MiR-142-3p May Be Involved in the Development of Solitary and Multiple Uterine Leiomyomasby Interacting with CTNNB1 and AXIN-2 Through Wnt Signaling Pathway

The pathogenesis and clinical behaviors between solitary uterine leiomyoma (SUL) and multiple uterine leiomyomas (MUL) vary, which lead to the difference in management for childbearing-aged patients. Herein, we aim to nd the potential miRNAs involved in the development of SUL and MUL. The top 5 differentially expressed miRNAs, Wnt signalling pathway and its two central molecules APC and CTNNB1 were screened out according to microarray analysis and bioinformatics. MiR-142-3p was selected for further exploration. In validation of qRT-PCR, MiR-142-3p was signicantly upregulated in SUL, while downregulated in MUL, CTNNB1 and sequencing target AXIN-2 were expressed at higher level in MUL than SUL. Overexpression of MiR-142-3p resulted in lower transcription level of CTNNB1 and AXIN-2, and lower cell proliferation level.


Conclusions
MiR-142-3p may be involved in the development of SUL and MUL by interacting with CTNNB1 and AXIN-2 through Wnt signaling pathway. MiR-142-3p could serve as a potential biomarker for individualized treatment between SUL and MUL in the future.

Background
Uterine leiomyoma (UL) is the most prevalent neoplasm in reproductive-age females. UL is associated with excessive menstrual bleeding, impaired fertility, recurrent abortion, and accounts for the most common cause of hysterectomy among gynaecological disorders (1). However, the understanding of UL pathobiology is unclear, thus alternative treatment options are limited. As hysterectomy is invasive and can result in permanent infertility, myomectomy has become one of the only options to preserve fertility.
Several studies have reported that recurrence of multiple uterine leiomyomas (MUL) occurs at higher rates than solitary uterine leiomyoma (SUL) (2)(3)(4). Moreover, clinical observation and epidemiology showed that SUL tended to grow in a larger diameter and rarely developed into multiple tumours. Cytogenetic studies have suggested recurrent and mutually exclusive mutations exist in UL (5), including the most frequently mutated gene mediator subcomplex 12 (MED12)(6) and a frequently rearranged allele of HMGA2 (7). Moreover, gene polymorphisms studies showed that carriage of higher CYP17A1 G allele frequency was correlated with MUL, while higher CYP17A1 A allele frequency was correlated with a healthy population and SUL (8). Taken together, these suggested the involvement of molecularly distinct pathobiology between SUL and MUL, encouraging further studies to reveal the differentiated molecular processes between SUL and MUL for novel perspective of UL pathobiology.
MiRNAs are negative regulators of gene expression through post-transcriptional gene silencing (9) and they have been found to take part in disease pathogenesis and embryonic development. Few studies have described miRNA dysregulation between UL versus corresponding myometrium, based on microarray analysis (10), which yielded some dysregulated miRNAs. However, the roles of dysregulated miRNAs in UL pathogenesis is still to be elucidated. Here, aiming to explore individualised therapeutic applications and molecular interventions for UL, we investigated miRNA-target networks between SUL and MUL compared to their corresponding myometrium. Taken together with previous studies, we found that β-catenin signalling was signi cantly differentially regulated between SUL and MUL, as compared to their corresponding myometrium, as a potential result of post-transcriptional negative regulation by miR-142-3p directly targeting β-catenin.

