Evaluation of NF1 and RASA1 gene expression in endometriosis.

Background: Endometrios affecting 6-10% of women of reproductive ages around the globe. Important pathways, including the MAPK and PI3K / Akt pathways, have been identified in the disease. The NF1 and RASA1 genes inactivate Ras by their own GTPase activity and controlled the high activity of these pathways. Objective: In this study, we measured NF1 and RASA1 gene expression in the endometrial tissues of patients (eutopic and ectopic tissues) compared to the control samples. Materials and methods: In our study, tissue samples were collected from 15 patients with endometriosis and 15 healthy women. We used quantitative polymerase chain reaction (qRT-PCR) to measure the NF1 and RASA1 gene expression levels in these samples.
Results: We observed a significant decrease in the expression level of the NF1 gene in both eutopic and ectopic samples of endometriosis patients compared to control samples, while the expression of the RASA1 gene was significantly reduced only in ectopic tissues.

Introduction

Endometriosis, as an estrogen-dependent disease, is one of the most common abnormalities among women. In this disease endometrial tissues, including the mesenchyme and stroma, grow outside the uterine cavity [1]. It affects approximately 10% of women of childbearing age and 20–50% of women with infertility problems [1], [2], [3]. These tissues grow in the ovaries, pelvis, peritoneal cavity, vaginal, anus wall and in rare cases are found in the diaphragm, pericardium and pleura. Some symptoms are common in endometriosis patients such as painful menstruation, chronic pelvic pain, abnormal bleeding from the uterus and reduced fertility and infertility [4], [5], [6]. Although the exact cause of this disease is not yet clear [7]. many studies have shown that endometriosis is a multifactorial disease and several factors are involved in its pathogenesis such as signaling pathways, hormonal activity, immune system problems, environmental factors and genetic factors [8]. The role of genetic factors including aberrant expression of genes and important signaling pathways has been demonstrated [9].

Some studies have shown that the MAPK(mitogen-activated protein kinase)pathway plays an important role as extracellular and intracellular signaling in endometriotic cells, then its abnormal activation can involve in the pathogenesis of endometriosis [10], [11]. One of the main pathways downstream of many EGF(epidermal growth factor)receptors is the RAS-mediated signal, which activates the MAPK pathway [12]. Ras proteins that are constantly rotating between active Ras-GTP and inactive Ras-GDP are located mainly on the inner surface of the plasma membrane [13]. Several pathways, including PI3K / AKT and MAPK, which play an important role in cell survival and growth, can be interconnected and activated by Ras linked to GTP [14]. RASA1 and NF1 proteins, which are RasGAPs (Ras-GTPase Activating Proteins), terminate the active Ras state by hydrolyzing GTP to GDP, thus negatively regulating the Ras pathway [15]. Accordingly, the development of various mutations and disruption of the expression of this tumor suppressor RasGAPs can lead to the formation of various types of cancers as well as endometriosis [16], [17].

In this study, our aim was to investigate the expression of NF1 and RASA1 genes in patients with endometriosis (both ectopic and eutopic samples) compared with healthy individuals.

Materials and methods

According to the ethical rules, the general consent of all patients was obtained consciously and freely. This study has also been approved by the Ethics Committee of Yazd Institute of Reproductive Sciences, Shahid Sadoughi University of Medical Sciences, Yazd, Iran. From September 2020 to February 2021,45 samples were collected from women, who referred to Madar and Shahid Sadoughi hospitals. Normal endometrium (15 samples) and endometriosis tissues (15 ectopic and 15 eutopic) were collected during surgery. Normal endometrium samples were collected from women who were hospitalized for other reasons such as fallopian tube closure and pelvic pain who did not have endometriosis. These samples were placed in microtubes containing RNA later solution (CinnaGen,Iran) and stored at − 80 °C. Inclusion criteria were that all women who underwent surgery were between 20 and 45 years old and did not receive any hormone therapy until 6 months before surgery, as well as exclusion criteria, included the presence of benign tumors polyps (fibroids), hyperplasia, carcinoma, and human papillomavirus infection.

Quantitative real-time PCR and RNA isolation

We extracted total RNA from the tissues of patients and healthy individuals using RNA xPlus Solution (Cinna Gen, Iran). We determined the RNA concentration and purity by using OD 260/280 and a spectrophotometer (NanoDrop, Thermo Fisher). cDNA (complementary DNA) of the NF1 and RASA1 was synthesized by using a cDNA synthesis kit (Yekta Tajhiz Azma, Iran) according to its instructions. Real-time PCR was performed separately for NF1 and RASA1 and run in a Rotor GeneQ device (Qiagen, Germany) to check their expression. We used GAPDH for the reference gene for NF1 and RASA1 (Table 1).

Table 1

Oligonucleotide primers.

