Ectopic expression of CYP24A1 circular RNA hsa_circ_0060927 in uterine leiomyomas

Abstract Background As a novel class of non‐coding RNAs, the role of circular RNAs (circRNAs) in tumor biogenesis and progression has been proved in a number of human tumors; however, up to now, the relation between circRNAs and uterine leiomyomas (ULM) remains unclear. Methods In this study, we have estimated the expression level of CYP24A1 hsa_circ_0060927 in uterine leiomyoma and adjacent tissues considering the mediator complex subunit 12 gene (MED12) mutation profile by quantitative real‐time polymerase chain reaction (qRT‐PCRs). Results Using Sanger sequencing method, somatic mutations in the MED12 exon 2 were detected in 14 (35.90%) ULM samples, including 10 (71.43%) missense mutations and 4 (28.57%) in‐frame deletions. Our results revealed that hsa_circ_0060927 was ectopically expressed in 33.33% of ULM tissues; although, this expression was independent of the MED12 mutation profile in the ULM samples. Conclusions Present results provide primary evidence for the role of circular RNAs in the leiomyoma development; however, further studies are essential to confirm the importance of these molecules as potential biomarkers for diagnosis and/or prognosis in ULM.

or novel therapeutic targets. According to the literature, leiomyomas have different genetic drivers, that the most common are rearrangements in HMGA2 (high mobility group AT-hook 2), inactivation of FH (fumarate hydratase), and mutations in the MED12 (mediator complex subunit 12). 6 Among these, somatic mutations in the MED12 exon 2 have a higher frequency in diverse populations. [7][8][9] Mehine et al 6 suggested that each genetic driver results in a different expression pattern in ULMs. Hence, molecular classification should be considered as a significant step in studying ULMs pathogenesis. Circular RNAs (circRNAs) are covalently closed loop non-coding RNA molecules, lacking 3' tail and 5'cap that widely exist in eukaryotic cells. Previously, these molecules were considered as byproducts of splicing errors; however, several circRNAs have been reported to have physiological functions. There are a great number of studies that validated the important correlation of circRNAs and different cancer types, including their role in initiation, progression, and metastasis of cancer. 10,11 However, to date, the association between ULMs pathology and circRNAs remains unclear.
Recently, there has been a greater focus on the vitamin D role in developing ULMs. 1α, 25(OH) 2 D 3, the active form of vitamin D, is present in most of human tissues such as myometrium. Vitamin D has an anti-tumorgenesis function and regulates the expression of many genes that play a role in cellular proliferation, differentiation, and apoptosis. CYP24A1 (Cytochrome P450 family 24 subfamily A member 1) is a mitochondrial enzyme that is responsible for neutralizing active vitamin D. 12,13 According to the previous studies, CYP24A1 functions as an oncogene and is upregulated in many tumors, such as ULMs 13,14 ; however, there is no evidence concerning the role of CYP24A1 related circRNAs in this disease. Due to the dysregulation of CYP24A1 in ULMs, we hypothesized that its related circRNA, hsa_circ_0060927 may also play a role in the disease development. Hence, the aim of the present study was to explore the relationship between the hsa_circ_0060927 expression level in ULM tissues, regarding the MED12 mutation profile.

| MATERIAL S AND ME THODS
Seventy-eight tissues, including 39 leiomyoma samples and 39 related adjacent tissues, were acquired from Iranian women with ULM who underwent myomectomy or hysterectomy in Taleghani hospital (Tehran, Iran). Surgery was performed within the first

| Patients
Genomic DNA was extracted from 30 mg of the tissues using CELL SV MINI kit (GeneAll) regarding the manufacturer procedure. The genomic DNA was amplified by PCR using two sets of specific primers (Table 1)    The amplification program was as follows: One cycle of 94°C for 15′, and 94°C for 20″, followed by 60°C for 45″ for 40 cycles, and the melt curve stage assessment. To check the primer specificity, melting curves analysis and agarose gel (2%) electrophoresis were performed. Table 1 shows the primer sequences for the studied genes. The hsa_circ_0060927 relative expression level was normalized to the β2M expression level.

| Statistical analysis
Prior to the expression analysis, the cycle threshold (C t ) data and amplification efficiency for each reaction were calculated using

| RE SULTS
The basic characteristics of the studied patients and the ULM samples are shown in Table 2

Circular RNAs have gained increasing attention in recent years.
Although there is little known about their role in human biological processes, the dysregulation of circRNAs is confirmed in several diseases including cancer. 11,16,17 Up to now, the relation between circular RNAs and ULMs remains unclear. We evaluated the hsa_ circ_0060927 expression level in uterine leiomyoma and adjacent tissue samples by qRT-PCR. Our results showed that this circular RNA was ectopically expressed in ULM tissues. We also showed that MED12 mutation did not have any significant effect on hsa_ circ_0060927 expression (p value = 0.775). To our knowledge, this is the first study about the relationship between circular RNAs and ULMs. Recent studies proved hypovitaminosis D as an important risk factor in ULM; and therefore, vitamin D metabolism plays an important role in the disease development. 18 (Figure 4).

| CON CLUS ION
We showed for the first time that the hsa_circ_0060927 was ec-

ACK N OWLED G M ENTS
This study has been extracted from the thesis written by Ms Elnaz