INTRODUCTION
Also known as fibroids, uterine leiomyomas are the most common benign gynaecological tumours in women in reproductive age. Averagely, between 10% and 30% of cases cause abnormal uterine bleeding, recurrent pregnancy loss, pelvic pain, preterm birth and infertility [1, 2]. Nulliparity, early menarche, late menopause and obesity enlarge lesion size. However, lesion shrinking after menopause makes us consider that oestrogen and progesterone have a significant role in leiomyoma pathogenesis [3]. Cellular and molecular pathogenesis of common leiomyomas has not been revealed yet. It has been suggested that each leiomyoma stems from a mutant myometrial smooth muscle stem cell [4].
In some studies that research genetic factors underlying uterine leiomyomas, it is claimed that, in 40–50% of uterine myomas, certain gene regulations including 7q deletion and some different chromosomal aberrations such as 12q15 and 6p21 are observed [5, 6]. In addition to chromosomal change, Makinen et. al. [7], found that, in 71% of uterine leiomyoma cases, tissues had mutation in mediator complex subunit 12 (MED12) exon 2. MED12 is situated on chromosome sub-band Xq13 and consists of 45 exons. However, it is observed that leiomyoma mutations are only existent in 36.–44. codons in exon 2 [8, 9]. The prevalence of MED12 mutation is studied with different ethnic groups and it is found that its prevalence ranges from 50% to 80%, depending on ethnic group [8, 10, 11]. MED12 mutation is also seen in leiomyosarcoma (52%) and uterine smooth muscle tumour of uncertain malignant potential (STUMPs) (8%) [12].
In some other studies, it is illustrated that microRNAs regulate gene expressions by degrading or repressing messenger RNAs [13–15]. A mature microRNAs complex, combined collaterally with target gene sequences, results in gene expression degradations or gene expression decrease [16]. It is also known that microRNAs act as oncogenesis and tumour suppressor gene [17]. Similarly, in leiomyoma cases, microRNA expressions at different levels and microRNA deregulation are reported. Therefore, they are mostly associated with leiomyoma pathogenesis by many researchers [18]. Although many microRNAs are identified to have a role in leiomyoma formation, microRNA-124, which, like MED12, is situated in Wnt pathway, has not yet been found to have any impact on leiomyoma formation [19].
In these contexts, in this study which consists of a population of some Turkish patients, it is aimed to compare microRNA-124 expression differences and MED 12 gene exon 2 region mutations between specimens of leiomyoma tissues and myometrium tissues of the patients.
MATERIAL AND METHODS
In this retrospective study, paraffin blocks were obtained from total abdominal hysterectomy specimens of patients with a diagnosis of uterine leiomyoma, who visited Gaziantep University, Medical School, Department of Obstetrics and Gynaecology between January 2013 and January 2016. Inclusion criteria were total abdominal hysterectomy due to leiomyoma resulting in irregular bleeding, pelvic pain and sensation of pelvic fullness without the diagnosis that have been reported among the exclusion criteria. Exclusion criteria were patients with other gynaecologic diseases (endometriosis, premalignant or malignant gynaecological disease), systemic disease or other system malignancies. Ethics committee approval for the study was obtained from the Clinical Research Ethics Committee of Gaziantep University (No: 255, 26th September 2016). The consent form was obtained from the patients and tissue samples were transferred to Department of Molecular Medicine, Istanbul University Aziz Sancar Institute of Experimental Medicine (ASDETAE). Experimental stages of the study were carried out in that laboratory. Demographic characteristics and histopathological diagnosis of the patients were all recorded. DNA isolation, exon 2 region of MED12 gene with Polimerase chain reaction (PCR) and Sequencing method and microRNA-124 levels of leiomyoma tissues (leiomyoma group) and intact eutopic myometrium tissues of the same patients (control group) were studied. Paraffin embedded uterine tissue samples were divided into two groups (Group 1: Leiomyoma group, Group 2: Control group).
DNA and Total RNA isolation from paraffin tissue
The pieces cut from the paraffin block with microtome were treated with xylol and the paraffin was dissolved to provide tissue cleaning. DNA and Total RNA was isolated from 30 leiomyoma and 30 intact eutopic myometrium tissues with the QIAzol Lysis Reagent®QIAGEN from ~ 30 mg sections of each sample to the different protocol [20, 21].
