Metformin as a potential agent for modulating the faulty endometriotic mesenchymal stem cells: A case-control study

Abstract Background According to stem cell theory, it seems that the proliferation/differentiation imbalance in endometrial mesenchymal stem cells (enMSCs) is the leading cause of endometriosis, so targeting them to modulate stemness-relevant factors seems to be a wise choice for endometriosis treatment. Objective We aimed to investigate the effects of metformin on stemness properties of enMSCs by evaluating the expression profile of stemness-related genes and microRNAs (miRNAs). Materials and Methods In this case-control study, MSCs were isolated from the eutopic endometrium of 3 endometriotic and 3 healthy women. After their characterization and culture, they were treated with 0.1, 1, and 10 mM metformin for 72 hr. Finally, the expression of octamer-binding transcription factor (OCT) 4A, OCT4B, OCT4B1, sex determining region Y-Box transcription factor 2, nanog homeobox, microRNA-200b, microRNA-145, and lethal-7b were analyzed by quantitative reverse transcription-polymerase chain reaction. Results Metformin modulated the expression of stemness-related genes and miRNAs, OCT4A, OCT4B, OCT4B1, sex determining region Y-Box transcription factor 2, nanog homeobox, microRNA-200b, microRNA-145, and lethal-7b in enMSCs, especially at 1 and 10 mM concentration. Notably, metformin had a paradoxical effect on normal enMSCs. Conclusion We showed that metformin could modulate the expression of deregulated genes and miRNAs in faulty enMSCs, and restore their skewed self-renewal/differentiation balance, so it might be a promising drug for endometriosis treatment. The paradoxical effect of metformin on enMSCs and normal enMSCs might be because of their different metabolic patterns, so it requires further investigation to illustrate.


Introduction
Endometriosis is a benign debilitating gynecologic disorder with a growing prevalence and affects approximately 10% of reproductiveaged women and 50% of infertile women (1).
There are several theories about the cause of endometriosis, and stem cell theory is the most contentious. This theory states that stem cells play a key role in endometriosis development.
Since retrograde menstruation has an important role in carrying the endometrial stem cells to the peritoneum; it also plays a crucial role in endometriosis development (2). Although retrograde menstruation frequently occurs in reproductive-aged women, endometriosis occurs in only 10%.
As a result, it seems that endometriotic mesenchymal stem cells (enMSCs) are different from normal stem cells: they have shorter doubling time, high proliferation activity, decreased decidual response (3,4), and it also appears that they have skewed proliferation/differentiation balance.
The pluripotency regulated transcription factors Indeed, increased expression of these transcripts enhances the self-renewal, proliferation, and motility of stem cells despite decreases their differentiation (7). Transcriptional analyses and in vitro assays help identify the skewed expression of stemness-related genes and miRNAs in eutopic and ectopic endometrium of endometriotic women (8)(9)(10).

Specimen sources
In this case-control study, human endometrial tissue was obtained from premenopausal women aged 30-45 yr (mean 34.8 ± 4.7) in the secretory phase. We recruited 3 healthy fertile women as the control group and another 3 with stage III and IV endometriosis who went under laparoscopy at the Rasoul Akram hospital of Iran Medical University, Tehran, Iran. Women with endometrial anomalies like polyps, hyperplasia, or cancer, besides those who take hormonal treatment and gonadotropinreleasing hormone agonists, were excluded from the study.

Isolation and culture of human endometriotic mesenchymal stem cells
The endometrium layer was separated from the myometrium and washed in phosphate-buffered saline (PBS), then minced into 1-2 mm 3 pieces in a medium containing Dulbecco modified Eagle medium. Ham's F-12 (DMEM.F-12; Invitrogen, UK) and 1% penicillin-streptomycin antibiotic solution (Invitrogen, USA) were used. Enzymatic digestion was done with collagenase type 3 (300 µg/ml; Sigma, Germany) at 37°C for 90 min to obtain the cell suspensions. The cell suspension was filtered through 150, 100, 40 mm wire sieve to remove undigested tissue and epithelial components. Endometrial stromal cells were then cultured in T25 culture flasks containing DMEM/F-12 (Invitrogen, UK), 1% penicillin-streptomycin solution, and 10% fetal bovine serum (FBS, Gibco, USA) (19).

