorating Smad and non-Smad signaling pathways in immortalized uterine fibroid cells. Fertil Steril 2012;98:178–84. [DOI] [PubMed] [Google Scholar]
  • 21.Arici A, Sozen I. Transforming growth factor-beta3 is expressed at high levels in leiomyoma where it stimulates fibronectin expression and cell proliferation. Fertil Steril 2000;73:1006–11. [DOI] [PubMed] [Google Scholar]
  • 22.Joseph DS, Malik M, Nurudeen S, Catherino WH. Myometrial cells undergo fibrotic transformation under the influence of transforming growth factor beta-3. Fertil Steril 2010;93:1500–8. [DOI] [PubMed] [Google Scholar]
  • 23.Lee BS, Nowak RA. Human leiomyoma smooth muscle cells show increased expression of transforming growth factor-beta 3 (TGF beta 3) and altered responses to the antiproliferative effects of TGF beta. J Clin Endocrinol Metab 2001;86:913–20. [DOI] [PubMed] [Google Scholar]
  • 24.Arici A, Sozen I. Expression, menstrual cycle-dependent activation, and bimodal mitogenic effect of transforming growth factor-beta1 in human myometrium and leiomyoma. Am J Obstet Gynecol 2003;188:76–83. [DOI] [PubMed] [Google Scholar]
  • 25.Ohara N, Morikawa A, Chen W, Wang J, DeManno DA, Chwalisz K et al. Comparative effects of SPRM asoprisnil (J867) on proliferation, apoptosis, and the expression of growth factors in cultured uterine leiomyoma cells and normal myometrial cells. Reprod Sci 2007;14:20–7. [DOI] [PubMed] [Google Scholar]
  • 26.Ciebiera M, Wlodarczyk M, Wrzosek M, Slabuszewska-Jozwiak A, Nowicka G, Jakiel G. Ulipristal acetate decreases transforming growth factor beta3 serum and tumor tissue concentrations in patients with uterine fibroids. Fertil Steril 2018;109:501–7.e2. [DOI] [PubMed] [Google Scholar]
  • 27.Lewis TD, Malik M, Britten J, Parikh T, Cox J, Catherino WH. Ulipristal acetate decreases active TGF-beta3 and its canonical signaling in uterine leiomyoma via two novel mechanisms. Fertil Steril 2019;111:806–15.e1. [DOI] [PubMed] [Google Scholar]
  • 28.Tanwar PS, Lee HJ, Zhang L, Zukerberg LR, Taketo MM, Rueda BR et al. Constitutive activation of Beta-catenin in uterine stroma and smooth muscle leads to the development of mesenchymal tumors in mice. Biol Reprod 2009;81:545–52. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 29.Chuang TD, Khorram O. Regulation of Cell Cycle Regulatory Proteins by MicroRNAs in Uterine Leiomyoma. Reprod Sci 2019;26:250–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 30.Chuang TD, Khorram O. miR-200c Regulates IL8 Expression by Targeting IKBKB: A Potential Mediator of Inflammation in Leiomyoma Pathogenesis. PLoS One 2014;9:e95370. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Chuang TD, Luo X, Panda H, Chegini N. miR-93/106b and their host gene, MCM7, are differentially expressed in leiomyomas and functionally target F3 and IL-8. Mol Endocrinol 2012;26:1028–42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Chuang TD, Khorram O. Expression Profiling of lncRNAs, miRNAs, and mRNAs and Their Differential Expression in Leiomyoma Using Next-Generation RNA Sequencing. Reprod Sci 2018;25:246–55. [DOI] [PubMed] [Google Scholar]
  • 33.Busk PK. A tool for design of primers for microRNA-specific quantitative RT-qPCR. BMC Bioinformatics 2014;15:29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Almeida TA, Quispe-Ricalde A, Montes de Oca F, Foronda P, Hernandez MM. A high-throughput open-array qPCR gene panel to identify housekeeping genes suitable for myometrium and leiomyoma expression analysis. Gynecol Oncol 2014;134:138–43. [DOI] [PubMed] [Google Scholar]
  • 35.Chuang TD, Xie Y, Yan W, Khorram O. Next-generation sequencing reveals differentially expressed small noncoding RNAs in uterine leiomyoma. Fertil Steril 2018;109:919–29. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Chuang TD, Ho M, Khorram O. The Regulatory Function of miR-200c on Inflammatory and Cell-Cycle Associated Genes in SK-LMS-1, A Leiomyosarcoma Cell Line. Reprod Sci 2015;22:563–71. [DOI] [PubMed] [Google Scholar]