Amplification and Sequencing of Exon 2 region of MED12 gene by PCR method
Primary sequences used to amplify the PCR fragment of Exon 2 region of MED12 gene are as follows: Forward: 5’-CCC CTT CCC CTA AGG AAA AA-3’, Reverse: 5’-ATG CTC ATC CCC AGA GAC AG-3’. Thermal cycler (T100™; BioRad, CA, USA) was used for PCR amplifications. PCR amplification was performed in a total volume of 25 µL with 1 µL of 150–200 ng DNA, 1 µL of forward and reverse primers (50 pmol/μL), 5 µL of dNTPs (1 mM), 1.5 µL of 10X Taq tampon, 1.5 µL of MgCl2 (25 mM), 0.3 µL of Taq DNA polymerase (5 u/μL) (GeneMarkGMbiolab Co., Ltd. Taichung, Taiwan) and 14.7 µL of distilled H2O. The PCR mixture was incubated for five minutes at 95°C, followed by 30 cycles of 45 seconds at 94°C, 45 seconds at 59°C and 45 seconds at 72°C and a final step at 72°C for five minutes. The synthesized PCR products were separated by agarose gel electrophoresis and it was checked whether the DNA fragment of the desired size was amplified. PCR products were purified, and DNA sequencing was performed to the Sanger sequencing method which was studied by Dogan et al. [22].
microRNA-124 expression analysis
Total RNAs were diluted to 1μg/5μL and QIAGEN miScript®microRNA-124 detection kit was used for complementary DNA (cDNA) synthesis. The PCR was carried out with a total volume of 20 µL containing 5 µL of Total RNA, 4 µL of 5 × miScriptHiFlex Buffer, 2 µL of 10 × miScript nucleic mixture, 2 µL of reverse transcriptase and 7 µL of RNase free water. The reactions were subjected to 25°C for 10 min, 37°C for 60 min, 95°C for 5 min, and 4°C hold. For quantitative real-time PCR (qPCR), the U6 gene was used as reference gene for the RNA expression detection. After cDNA synthesis, Real-Time PCR analysis was performed with QIAGEN miScript SYBR ® Green PCR kit. The qPCR reactions were carried out with a total volume of 12.5 µL containing 6.25 µL of 2x Quantitech SYBR Green PCR Master Mix, 1.25 µL of 10x miScript Universal Primer, 1.25 µL of 10x miScript Primer Assay, 1,25 µL of cDNA, and 2.5 µL of RNase free water. The qPCR reactions were subjected to hot start at 95°C for 15 min, followed by 40 cycles of denaturation at 94°C for 15 s, annealing 55°C for 30 s, and extension at 70°C for 30 s using the real-time detection system. Real-time PCR was performed twice for each sample. CT was obtained as a real-time PCR result for each repetition of each gene. The mean CT value was calculated by averaging the two results. The expression of genes was quantified by measuring the cycle threshold (Ct) values and normalized using the 2-∆∆CT method relative to the U6 RNA [23].
Statistical analysis
Statistical analyses were performed using the IBM Statistical Package for Social Sciences 22nd version. The Kolmogorov-Smirnov, Student’s t test and Mann–Whitney U tests were used in the statistical analysis of the data. The value of p < 0.05 was considered as statistically significant.
RESULTS
There were a total 60 tissue samples belonging to 30 patients that were included in the study. Samples taken from the leiomyoma tissues of the patients were compared with the samples taken from the patients’ own eutopic myometrium tissues. The mean age of the patients included in the study was 46.67 ± 5.42 (min: 38, max: 68). The mean size of leiomyomas was 6.17 ± 4.74 cm (min: 1 cm, max: 23 cm) and the mean number of leiomyomas was 4.90 ± 5.62 (min: 1, max: 25). The levels of white blood cells (WBC), hemoglobin, platelets, glucose, blood urea nitrogen (BUN), creatinine, aspartate transaminase (AST), and alanine transaminase (ALT) of the patients were 7.43 ± 1.85 (106/mL) (min: 3, max: 11), 11.53 ± 2.42 (gr/dL) (min: 4, max: 16), 36.23 ± 6.35 (%) (min: 15, max: 46), 304.93 ± 75.57 (106/mL) (min: 166, max: 473), 111.27 ± 55.95 (mg/dL) (min: 75, max: 377), 27.23 ± 7.68 (mg/dL) (min: 15, max: 48), 0.9 ± 0.40 (mg/dL) (min: 0, max: 2), 20.17 ± 10.76 (U/L) (min: 10, max: 60), and 17.53 ± 9.00 (U/L) (min: 7, max: 41); respectively. Thirteen pateitns included in the study had one leiomyoma (43.3%) whereas 17 had more than one leiomyoma (56.7%). Two patients had only subserous leiomyomas, three had only intraligamentary leiomyomas, four had only submucous leiomyomas, and 11 had only intramural leiomyomas. Other patients had leiomyomas located in two or three different regions.