Gene	Primer sequence
NF1	Forward: 5′- CGAATGGCACCGAGTCTT AC-3`Reverse:5′-GACCAGTTGGACGAGCCC − 3′
RASA1	Forward:5`-CCAACGCCAAACAATCAG − 3`Reverse: 5`- ATTTCCTTGCCATCCACT − 3`
GAPDH	Forward: 5`-CAAGAGCACAAGAGGAAGAGAGAG-3`Reverse: 5`-TCTACATGGCAACTGTGAGGAG-3`

Statistical analysis

Statistical analysis was done by one-way ANOVA using GraphPad Prism software (Version 7.3). We considered p < 0.05 for a significant difference. The figures in this article are also drawn by Prism7.3 software.

Result

Real-time PCR data analysis showed that NF1 gene expression was significantly different between patients and control samples. NF1 gene, in the ectopic group compared to the control group, showed a significant decrease (p = 0.0024), while, its expression did not change much compared to the eutopic group (p = 0.8773). The expression level of the NF1 gene in the eutopic samples also had a significant decrease compared to the control samples (p = 0.0093) (Fig. 1A).

Fig. 1

comparison of NF1 and RASA1 gene expression with GAPDH gene expression in three ectopic, eutopic and control tissues. The ratio of NF1 to GAPDH (A) and the ratio of gene expression of RASA1 to GAPDH (B).

RASA1 expression in the ectopic group showed a significant decrease compared to the control group (p = 0.0346), while, its expression did not change significantly compared to the eutopic group (p = 0.3470). The expression of this gene in the eutopic group there was no significant difference compared to the control (p = 0.4708) (Fig. 1B).

Discussion

Endometriosis originates from the uterus, which is a reproductive tissue of women [18]. Endometriosis tissues located at other parts of the body, especially in the ovaries [19]. Some processes in endometriosis such as transmission to other tissues, growth and proliferation is similar to female reproductive cancer. In fact, it is a precancerous condition and several studies have been performed to investigate its molecular processes [20], [21]. The MAPK signaling pathway is very important in regulating cellular processes such as survival, differentiation, proliferation, apoptosis, inflammation and innate immune processes [22]. Therefore, the impaired activity of any of the components of this pathway can cause metastasis, invasion and formation of various tumors [23]. In addition, PI3K / Akt signaling, which is involved in processes such as angiogenesis, cell proliferation, and survival, has been shown to be an important pathway in the development of endometriosis [24]. Ras in its active state (connected to GTP) activates the MAPK and PI3K / Akt paths [25]. NF1 and RASA1 genes are tumor suppressors that inhibit RAS activity by acting as GAP [26]. As a result, the activity of the MAPK and PI3K / Akt pathways is either reduced or inhibited [26].

The RASA1 gene plays an important role in controlling the angiogenesis process [27]. Studies have shown that reducing the function of the RASA1 gene interferes with the regulation of the angiogenesis process and causes various cancers, such as breast cancer [16], [28]. It has been shown that increasing RASA1 levels by suppressing miR-145 can reduce the progression of ovarian cancer [29]. In a study by Zheng et al. on hepatocellular carcinoma, it was found that in hypoxic condition, decreased RASA1 expression increased angiogenesis [30]. In the development of advanced endometriosis, increased angiogenesis can have a significant effect and cause the disease to spread to different organs [31]. The results of this study showed that RASA1 expression level in ectopic tissues was reduced compared to control and eutopic tissues. Based on this, it can be concluded that decreased expression of the RASA1 gene can lead to the formation of endometriosis, especially its advanced stage.

NF1 (encoding the neurofibromin protein 1) is the major mutated gene in the germline of patients with neurofibromatosis type 1, which disrupts the regulation of the RAS / MAPK pathway [32]. Studies have shown that people with the disease with NF1 loss-of-function mutation, have a 5 times higher risk of breast cancer than the general population [33]. Jiang et al.'s study on ovarian cancer showed that reducing NF1 gene expression as a tumor suppressor stopped apoptosis and increased the growth and proliferation of cancer cells, and in a separate study, the NF1 gene mutation was found only in endometriosis-related ovarian cancer [34], [35]. Our study showed that NF1 gene expression was decreased in ectopic and eutopic samples of patients compared with control samples of healthy individuals. According to this result, we can conclude that the NF1 gene plays an important suppressive role both in the early stages of endometriosis and in its advanced stages, and reducing its expression stops apoptosis, increases the proliferation and invasion of endometriosis cells.

Since no study was performed on the expression of RASA1 and NF1 genes in endometriosis and due to the regulatory role of these two genes in the MAPK and PI3K / Akt pathways, in this study we examined the level of expression of these genes in endometriosis tissues (ectopic and eutopic) and normal endometrial tissue.

Conclusion

The results of this study showed that NF1 and RASA1 genes play a role in tumor suppression in endometriosis and can control activated signaling pathways (MAPK and PI3K / Akt) in this disease through negative regulation. Decreased expression of NF1 and RASA1 genes may be a new biomarker for the prognosis of the disease if patients' clinical outcomes and expression characteristics of NF1 and RASA1 genes are determined.