Results
The top ve dysregulated miRNAs between SUL and MUL were ltered by microarray analysis To determine potentially dysregulated microRNAs between SUL and MUL, we rstly performed a miRNA microarray analysis of 12 samples, including 3 pairs of SUL with corresponding MSUL and 3 pairs of MUL with corresponding MMUL (Fig. 1). The top 5 ranked miRNAs: miR-142-3p, miR-146a-5p, miR-146b-5p, miR-136-3p and miR-608( Fig. 1 and Additional le 1) were ltered for further validation in enlarged samples. miR-142-3p was the most dysregulated miRNAs between SUL and MUL Secondly, qRT-PCR was used to validate the top ve miRNAs in enlarged UL samples (13 pairs of SUL vs. MSUL and 19 pairs of MUL vs. MMUL, total 64 tissues) (Fig. 2). The expressions of miR-142-3p, miR-146b-5p and miR-136-3p were signi cantly different (Fig. 2a). MiR-142-3p was signi cantly upregulated in SUL group, while downregulated in MUL group. Moreover, the baseline expression of miR-142-3p in MSUL was signi cantly lower than MMUL, and the expression level in SUL was signi cantly higher than MUL (Fig. 2b). Similarly, miR-146b-5p was upregulated in SUL group, while downregulated in MUL group. Varying from miR142-3p, baseline expression of miR-146b-5p in MSUL and MMUL showed no statistical difference (Fig. 2c). MiR-146a-5p, which harbours the identical seed region with miR-146b-5p, was downregulated in both groups, respectively. However, no signi cant interaction between groups and sites was found between SUL group and MUL group (Fig. 2d). MiR-136-3p was signi cantly upregulated between SUL group and MUL group, respectively, due to a lower baseline expression in MSUL and similar upregulated levels in SUL and MUL (Fig. 2e). No statistical difference was found in miR-608 (Fig. 2f).
Above all, miR-142-3p was the most dysregulated one between SUL and MUL. Targets and pathway analysis of miR-142-3p After microarray analysis and qRT-PCR validation, we focused on researching miR-142-3p including its target genes and pathway analysis. Based on target gene prediction and functional annotation clustering analysis and a previous miR-142-3p target gene signalling prediction conducted through miRTar by Carraro et al (11), the Wnt signalling pathway was found the rst signi cantly enriched signalling pathway. Interestingly, activation of Wnt signalling was reported to resulted in myometrium cell hyperplasia and development of leiomyoma-like tumours in a Cre-recombinase mouse model (12). Further literature retrieval found that the regulation of canonical Wnt signalling by miR-142-3p was explored in several studies. However, involvement of different target genes showed contrary regulation results. Isobe et al. (13) and Carraro et al. (11) reported that miR-142-3p activated Wnt signalling by targeting APC, a member of the β-catenin (CTNNB1) destruction complex. Hu et al. (14) reported that miR-142-3p inactivated Wnt signalling by directly targeting CTNNB1. Further, Shrestha et al (15) reported that β-catenin protein levels increased, while the CTNNB1 gene was not altered in miR-142 knockout mice. APC and β-catenin were both validated as miR-142-3p targets by luciferase assay (11,13,14). Several studies have suggested that the intracellular β-catenin pathway of Wnt signalling is constitutively active in the majority of LM cells (12,16). Therefore, APC and CTNNB1, two central molecular of Wnt signalling and validated gene targets of miR-142-3p, were selected for further validation.
Contrary to the expression of miR-142-3p, CTNNB1 and AXIN2 were signi cantly upregulated in MUL APC and CTNNB1, potential dysregulated target genes of miR-142-3p (Fig. 3a) and central members of the canonical Wnt/β-catenin signalling pathway, and AXIN2, a β-catenin signalling target gene, were further validated in UL samples (10 pairs of SUL and 10 pairs of MUL, a total of 40 tissues included in previous miRNA validation). The results showed that CTNNB1 was upregulated in both SUL group and MUL group, but with a signi cantly higher FC value of 12.327 in MUL group (Fig. 3a). Furthermore, the baseline expression of CTNNB1 between MSUL and MMUL, and upregulated levels between SUL and MUL, were signi cantly different (Fig. 3c), consistent with the miR-142-3p expression pro le. Conversely, the expression of APC showed no differences in any statistical comparisons (Fig. 3b, e). AXIN2, representing the activated level of β-catenin signalling, was signi cantly upregulated at 2.303 FC value in MUL group. The detailed expression pro le was consistent with CTNNB1 ( Fig. 3b, d).

MiR-142-3p overexpression downregulated CTNNB1 and AXIN2 and inhibited cell proliferation in vitro
To further investigate the function of miR-142-3p on β-catenin signalling, we detected the expression changes of the predictive target genes and subsequent downstream genes of β-catenin signalling after overexpressed miR-142-3p in Ishikawa cells (Fig. 4a). The results revealed that CTNNB1 and AXIN-2 were signi cantly downregulated (Fig. 4c, d), while APC showed no difference (Fig. 4b), consistent with the expression in human leiomyoma samples. Furthermore, the inhibition rate of cell proliferation was lower (Fig. 5).

