Uterine leiomyoma tissue samples were obtained from 18 female patients (mean age, 45.7±3.03 years; age range, 37–50 years) who underwent myomectomy or hysterectomy for uterine leiomyoma at Hanyang Hospital in Seoul (South Korea) between October 2017 and June 2019; all patients had multiple leiomyoma. Normal myometrium tissues were obtained from 13 of 18 patients. Inclusion criteria included the presence of a symptomatic myoma. Patients with organ dysfunction, for example, in the liver, kidney and lungs, were excluded. Samples taken from the centers of leiomyoma and from adjacent normal myometrium were immediately snap-frozen and stored in liquid nitrogen. This study was approved by the Institutional Review Board of Hanyang Hospital, Hanyang University College of Medicine (approval no. 201707012), and the requirement for informed consent was waived.

Fresh tissue homogenized using a disposable homogenizer (BioMasher; Nippi, Inc.) was incubated with 20 µg DNase- and protease-free RNase A (Thermo Fisher Scientific, Inc.) at 37°C for 1 h in 500 µl lysis buffer (10 mM Tris-HCl; pH 8.0; 100 mM EDTA; 0.5% (w/v) SDS), then further digested at 50°C overnight with 50 µg proteinase K (Invitrogen; Thermo Fisher Scientific, Inc.). Genomic DNA was extracted using phenol/chloroform/isoamyl alcohol (PCI; Bioneer Corporation) a total of three times. MaXtract High-Density tubes (Qiagen GmbH) were used for PCI extractions to prevent carryover of organic solvent, proteins, and other contaminants. The supernatant was supplemented with ammonium acetate to a final concentration of 2.5 M and one volume of isopropanol. Genomic DNA spooled on a pipette tip was washed with 70% ethanol three times, air-dried and resuspended in water. Quantification of DNA was performed using a NanoDrop™ 2000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.).

Oligonucleotides were labeled at the 3′-end by incorporation of a single DIG-labeled ddUTP (Roche Diagnostics). Briefly, 100–200 pmol oligonucleotides were incubated at 37°C for 60 min with 1 µl of 1 mM DIG-ddUTP and 20 units terminal deoxynucleotidyl transferase (Roche Diagnostics) in 1X reaction buffer and 5 mM CoCl₂ in a total volume of 20 µl, and the reaction was stopped by addition of 2 µl of 0.2 M EDTA (pH 8.0).

Southern hybridization was performed to measure telomere length. Briefly, 5 µg HinFI-cut DNA was fractionated on an 0.8% (w/v) agarose gel and blotted by capillary transfer onto a nylon membrane (Hybond™ N+; GE Healthcare Biosciences) in 10X saline-sodium citrate (SSC) buffer. Blots were UV-crosslinked (Spectronics), pre-hybridized in DIG Easy Hyb (Roche Diagnostics) at 45°C for 1 h, and hybridized with DIG-labeled d(TTAGGG)₄ at the same temperature overnight. The membrane washing and antibody reaction were carried out at room temperature as follows. The membranes were washed in 2X SSC with 0.1% SDS for 15 min twice, in 0.5X SSC with 0.1% SDS for 15 min twice, and then in 1X maleic acid buffer (MAB; 0.1 M maleic acid; 0.15 M NaCl; pH 7.5) for 5 min. The blots were immersed in 1X Blocking Solution (Roche Diagnostics; diluted in 1X MAB) for 30 min, then incubated with an anti-DIG antibody conjugated to alkaline phosphatase (cat. no. 11093274910; 1:10,000; Roche; MilliporeSigma; Merck KGaA) in 1X Blocking Solution for 60 min. The membranes were washed in 1X washing buffer (0.3% Tween-20 in 1X MAB) for 15 min twice, then in detection buffer (0.1 M NaCl; 0.1 M Tris-HCl, pH 9.5) at room temperature for 5 min. CDP-Star (Roche Diagnostics) was used as a chemiluminescence substrate, and the hybridization signal was detected by scanning using a ChemiDoc XRS+ image analysis system (Bio-Rad Laboratories, Inc.). For the next round of hybridization, the membrane was rinsed thoroughly with sterile water, washed at 37°C in stripping buffer (0.2 N NaOH; 0.1% SDS) for 15 min twice, then rinsed with 2X SSC buffer for 5 min. The de-probed blot was rehybridized with the DIG-labeled d(CAC)₈ probe. Telomere length (TL) was calculated as previously described (26). Briefly, the telomere signal in each lane was quantified in a grid object defined as a single column with 20 rows (1×20 grid over the lane ranging from 3.5 to 21 kb), using the Image Lab software (version 6; Bio-Rad Laboratories, Inc.), and the TL was defined as ∑(MW_i × OD_i)/∑(OD_i), where OD_i is the optical density and MW_i is the molecular weight of the DNA at the i^th position.

