MiR-142-3p could serve as a potential biomarker for individualized treatment of solitary and multiple leiomyomas

Background: The pathogenesis and clinical behaviors between solitary leiomyoma (SL) and multiple leiomyomas (ML) vary, which lead to the difference in management for childbearing-aged patients. Herein, we aim to find the potential miRNA biomarkers for optimizing the individualized management between SL and ML. Methods: A microarray analysis was conducted to screen out the potentially dysregulated miRNAs. Target genes and signaling pathway potentially involved in UL pathogenesis were predicted by bioinformatics. The effect of miRNA was examined by Cell Counting Kit-8 proliferation assay and qRT-PCR after transfection of miRNA mimics Results: 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 significantly upregulated in SL, while downregulated in ML, CTNNB1 and sequencing target AXIN-2 were expressed at higher level in ML than SL. 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 SL and ML by interacting with CTNNB1 and AXIN-2 through Wnt signaling pathway. MiR-142-3p could serve as a potential biomarker for individualized treatment between SL and ML in the future. used instead of primary uterine leiomyoma cells due to growth failure. Ishikawa cells were cultured in Dulbecco’s modified 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 1 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) according to the manufacturer’s instructions. Cells were treated with 30 M LiCl 12 h after transfection and incubated for another 24 h. Further, the transfection effects of miR-142-3p mimics and CTNNB1, APC, and AXIN-2 gene transcripts 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 Scientific) at a wavelength of 450 nm. Two-tailed

United States [1]. However, the understanding of UL pathobiology is still in its infancy, thus alternative treatment options (other than surgical interventions) 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 leiomyomas (ML) occurs at higher rates than solitary leiomyoma (SL) [2-13].
However, a significant difference was not found in some studies [14,15], and one study reported contradicting results [16]. Moreover, clinical observation and epidemiology showed that SL 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 [17], including the most frequently mutated gene mediator subcomplex 12 (MED12)[18] and a frequently rearranged allele of HMGA2 [19]. Moreover, gene polymorphisms studies showed that carriage of higher CYP17A1 G allele frequency was correlated with ML, while higher CYP17A1 A allele frequency was correlated with a healthy population and SL [20,21]. Taken together, these clinical epidemiology and cytogenetic studies suggested the involvement of molecularly distinct pathobiology between SL and ML, encouraging further study to reveal the differentiated molecular processes between SL and ML for novel perspective of UL pathobiology.
MiRNAs are negative regulators of gene expression through post-transcriptional gene silencing [22] 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 [23][24][25], which yielded some dysregulated miRNAs (miR-let7 family, miR-21, miR-34a, etc.). 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 SL and ML compared to their corresponding myometrium.
Taken together with previous studies, we found that β-catenin signalling was significantly differentially regulated between SL and ML, as compared to their corresponding myometrium, as a potential result of post-transcriptional negative regulation by miR-142-3p directly targeting β-catenin.

Methods
Study samples: Tissues of uterine leiomyomas and corresponding normal myometrium were collected from 32 women with symptomatic leiomyomas, who underwent operations at West China Second University Hospital (Chengdu, P.R. China), and were stored in liquid nitrogen immediately after surgeries. 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 defined 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.
MiRNA microarray analysis: MiRNA microarray analysis was performed on three pairs of solitary leiomyoma and corresponding myometrium, and three pairs of multiple leiomyomas and corresponding myometrium. The frozen tissues were cryopulverised to fine 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 purified using the RNeasy Mini Kit (Qiagen). The quality of the total RNA was verified by spectrophotometry using NanoDropND-1000 (ND-1000, Nanodrop Technologies). The integrity of RNA was evaluated by denaturing agarose gel electrophoresis. RNA labelling and array hybridisation was performed according to the manufacturer's protocol (Exiqon). Total RNA (1μg) was labelled with Hy5 fluorescent and Hy3 fluorescent probes using the miRCURY Array Power Labeling kit (Exiqon). The miRCURY LNA Array version 7th generation (Exiqon) was used to hybridise the labelled RNA. Subsequently, the hybridised slides were scanned using the Axon GenePix 4000B microarray scanner (Axon), and image reading was performed using GenePix pro V6.0 (Axon). gene targets of validated differentially expressed miRNAs in this study were identified using the same search methodology in PubMed. Candidate miRNAs were rated in four aspects: 1) The false discovery rate (FDR): 3 points for FDR < 0.05, 2 for 0.05 < FDR < 0.1, and 1 for FDR > 0.1; 2) expression condition in another group: 3 points for contrarily regulated in 2 pairs of leiomyoma vs. myometrium in another group, 2 for contrarily regulated in 1 pair of leiomyoma vs. myometrium in another group, 1 for not found dysregulated in another group, and -2 for regulated in same way in 1 pair of leiomyoma vs. myometrium in another group; 3) expression level (normalised signal intensity (NSI)): 3 points for NSI > 5, 2 for 5 > NSI > 1 and 1 for NSI < 1. 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 SL and ML, MSL and MML. Detailed statistics are shown in Supplementary file 1-2. SPSS 23.0 (SPSS) was used for analyses. GraphPad Prism 6.0 (GraphPad Software, Inc) was used for figure drawing. Statistical significance was defined as P < 0.05.