Discussion
Canonical Wnt/β-catenin signalling pathway has been reported to be involved in UL pathobiology.
Selective overexpression of constitutively activated β-catenin in embryonic uterine mesenchyme and in the uterus of adult mice leads to growth of leiomyoma-like tumours, while selective deletion of β-catenin in embryonic uterine mesenchyme signi cantly reduces uterine size and replaces normal myometrium smooth muscle cells with adipocytes (12). More recently, Ono and colleagues demonstrated that Wnt/βcatenin signalling is involved in proliferation of leiomyoma stem cells (representing 1% of tumour cells) through Wnt paracrine behaviour. The co-treatment of oestrogen and progesterone could induce expression of Wnt11 and Wnt16 in mature leiomyoma cells. Sequentially, through paracrine effects, Wnt11 and Wnt16 bind to leiomyoma stem cell-surface FZD family receptors (FZD1 and FZD7), leading to activation and nuclear tranSULocation of β-catenin in leiomyoma stem cells with subsequent AXIN2 transcription and stem cell proliferation (Fig. 6). Interestingly, this study validated that AXIN2 transcription was constitutively activate in mature leiomyoma cells, which was not dependent on Wnt secretion. Based on our results, we found that β-catenin signalling was upregulated in UL, and at a notable higher level in MUL vs. MMUL than SUL vs. MSUL. Further, miR-142-3p, the validated negative regulator of β-catenin, was found negatively correlated to β-catenin expression. Moreover, APC, another potential target gene of miR-142-3p, showed no difference in our results, thus was not regulated by miR-142-3p in leiomyomas and myometrium. However, APC as a member of the β-catenin destruction complex might not be involved in β-catenin activation in UL pathobiology. Taken together with previous studies, we propose that βcatenin is initially expressed at high transcriptional level in both SUL and MUL. However, in SUL, the highly expressed β-catenin transcripts are silenced partially by upregulated miR-142-3p, as the biological function of miRNAs is post-transcriptional negative regulation of target genes. Conversely, the lower level of miR-142-3p in MUL than MMUL enhances β-catenin upregulation in MUL, resulting in upregulation of β-catenin in MUL vs. MMUL at a notable higher level than SUL vs. MSUL. Further, MED12 gene, which is reported to regulate canonical Wnt signalling through direct binding to β-catenin, (17) is altered in about 70% of leiomyomas (6) and is correlated to higher possibility of MUL.(18) However, it is unknown whether the mutated MED12 in leiomyoma cells leads to β-catenin signalling alternation. However, the difference between MSUL and MMUL is hardly explained by MED12 alternation in leiomyomas. In addition, Markowski et al. reported that Wnt4b was expressed at higher level in MED12 altered UL cells, (19) leading to the speculation that MED12 mutation in UL is more likely to be involved in paracrine signalling of stem cell activation. Taken together, evidence suggests that β-catenin activation in leiomyomas is regulated at least partially by miR-142-3p negatively regulation during post-transcriptional process. Further studies to elucidate the regulatory mechanism of miR-142-3p targeting CTNNB1 and APC are expected in the future.

Conclusion
In the uterus of MUL patients, it is impossible to thoroughly remove undetected leiomyomas during myomectomy, possibly contributing to recurrence. However, SUL rarely spreads into multiple areas during the course of the disease. More interestingly, differential expression of miRNAs (miR142-3p and miR-136-3p) and β-catenin signalling in the myometrium of SUL and MUL, together with exclusive gene subgroup correlations with SUL and MUL, indicates that MUL is more similar to "myometrial disease" than simple leiomyoma disease. Thus, with the gradually deepening understanding of UL pathobiology, individualised clinical decision of myomectomy or hysterectomy could be guided by evidence of speci c disease grading biomarkers, and more importantly, molecular intervention for leiomyoma could become possible.

Methods
Study samples: Tissues of UL and corresponding myometrium were collected from women with symptomatic leiomyomas, who underwent operations at West China Second University Hospital (Chengdu, P.R. China). Written informed consents were obtained from all patients, and the Institutional Review Board of West China Second University Hospital approved the study protocol. During tissue collection, eligible subjects and tissue specimens were de ned as: 1) no GnRH-a or oral contraception used before surgery; 2) pathological diagnosis of leiomyoma; 3) no accompanying adenomyosis or endometriosis; 4) minimum number of leiomyomas involved in multiple leiomyomas was 5; and 5) leiomyomas intraoperatively revealed as multiple leiomyomas fused as one were excluded from solitary leiomyoma group. 6) SUL was de ned as one leiomyomas identi ed by both ultrasound and surgery ndings.

MiRNA microarray analysis
MiRNA microarray analysis was performed on three pairs of SUL with corresponding myometrium(MSUL), and three pairs of MUL and myometrium(MMUL). The frozen tissues were cryopulverised to ne powder with the BioPulverizer (BioSpe, America). The powdered tissue was homogenised with TRIzol Reagent (Invitrogen) using the Mini-beater-16 (BioSpec), and total RNA was puri ed using the RNeasy Mini Kit (Qiagen). The quality of the total RNA was veri ed by spectrophotometry using NanoDropND-1000 (ND-1000, Nanodrop Technologies). The integrity of RNA was evaluated by denaturing agarose gel electrophoresis. Total RNA (1 µg) was labelled with Hy5 uorescent and Hy3 uorescent probes using the miRCURY Array Power Labeling kit (Exiqon). The miRCURY LNA Array version 7th generation (Exiqon) was used to hybridise the labelled RNA.
Microarray analysis: With a cut-off fold-change (FC) value of 2, miRNAs expression pro les were yielded. Dysregulated miRNAs were ltered by taking intersection. The validated functional information of each ltered miRNA was conducted using miRTarBase 6.0 and search of published studies in PubMed. Candidate miRNAs were rated in four aspects: 1) The false discovery rate (FDR); 2) expression condition in another group; 3) expression level (normalised signal intensity (NSI)). 4). Validated function potentially related to leiomyoma genesis (Wnt signalling/ brogenesis/TGF/hormone synthesis or metabolism (20)).

