Cell cycle gene expression is restored in eutopic primary stromal cells after endometriosis surgery

doi:10.21203/rs.3.rs-3010156/v1

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Cell cycle gene expression is restored in eutopic primary stromal cells after endometriosis surgery

https://doi.org/10.21203/rs.3.rs-3010156/v1

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The role of the endometrium in the pathogenesis of endometriosis has assumed prominence. Abnormality of gene and protein expression, apoptosis and changes in cell cycle have been extensively studied in endometriosis. We evaluated the cell cycle genes expression on primary stromal endometrial cells (EC) isolated from eutopic endometrium in two different moments: before and after deep infiltrating endometriosis (DIE) surgical treatment. We analysed five ECs from healthy patients (group control) and 9 from women with diagnosis of DIE. Were identified 7 cell cycle genes (p53, TFDP1, TFDP2, KPNA2, RB1, RBL2, SERTAD1) differentially expressed between pre, post-operative and controls. The p53 and KPNA2 genes were downregulated 3.34 fold (p = 0.006) and 2.62, (p = 0.042), respectively, in the endometrium of DIE compared to control. Both were upregulated (p53 - Fold 2.22, p = 0.157; KPNA2 - Fold 4.36, p = 0.017) in the post-operative DIE group in comparison to pre-operative one. Also, the RB1 gene was downregulated 9.36 fold (p = 0.029) in the DIE-post group in comparison to DIE-pre group, having no difference between DIE-pre and control group (p = 0.311). The proteins coded by these genes have association between each other, indicating that the surgical treatment could change the cell cycle regulation in the endometrium of women with endometriosis and that the changes remain after the cell isolation from the tissue.

Endometriosis

cell cycle genes

p53

endometrium

surgery

The endometriosis pathogenesis studies have revealed that the eutopic endometrium is innately aberrant and plays an important role in the pathophysiology of the disease [1].The eutopic endometrium of women with endometriosis have an increased cell proliferation and decreased programmed cell death [2]. Analysing the expression of cell cycle molecules in endometrial biopsies, our group demonstrated that endometriotic tissues have lower levels of p27 protein compared with those of healthy control [3–5].

The eutopic endometrium shares changes with ectopic tissue, which are not found in the eutopic endometrium of women without the disease, corroborating the thesis that the main defect in endometriosis must be primarily in the eutopic endometrium[1]. However, it is still unclear whether abnormal findings in the eutopic endometrium of patients with the disease is related to the ectopic inflammation due to the ectopic tissue or primary of the uterine mucosa [6].

Researchers suggest that foci of endometriosis can activate transduction signals that alter the gene expression of the eutopic endometrium [7] and may be an explanation for presenting better rates of embryo implantation in infertile patients when submitted to surgical treatment [8]. According to this, we have evaluated the cell cycle-related genes expression on primary eutopic endometrial cells obtained before and after endometriosis surgical treatment to understand if the alterations of endometriosis are primary or secondary to the endometrial tissue, that is, if the change of the peritoneal environment through the surgical treatment would provide reestablishment of the normal conditions of the endometrial environment.

Patients’samples

The study was approved by the Research Ethics Committee of the Federal University of São Paulo (Ref. 226536/2013), and free informed consent was obtained from all of the participants. ECs were isolated from pipelle biopsies of the eutopic endometrium from 14 women, aged between 20 and 35 years, undergoing surgical treatment for the diagnosis of benign ovarian cyst (control) or endometriosis. None of the participants had received hormonal preparations or used GnRH analogues in the three months before the endometrial collection. Were excluded the patients that had childbirth or breastfed in the six months before enrollment in the study, those with menstrual irregularities, teratoma, myoma, endometrial polyp or adenomyosis.

Cell isolation and RNA extraction

ECs were isolated according to Pelvic Pain Unit standard protocol and cultured until the second passage and then cryopreserved [9]. mRNA was extracted with TRIzol (Invitrogen – LifeTechnologies - Carlsbad, CA, USA) according to manufacture’s protocol. Quantification and quality control (260/280 ratio) were evaluated on Nanodrop (Nanodrop Technologies Inc., Rockland, DE).

Real-time PCR analysis

The cDNA was synthesized with RT2 First Strand Kit (Qiagen, Hilden, Germany). The gene expression was evaluated by the SYBR Green RT-PCR method with QuantiTect Primer Assay (Qiagen, Hilden, Germany) using the PAHS-020A (Qiagen, Hilden, Germany) plate following the manufacturer’s instructions. The groups studied were divided into: Control (n = 5, patients without endometriosis), Pre-operative DIE - DIE-Pre (n = 6) and Post-operative Endometriosis - DIE-Post (n = 3). The analysis included two housekeeping genes: GAPDH and β-Actin. Triplicates were performed for all samples, and reactions carried-out on Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA).

Statistical analysis

The results were analyzed in the Data Analysis V3.5 PCR software (Qiagen) and p-values calculated using Student’s t-test. A value greater than one indicated an upregulation, and a value less than one indicated a downregulation. A comparison was considered as statistically significant if the p values were less than 0.05.