DNA sequences obtained from leiomyoma and myometrium tissues of 30 patients were analyzed by Sanger sequencing method. Exon 2 region of MED12 gene c.130G>T, p.G44C mutation was found in 14 leiomyomas (46.6%) (p < 0.001) (Fig. 1), and c.131G>A (p.G44A) mutation was observed in 6 of leiomyoma tissues (p = 0.01) (Fig. 2). No such mutation was observed in healthy myometrium tissues of same patients. According to the results of our study, c.130G>T, p.G44C mutation and c.131G>A (p.G44A) mutation of MED 12 gene exon 2 were found significantly higher in leiomyoma tissue.
The expression levels of microRNA-124 were calculated according to the 2-ΔΔCT method (Tab. 1). There was a 3.7-fold decrease in the expression of microRNA-124 in leiomyoma tissues compared to intact eutopic myometrium tissues, but this difference was not statistically significant (p = 0.109) (Fig. 3).
|
Table 1. Expression values for microRNA-124 |
||
|
Leiomyoma |
Myometrium |
|
|
ΔCT |
–0.938 |
–2.835 |
|
ΔΔCT |
1.897 |
|
|
2−ΔΔCT |
0.27 |
|
|
Fold |
–3.7 |
|
|
p |
0.109 |
|
There was no significant correlation between miR-124 expression levels and the MED12c.130G>T, p.G44C mutation in tissues with the mutation (p = 0.125).
DISCUSSION
Uterine leiomyomas are the most common benign mesenchymal neoplasm in women [8, 9]. Along with underlying etiological factors, genetic factors that might lead to leiomyoma formation have recently attracted many researchers. Therefore, there are now many studies regarding this subject.
In recent studies, the function of MED12 mutation in leiomyoma pathogenesis has been studied. For example, Di Tomasso et al. analysed MED12 mutation on leiomyoma, myometrium and pseudocapsule and reported both distinguished mutation profiles and IGF-2 levels between leiomyoma and pseudocapsule [24, 25]. It is also demonstrated that MED12 mutation disrupts the relation between MED12 and Cyclin C, CDK8/19, and that it also harms mediator-associated CDK kinase activity [26]. In some other studies, the relation between MED12 and β-catenin/Wnt pathway is revealed [7, 27]. Wnt pathway’s selective inactivation decreases normal uterine myometrial cell formation and supports its conversion into a small but fart-rich uterus. This shows us the significance of Wnt signal for normal uterus development [28].
MED 12 is a 25-subunit protein complex that ensures cell development and durability by regulating RNA polymerase 2 in association with CDK8 [29]. The MED12 mutation, which is effective in Wnt pathway, is seen uterine leiomyomas (30–52%), leiomyosarcoma (14-20%) and unclear smooth muscle tumours with a potential malignity (8%) [12, 30, 31]. MED12 is situated on chromosome sub-band Xq13 and consists of 45 exons. However, it is observed that leiomyoma mutations are only existent in 36.–44. codons in exon 2 [8, 9]. The prevalence of MED12 mutation ranges from 50% to 80%, depending on ethnic group [8, 10]. Je et al., examined 1862 tumour tissues that include carcinoma, leukaemia and stromal tumours. They reported that MED12 mutation was seen in 52.2% of uterine leiomyoma cases and 0.3% of colon carcinomas [12]. In these contexts, in our study, cDNA sequence, which is obtained from leiomyoma and myometrium tissues of 30 patients via Sanger sequencing method, is analysed. Findings of the study illustrate that 67% of the patients have MED12 exon 2 mutations. In 14 leiomyoma tissues, c.130G>T (p.G44C) mutation was seen in exon 2 region of MED12 gene (70%). On the other hand, in six cases, c.131G>A (p.G44A) mutation was observed (30%).