The top five dysregulated miRNAs between SL and ML were filtered by microarray analysis
To determine potentially dysregulated microRNAs between SL and ML, we firstly performed a miRNA microarray analysis of 12 UL samples, including 3 pairs of SL with their corresponding MSL and 3 pairs of ML with corresponding MML. Only patients with more than 4 leiomyomas were included in the ML group, according to the meta-analysis results. and assigned into ML up-regulated group. In a similar fashion, 16 miRNAs were in SL downregulated group and 4 miRNAs were in ML down-regulated group (Fig. 1).
Then the differentially expressed miRNAs between SL groups and ML groups were further filtered. 10 miRNAs in SL up-regulated group were excluded for they also up-regulated in at least 2 pairs of tissues in ML up-regulated group. Similarly, 3, 2, and 2 miRNAs were excluded from SL down-regulated group, ML up-regulated group and ML down-regulated group, respectively (Table 1. 1-1.4). The remaining 28, 15, 13, and 1 miRNAs were considered to be potentially dysregulated miRNAs among groups and were ranked based on the following four items: false discovery rate, expression condition in another group, expression level and stability, validated function potentially related to leiomyoma genesis.
Based on microarray analysis, the expressions of miR-142-3p, miR-146b-5p and miR-136-3p were significantly different between SL groups and ML groups (Fig. 2a). MiR-142-3p was significantly upregulated in SL group by 2.236 FC value, while downregulated in ML group by -1.757 FC value. Moreover, the baseline expression of miR-142-3p in MSL was significantly lower than MML, and the expression level in SL was significantly higher than ML (Fig.2b). Similarly, miR-146b-5p was upregulated in SL group by 1.858 FC value, while downregulated in ML group by -1.398 FC value. Varying from miR142-3p, baseline expression of miR-146b-5p in MSL and MML showed no statistical difference (Fig. 2c). MiR-146a-5p, which harbours the identical seed region with miR-146b-5p, was downregulated in both groups by -2.263 FC and -1.700 FC, respectively. However, no significant interaction between groups and sites was found between SL group and ML group (Fig. 2d).
MiR-136-3p was significantly upregulated by 3.208 FC and 1.270 FC between SL group and ML group, respectively, due to a lower baseline expression in MSL and similar upregulated levels in SL and ML (Fig. 2e). No statistical difference was found in miR-608 (Fig. 2f).
Above all, miR-142-3p was the most dysregulated one between SL and ML. Although miR-146b-5p showed a similar expression profile with miR-142-3p, miR-146a-5p shared identical seed regions with miR-146b-5p and showed a different expression profile.
Further, Shrestha et al [34] reported that β-catenin protein levels increased, while the Contrary to the expression of miR-142-3p, CTNNB1 and AXIN2 were significantly upregulated in ML 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 another UL samples (10 pairs of SL and showed that CTNNB1 was upregulated in both SL group and ML group, but with a significantly higher FC value of 12.327 in ML group (Fig. 3a). Furthermore, the baseline expression of CTNNB1 between MSL and MML, and upregulated levels between SL and ML, were significantly different (Fig. 3c), consistent with the miR-142-3p expression profile.
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 significantly upregulated at 2.303 FC value in ML group. The detailed expression profile 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 significantly 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  (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 ML vs. MML than SL vs. MSL. 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 β-catenin is initially expressed at high transcriptional level in both SL and ML. However, in SL, the highly expressed β-catenin transcripts are silenced partially by upregulated miR-142-3p, as the biological function of miRNAs is posttranscriptional negative regulation of target genes. Conversely, the lower level of miR-142-3p in ML than MML enhances β-catenin upregulation in ML, resulting in upregulation of β-catenin in ML vs. MML at a notable higher level than SL vs. MSL. Further, MED12 gene, which is reported to regulate canonical Wnt signalling through direct binding to β-catenin, [36] is altered in about 70% of leiomyomas[18] and is correlated to higher possibility of ML. [37] However, it is unknown whether the mutated MED12 in leiomyoma cells leads to βcatenin signalling alternation. However, the difference between MSL and MML 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, [38] 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
After myomectomy, women with ML suffer greater recurrence probability than SL. In the uterus of ML patients, it is impossible to thoroughly remove undetected leiomyomas during myomectomy, possibly contributing to recurrence. However, SL 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 SL and ML, together with exclusive gene subgroup correlations with SL and ML, indicates that ML 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 specific disease grading biomarkers, and more importantly, molecular intervention for leiomyoma could become possible.

Ethics approval and consent to participate
Written informed consents were obtained from all patients, and the study protocol was approved by Institutional Review Board of West China Second University Hospital.

Availability of data and material
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.

Funding
The microRNA microarray work and analysis of the data of this study were supported by National Natural Science Foundation of China (Grant Number: 81170592/H0420).      Over-expression of miR-142-3p resulted in CTNNB1(β-catenin) and β-catenin signaling down-regulation in vitro. Transfection of miR-142-3p mimics (25nM or 250nM) and its negative control (NC) was carried out in Ishikawa cells, then βcatenin signaling was activated by treating with 30mM LiCl, and the transfection effects of miR-142-3p mimics and β-catenin signaling expression were examined by qRT-PCR. a, MiR-142-3p was validated to be over-expressed after mimic transcription. b, APC mRNA level had no significant differences after overexpression of miR-142-3p. c, CTNNB1 mRNA level was significantly lower after 28 over-expression of miR-142-3p. d, AXIN-2 mRNA level, the down-stream gene of βcatenin signaling, was significantly lower after over-expression of miR-142-3p.

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.