MiRNA gene targets prediction and pathway analysis
Target gene prediction of miRNAs was conducted using intersections of results yielded from four databases (miRWalk 2.0, miRanda, TargetScan, and RNA22). Yielded genes were uploaded to the Database for Annotation, Visualization and Integrated Discovery (DAVID) v6.8 to perform functional annotation clustering an KEGG PATHWAY. We further searched miRTarBase 7.0 and PubMed for validated functional information of miR-142-3p.
Quantitative RT-PCR Quantitative RT-PCR (qRT-PCR) was performed on 13 pairs of SUL and MSUL and 19 pairs of MUL and MMUL to quantify miRNAs (miR-142-3p, miR-146b-5p, miR-146a-5p, miR-136-3p, and miR-608). Further, 10 pairs of SUL and MSUL and 10 pairs of MUL and MMUL (all included in those tissues used for miRNA quanti cation) were measured to quantify gene transcripts (CTNNB1, APC, and AXIN2). Total RNA was extracted and veri ed as before. Reverse transcription (RT) to complementary DNA (cDNA) was conducted using the miScript II RT Kit with miScript HiSpec Buffer (Qiagen) on the GeneAmp® PCR System 9700 (Applied Biosystems, USA). U6 was used as the housekeeping primer for miRNAs detection and ACTB for gene transcripts. The primer sequences were designed and synthesised by Generay Biotech (Generay, PRC) based on the miRNA sequences obtained from the miRBase database. Reactions were carried out in an ABI Prism 7900 HT Sequence Detection System (Applied Biosystems) using the miScript SYBR Green PCR Kit (Qiagen). Each tissue was run in independent experiments in triplicate. After PCR ampli cation, the relative expression of miRNAs and mRNAs was calculated based on the 2 −ΔCt methods. Cell culture, transfection of miRNA mimics, signalling detection, and cell proliferation assay

Ishikawa cells (acquired from Key Laboratory of Birth Defects and Related Diseases of Women and
Children, Sichuan university, Chengdu, China) were used instead of primary uterine leiomyoma cells due to growth failure. Ishikawa cells were cultured in Dulbecco's modi ed Eagle's medium (DMEM, Gibco, Carlsbad, CA) mixed with 10% foetal bovine serum (FBS, Gibco) and 1% penicillin and streptomycin (Gibco). All cells were cultured in a 37℃incubator containing 5% CO 2 . Ishikawa cells were seeded at a density of 10 6 /well in a 24-well dish and cultured for 12 h. Following, transfection of miR-142-3p mimics (25 nM and 250 nM) and its negative control (RiboBio, Guangzhou, China) was performed using Lipofectamine 3000 (Invitrogen). Cells were treated with 30M LiCl 12 h after transfection and incubated for another 2 h. Further, the transfection effects were examined by qRT-PCR. The inhibition rate of cell proliferation was measured by CCK8 (Jikai Gene, China) at 12, 24, 36, and 48 h after transfection according to the manufacturer's instructions. Optical density (OD) was measured by Varioskan Flash (Thermo Scienti c) at a wavelength of 450 nm.

Statistical analysis
Quantitative data of each miRNA and mRNA were recorded as mean ± S.E.M. A two-way ANOVA with repeated measures was used for integral data analysis as experimental group was one factor (two groups based on number of leiomyomas) and site was a repeated measure (paired leiomyoma with corresponding myometrium). Two-tailed paired Student's t-tests were performed for RNA dysregulated level between paired leiomyomas and myometrium. Two-tailed independent Student's t-tests were performed between SUL and MUL, MSUL and MMUL. Detailed statistics are shown in Supplementary le 1-2. SPSS 23.0 (SPSS) was used for analyses. GraphPad Prism 6.0 (GraphPad Software, Inc) was used for gure drawing. Statistical signi cance was de ned as P < 0.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.   Target genes of miR-142-3p involved in β-catenin signaling between paired SUL and paired MUL samples.

Figure 5
MiR-142-3p over-expression resulted in inhibition of cell proliferation in vitro. The inhibiton rate of cell proliferation was measured by at 12h, 24h, 36h and 48h after transfection of miR-142-3p mimics.

Supplementary Files
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