Endometrial stromal cells flow cytometry analysis
Isolated stromal cells were trypsinized and centrifuged. Cell pellets were resuspended in PBS containing 5% FBS and incubated for 45 min on ice then centrifuged. Cell pellets were resuspended in PBS containing 5% FBS and incubated with PE or FITC-conjugated antibodies for 30 min at 4°C in the dark. Hematopoietic stem cells specific antibodies served as negative controls (FITC-conjugated antihuman CD45 [BD Bioscience, USA] and CD34 [IMMUNOSTEP, Spain]), and PE-conjugated antihuman CD90, CD105, CD73, and CD146 (BD Bioscience, USA) were used as MSCs specific antibodies. Fluorescence-activated cell sorting (FACS) was done on the FACS Calibur apparatus (Becton Dickinson, USA), while data analysis was done with the FlowJo 7.6 software.

RNA extraction and cDNA synthesis
We used TRIzol (Sigma Germany) to extracte the cellular RNA, then evaluated the quality and

Real-time PCR for mRNAs and miRNAs expression detection
To determine the expression of OCT4A, OCT4B, OCT4B1, SOX2, NANOG, miR-200b, miR-145, and let-7b, we used the SYBR Green Assay kit (Applied Biosystems, UK), following the manufacturer protocol. Sequences of PCR primers were presented in our earlier studies (11,12). We accomplished real-time PCR reactions in 10 μL of the reaction mixture by AB StepOne real-time PCR System (Applied Biosystems, UK), followed by analyzing the data by the Pfaffl method. GAPDH and RNU44 were used to normalize the expression value of genes and miRNAs, respectively.

Ethical considerations
Ethics approval for this study was obtained by the Ethics Committee of Medical Faculty of Tarbiat Modares University, Tehran, Iran (No. 1395.409).
Written informed consent was obtained from each participant.

Statistical analysis
Statistical significance of variances between group means was analyzed using either Student t test or ANOVA using GraphPad Prism version 6.0.0 for Windows, GraphPad Software, San Diego, California USA. Results considered significant at a p < 0.05.

Metformin treatment repressed the gene expression of SOX2 and NANOG in a dose-dependent manner
Seventy-two hr treatment of normal enMSCs with 1 and 10 mM metformin compared to untreated MSCs showed significantly increased expression of SOX2 and NANOG ( Figure 3A) (Table I) Indicating that metformin had a modulator effect on NANOG expression in enMSCs ( Figure 3G).

Metformin mediates downregulation of miR-200b in enMSCs
In normal enMSCs, all metformin concentrations had an increasing effect on miR-200b expression after 72 hr compared to untreated-enMSCs ( Figure 4A) (Table II)

Metformin up-regulates the expression of miR-145 and let-7b in enMSCs
In normal enMSCs, all metformin concentrations decreased the let-7b expression compared to untreated-enMSCs, but the miR-145 expression was not influenced by any metformin doses  The results are presented as the fold change relative to 0 hr. Data were presented as the Mean ± standard deviation, analyzed using one-way analysis of variance and then compared among groups using Student's t test. A significance threshold of p < 0.05 was used Table II The results are presented as the fold change relative to 0 hr. Data were presented as the Mean ± standard deviation, analyzed by one-way analysis of variance and then compared among groups using Student's t test. A significance threshold of p < 0.05 was used

Discussion
Stem

Conclusion
In the current study, we showed that metformin could modulate the expression of deregulated genes and miRNAs in enMSCs, and may correct their skewed self-renewal/differentiation balance.
It might be a promising drug for endometriosis treatment.