We have observed seven (p53, TFDP1, TFDP2, KPNA2, RB1, RBL2, SERTAD1) differentially expressed genes in the ECs comparing all three groups (Fig. 1). These genes are strongly related to the homeostasis and control of the cell cycle indicating that the surgery can implicate in the gene expression of the endometrial cells, confirmed by the interaction between the protein coded by these genes and the clusterization observed for their expression (Fig. 2).

The differentially expressed genes found in this work codes for proteins which have an association between each other (Fig. 3). The cell cycle is strictly regulated by extracellular signals and the check-points (cell cycle arrest points) provides cells with the opportunity to repair damages before division, preventing the transmission of genetic errors to daughter cells. Also, these check-points are responsible for the recovery of the cells from damages preventing premature cell death[10]. The disrupted cell cycle and aberrant inflammatory molecules expression have been already described in the endometrium of women with DIE [11]. These differentially expressed genes found codes for proteins which have an association between each other and also with some endometriosis cell cycle key protein (p27 and p21) [4, 5], according to the following diagram (Fig. 3). Although these studies have considered only p27 protein levels and not the mRNA levels. In this study,we did not observe differences in the expression of p27 between DIE samples and controls (FC -1.58; p = 0.3768). Indicating that possible a post-translational mechanism regulates the p27 protein content in the endometriosis samples.

This can be explained by the Karyopherin alpha 2 (KPNA2) expression, which is 2.62 times downregulated in the endometriosis groups compared to control and 4.36 times upregulated after surgery in comparison to pre-surgery ECs. KPNA2 is known as a selective nucleocytoplasmic carrier of transcription factors and proteins related to DNA repair and cell cycle regulation [12]. It is directly related to the translocation of p27 in and out of the nucleous [13]. Exacerbated expression of KPNA2 has been reported in malignant tumours and its knockdown causes the accumulation of p53 protein in the cytoplasm[14]. We demonstrate an up-regulation of KPNA2 in the ECs from patients submitted to surgical treatment.

p53 protein is known as a general stress sensor, not only important to inhibit cancer progression, but also to respond during viral infection, starvation or oxidative stress, reducing cell proliferation, altering cellular metabolism and inhibiting survival[15]. The gene that codes to p53 (TP53) is 3.34 less expressed in the endometriosis group than in the control group and no statiscally significant difference was found between post-operative and control groups (fold change − 1.51, p = 0.3590). p53 is downregulated in the ECs of DIE patients compared to control and its expression is restored after surgical removal of ectopic endometriosis foci.

Post-operative ECs shows other sign of a tentative regulation of the G1/S transition. The gene RBL2, which encodes for a retinoblastoma (Rb) protein, was found to be 2.23 times more more expressed in endometriosis post-operative than in control group. RBL2 proteins intereact with CDKs complexes to promote a proper G1/S transition[16]. Another gene involved in controlling early cell cycle events, from G1 to S phases, is the transcription factor DP-1 (TFDP1). TFDP1 is downregulated 2.05 fold in the DIE-pre than in Control ECs. TFDP1 or TFDP2 forms a heterodimeric complex with the tumour suppressor RB1 (downregulated 9.36 and 14.28 fold in the DIE-post in comparison to DIE-pre (p = 0.0295) and Control (p = 0.0211), respectively) binded to E2F transcription factors. This interaction results in the prevention of G1/S transition [16]. In previous work, we observed the downregulation of TFDP1 in the endometrium of women with DIE, but no changes were observed on TFDP1 expression between pre and postoperative ECs (FC 1.55, p = 0.2475).

By the results presented, we infer that surgical removal of ectopic endometriosis foci leads the eutopic endometrial cells to a cell cycle genes pattern more similar to the control group. Acting directly on the cell cycle arrest, blocking or at least by, reducing the proliferation and development of endometriosis cells. To the best of our knowledge this is the first report of endometrial cells expression changes after endometriosis surgery. In spite of the limited number of samples, this study can highlight the importance of the study of post-operative endometrium cells for the understanding of endometriosis pathogenesis.

Ethical Approval and consent to participate

The study was approved by the Research Ethics Committee of the Federal University of São Paulo (Ref. 226536/2013), and free informed consent was obtained from all of the participants.

Competing interests

The authors declare that they have no affiliations that would constitute a financial conflict of interest regarding the subject matter presented in this study.

Authors' contributions

ALI, FYA and CVC: study design, acquisition of data, analysis and interpretation of the data.GK and AK: acquisition of data and interpretation of the data. ES: study design and interpretation of the data. All authors contributed with the drafting of the article, critical revision and have approved the final version of the manuscript.

Funding

This work received support of the National Council for Scientific and Technological Development (CNPQ) - 134111/2013-13 as a master scholarship to FYA.

Availability of data

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

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No competing interests reported.

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Version 1

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Cell cycle gene expression is restored in eutopic primary stromal cells after endometriosis surgery

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Version 1

Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Patients’samples

Cell isolation and RNA extraction

Real-time PCR analysis

Statistical analysis

RESULTS AND DISCUSSION

Declarations

References

Additional Declarations

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