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 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. 6) SUL was defined as one leiomyomas identified by both ultrasound and surgery findings.
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 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. Total RNA (1 µg) 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 SULides were scanned using the Axon GenePix 4000B microarray scanner (Axon), and image reading was performed using GenePix pro V6.0 (Axon).
Microarray analysis: With a cut-off fold-change (FC) value of 2, miRNAs expression profiles were yielded. Dysregulated miRNAs were filtered by taking intersection. The validated functional information of each filtered 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/fibrogenesis/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 (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 quantification) were measured to quantify gene transcripts (CTNNB1, APC, and AXIN2). Total RNA was extracted and verified 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 amplification, the relative expression of miRNAs and mRNAs was calculated based on the 2−ΔCt methods.(21) The relative fold-change of miRNAs and mRNAs between SUL vs MSUL and MUL and MMUL was calculated based on the 2−ΔΔCt methods.(22)
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 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% CO2. Ishikawa cells were seeded at a density of 106/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 Scientific) at a wavelength of 450 nm.
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 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.
alysis. Signalling pathway enrichment was carried out based on