Total RNA was isolated from tissues using Tri-RNA (Favorgen) or TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Then, 1 µg RNA was incubated with 2 units RNase-free DNase I (New England Biolabs, Inc.) and 40 units RNase inhibitor (Takara Bio, Inc.) with 2 mM DTT in a total volume of 1 µl at 37°C for 30 min, heat-inactivated at 75°C for 10 min, then subjected to cDNA synthesis. First-strand cDNA synthesis was carried out in 20-µl volumes containing 6 µM random hexamers (Takara Bio, Inc.) and 200 units Moloney murine leukemia virus (M-MLV) reverse transcriptase (Thermo Fisher Scientific, Inc.) at 37°C for 50 min. To ensure that genomic DNA was completely removed, the RT reaction mixture without M-MLV reverse transcriptase was subjected to PCR with TERC-specific primers. PCR was performed in duplicate with 1 µl cDNA template, 0.2 µM primers (Macrogen), and 1X LightCycler^® 480 SYBR-Green I Master mix (Roche Diagnostics) using the LightCycler^® 480 system (Roche Diagnostics). Thermocycling conditions were as follows: i) 95°C for 5 min; ii) 40 cycles of 95°C for 15 sec and 60°C for 1 min; iii) a dissociation stage of 95°C for 5 sec; 65°C for 1 min; and iv) 40°C for 30 sec. GAPDH was used as the reference gene, and sequences of the primers used in the present study are listed in Table I (27–30). Expression levels were quantified using the 2^−∆∆Cq method (31).

The data were obtained from a single experiment and data are presented as the mean ± standard deviation. Statistical analysis was performed using SPSS software (v. 24, IBM, Corp.) and assessed using an unpaired Student's t-test and the Pearson correlation. P<0.05 was considered to indicate a statistically significant difference.

In this study, terminal restriction fragment lengths were measured using Southern blotting in leiomyoma (n=18) and adjacent normal myometrium (n=13) tissue. Of note, five of the leiomyoma did not have matched normal tissues (Table II). Hybridization with the telomere-specific 3′-DIG-labeled d(TTAGGG)₄ probe indicated that telomere length ranged from 10.4 to 14.9 kb in normal myometrium and from 8.7 to 12.5 kb in leiomyoma (Fig. 1 and Table II). Leiomyoma samples presented shorter telomeres than the normal myometrium (Fig. 1B). Mean telomere lengths in normal and leiomyoma tissue samples were 12.5±1.11 (n=13) and 11.1±1.04 kb (n=18), respectively (P<0.001; Fig. 1B and Table II). There were no significant differences in telomere length related to patient age, possibly due to the narrow range of patient ages (37–50 years) (data not shown). Telomere lengths in multiple leiomyoma samples from a single patient (Table II; patients no. 7, 8, 9) were also measured, but no obvious variations in lengths was observed among measurements. Membranes re-probed with a 3′-DIG-labeled d(CAC)₈ probe for minisatellite DNA confirmed the integrity of genomic DNA. A minisatellite is a tract of repetitive DNA sequences that are present throughout the entire genome and often used for the fingerprinting of DNA (32). Indeed, hybridization with d(CAC)₈ probe showed different hybridization patterns by patients (Fig. 1A).

TRF1 and TRF2, components of shelterin, protect telomeres and regulate telomere length. Depletion or deletion of these genes induces a persistent DNA damage response at telomeres, resulting in cessation of cell division and induction of apoptosis or senescence (16,33–35). Meanwhile, overexpression of TRF1, TRF2 and PINX1 results in telomere shortening in telomerase-positive cells (9,10,36), ultimately leading to induction of cell crisis which is characterized by the reduction in growth rate. To determine whether the expression of TRF1, TRF2 and PINX1 varied during the development of leiomyoma, the mRNA levels of these genes in leiomyoma (n=17) and normal tissues (n=12) were determined. RT-qPCR results suggested that these genes were expressed at similar levels in normal and leiomyoma nodules (Fig. 2). To determine whether there was a possible association between gene expression and telomere length, Pearson's correlation was used (Fig. 3). The expression of TRF1, TRF2 and PINX1 did not correlate with telomere length in either normal myometrium or leiomyoma (Fig. 3). Moreover, the mRNA levels of TERT and TERC, the essential components of telomerase, were also measured. TERC was expressed at comparable levels in leiomyoma and normal tissue samples, independently of telomere length (Figs. 2 and 3), whereas TERT expression was negative in all samples tested (data not shown).