  • 37.Chuang TD, Khorram O. Glucocorticoids regulate MiR-29c levels in vascular smooth muscle cells through transcriptional and epigenetic mechanisms. Life Sci 2017;186:87–91. [DOI] [PubMed] [Google Scholar]
  • 38.Li LC, Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics 2002;18:1427–31. [DOI] [PubMed] [Google Scholar]
  • 39.Poli E, Zhang J, Nwachukwu C, Zheng Y, Adedokun B, Olopade OI et al. Molecular Subtype-Specific Expression of MicroRNA-29c in Breast Cancer Is Associated with CpG Dinucleotide Methylation of the Promoter. PLoS One 2015;10:e0142224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Commandeur AE, Styer AK, Teixeira JM. Epidemiological and genetic clues for molecular mechanisms involved in uterine leiomyoma development and growth. Hum Reprod Update 2015;21:593–615. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Lagana AS, Vergara D, Favilli A, La Rosa VL, Tinelli A, Gerli S et al. Epigenetic and genetic landscape of uterine leiomyomas: a current view over a common gynecological disease. Arch Gynecol Obstet 2017;296:855–67. [DOI] [PubMed] [Google Scholar]
  • 42.Chuang TD, Panda H, Luo X, Chegini N. miR-200c is aberrantly expressed in leiomyomas in an ethnic-dependent manner and targets ZEBs, VEGFA, TIMP2, and FBLN5. Endocr Relat Cancer 2012;19:541–56. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Feng X, Wang Z, Fillmore R, Xi Y. MiR-200, a new star miRNA in human cancer. Cancer Lett 2014;344:166–73. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Rokavec M, Wu W, Luo JL. IL6-mediated suppression of miR-200c directs constitutive activation of inflammatory signaling circuit driving transformation and tumorigenesis. Mol Cell 2012;45:777–89. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Dawson K, Wakili R, Ordog B, Clauss S, Chen Y, Iwasaki Y et al. MicroRNA29: a mechanistic contributor and potential biomarker in atrial fibrillation. Circulation 2013;127:1466–75, 75e1–28. [DOI] [PubMed] [Google Scholar]
  • 46.van Rooij E, Sutherland LB, Thatcher JE, DiMaio JM, Naseem RH, Marshall WS et al. Dysregulation of microRNAs after myocardial infarction reveals a role of miR-29 in cardiac fibrosis. Proc Natl Acad Sci U S A 2008;105:13027–32. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 47.Sengupta S, den Boon JA, Chen IH, Newton MA, Stanhope SA, Cheng YJ et al. MicroRNA 29c is down-regulated in nasopharyngeal carcinomas, up-regulating mRNAs encoding extracellular matrix proteins. Proc Natl Acad Sci U S A 2008;105:5874–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Wang H, Wang B, Zhang A, Hassounah F, Seow Y, Wood M et al. Exosome-Mediated miR-29 Transfer Reduces Muscle Atrophy and Kidney Fibrosis in Mice. Mol Ther 2019;27:571–83. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Yang Q, Mas A, Diamond MP, Al-Hendy A. The Mechanism and Function of Epigenetics in Uterine Leiomyoma Development. Reprod Sci 2016;23:163–75. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Luna C, Li G, Qiu J, Epstein DL, Gonzalez P. Cross-talk between miR-29 and transforming growth factor-betas in trabecular meshwork cells. Invest Ophthalmol Vis Sci 2011;52:3567–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 51.Lan HY, Chung AC. Transforming growth factor-beta and Smads. Contrib Nephrol 2011;170:75–82. [DOI] [PubMed] [Google Scholar]
  • 52.Qin W, Chung AC, Huang XR, Meng XM, Hui DS, Yu CM et al. TGF-beta/Smad3 signaling promotes renal fibrosis by inhibiting miR-29. J Am Soc Nephrol 2011;22:1462–74. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 53.Garzon R, Liu S, Fabbri M, Liu Z, Heaphy CE, Callegari E et al. MicroRNA-29b induces global DNA hypomethylation and tumor suppressor gene reexpression in acute myeloid leukemia by targeting directly DNMT3A and 3B and indirectly DNMT1. Blood 2009;113:6411–8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Qin H, Zhu X, Liang J, Wu J, Yang Y, Wang S et al. MicroRNA-29b contributes to DNA hypomethylation of CD4+ T cells in systemic lupus erythematosus by indirectly targeting DNA methyltransferase 1. J Dermatol Sci 2013;69:61–7. [DOI] [PubMed] [Google Scholar]