It is demonstrated that MicroRNAs can be used as biomarkers for many malign and benign diseases [32]. MicroRNAs are key molecules in post-transcriptional regulation of gene expression and aberrant gene expression-oriented microRNA-124 expression alterations. Irregular microRNA expression is associated with tumuorigenesis, tumour progression, fibrosis, and tumour immunity. Similarly, microRNA-124 expression is observed in various sarcoma cases including GIST [33] and uterine leiomyoma cases [34]. Some microRNAs are expressed at different ratios in leiomyoma cases seen in different races [35]. These racial differences regarding microRNA expression play a significant role determining biomarkers, molecular pathogenesis and potential leiomyoma treatments.
Some inhibitors of Wnt signal pathway are run by microRNAs, and microRNA-124 is one of them [36]. MicroRNA-124’s role in tumour progression, motility and angiogenesis has been studied and it is identified as a tumour suppressor microRNA [37, 38]. It is also demonstrated that microRNA-124 suppresses some other tumour functions such as proliferation, activation, invasion, metastasis, and migration. In colorectal carcinomas, breast cancers, osteosarcoma, lung cancers and gastric cancers, abnormal expression of microRNA-124 is observed, as well [39–43]. On the other hand, microRNA-124 expression is reported to be decreasing in ovarian cancer [38], cervical cancer [44] and endometrial cancer [45]. There are some other studies illustrating that it influences proinflammatory cytokine leiomyomas [46–49]. For example, it is reported that microRNA-124 regulates cholinergic anti-inflammatory effect by inhibiting LPS-induced proinflammatory cytokines [50–53]. In the present study, it is also found that leiomyoma tissue has decreased 3.7 times as much as a healthy myometrium tissue since it has microRNA-124 expression. However, this difference is not determined to be statistically significant (p = 0.109). There are several studies found the relation between Wnt pathway and miR-124 expression. Hu et al. [54], found that miR-124 had lower expression levels in nasopharyngeal carcinoma compared to normal nasopharyngeal cells. They indicated that miR-124 suppresses of proliferation and invasion of nasopharyngeal carcinoma cells targeting Capn4 and the components of the Wnt/β-catenin signaling pathway [54]. Another study showed that overexpression of miR-124 suppressed the activity of non-canonical Wnt signaling, downstream of ROR2 and they suggested that miR-124 expression might inhibits osteosarcoma metastasis [55]. Furthermore, miR-124 might be a strategy for multiple-drug resistance. Long et al., reported that Wnt signaling is triggered by binding of Wnt ligands to Frizzled receptor proteins and this receptor/protein kinase C (PKC) signaling is responsible for the elevation of P-glycoprotein which is associated with multiple-drug resistance and cancer cell survival. They found miR-124 targeted to Frizzled receptor and ad significant inhibitory effects on P-glycoprotein expression. Thus, they suggested that miR-124 expression might be new therapy strategy to overcome P-glycoprotein mediated multiple drug resistance [56].
CONCLUSIONS
This study is unique in that it is the first study in Turkey that has been carried out about uterine leiomyoma cases regarding somatic mutations on exon 2 of MED12 complex and microRNA-124 gene expressions.
Age range of patients included in our study was between 38 and 68, so at least one patient was postmenopausal and expression of mir124 might be dependent on steroidal hormones, ideally all tested sample should be either pre-menopausal or post-menopausal for a homogeneous conclusion. This is clearly a limitation of our study. Also, the relatively small sample size may be another limitation.
This study demonstrates that both MED12 exon 2 mutations and microRNA-124 gene expressions might contribute to uterine leiomyoma pathology. When the prevalence of uterine leiomyomas and its impact on the quality of life are taken into consideration, it can be argued that, in the future, MED12 and microRNA-124 will be main focus of potential treatment strategies.
Ethics committee approval
Ethics committee approval was received for this study from the ethics committee of Gaziantep University.
Financial disclosure
The authors declared that this study received no financial support.
Conflict of interest
No conflict of interest was declared by the authors.