  • 55.Li S, Chiang TC, Richard-Davis G, Barrett JC, McLachlan JA. DNA hypomethylation and imbalanced expression of DNA methyltransferases (DNMT1, 3A, and 3B) in human uterine leiomyoma. Gynecol Oncol 2003;90:123–30. [DOI] [PubMed] [Google Scholar]
  • 56.Yamagata Y, Maekawa R, Asada H, Taketani T, Tamura I, Tamura H et al. Aberrant DNA methylation status in human uterine leiomyoma. Mol Hum Reprod 2009;15:259–67. [DOI] [PubMed] [Google Scholar]
  • 57.Tittle RK, Sze R, Ng A, Nuckels RJ, Swartz ME, Anderson RM et al. Uhrf1 and Dnmt1 are required for development and maintenance of the zebrafish lens. Dev Biol 2011;350:50–63. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Cardenas H, Vieth E, Lee J, Segar M, Liu Y, Nephew KP et al. TGF-beta induces global changes in DNA methylation during the epithelial-to-mesenchymal transition in ovarian cancer cells. Epigenetics 2014;9:1461–72. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 59.Pan X, Chen Z, Huang R, Yao Y, Ma G. Transforming growth factor beta1 induces the expression of collagen type I by DNA methylation in cardiac fibroblasts. PLoS One 2013;8:e60335. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Esteller M Cancer epigenomics: DNA methylomes and histone-modification maps. Nat Rev Genet 2007;8:286–98. [DOI] [PubMed] [Google Scholar]
  • 61.Vaissiere T, Sawan C, Herceg Z. Epigenetic interplay between histone modifications and DNA methylation in gene silencing. Mutat Res 2008;659:40–8. [DOI] [PubMed] [Google Scholar]
  • 62.Inui M, Martello G, Piccolo S. MicroRNA control of signal transduction. Nat Rev Mol Cell Biol 2010;11:252–63. [DOI] [PubMed] [Google Scholar]
  • 63.Jiang X, Tsitsiou E, Herrick SE, Lindsay MA. MicroRNAs and the regulation of fibrosis. Febs j 2010;277:2015–21. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 64.Navarro A, Yin P, Monsivais D, Lin SM, Du P, Wei JJ et al. Genome-wide DNA methylation indicates silencing of tumor suppressor genes in uterine leiomyoma. PLoS One 2012;7:e33284. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 65.Luo X, Chegini N. The expression and potential regulatory function of microRNAs in the pathogenesis of leiomyoma. Semin Reprod Med 2008;26:500–14. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Wang T, Zhang X, Obijuru L, Laser J, Aris V, Lee P et al. A micro-RNA signature associated with race, tumor size, and target gene activity in human uterine leiomyomas. Genes Chromosomes Cancer 2007;46:336–47. [DOI] [PubMed] [Google Scholar]
  • 67.Marsh EE, Lin Z, Yin P, Milad M, Chakravarti D, Bulun SE. Differential expression of microRNA species in human uterine leiomyoma versus normal myometrium. Fertil Steril 2008;89:1771–6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Bjorkman S, Taylor HS. MicroRNAs in endometriosis: biological function and emerging biomarker candidatesdagger. Biol Reprod 2019;100:1135–46. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 69.Chen B, Xu P, Wang J, Zhang C. The role of MiRNA in polycystic ovary syndrome (PCOS). Gene 2019;706:91–6. [DOI] [PubMed] [Google Scholar]
  • 70.Chiofalo B, Lagana AS, Vaiarelli A, La Rosa VL, Rossetti D, Palmara V et al. Do miRNAs Play a Role in Fetal Growth Restriction? A Fresh Look to a Busy Corner. Biomed Res Int 2017;2017:6073167. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 71.Murphy MS, Tayade C, Smith GN. Maternal Circulating microRNAs and Pre-Eclampsia: Challenges for Diagnostic Potential. Mol Diagn Ther 2017;21:23–30. [DOI] [PubMed] [Google Scholar]

RESOURCES