LncRNA SNHG4 promotes the increased growth of endometrial tissue outside the uterine cavity via regulating c-Met mediated by miR-148a-3p

https://doi.org/10.1016/j.mce.2020.110887Get rights and content

Highlights

  • SNHG4 was upregulated in ectopic endometrium, while miR-148a-3p was decreased.

  • MiR-148a-3p was the direct target of SNHG4.

  • Silencing SNHG4 could downregulate the c-Met level by enhancing miR-148a-3p expression.

  • Silencing SNHG4 suppressed endometriotic lesions growth in vivo.

Abstract

Long noncoding RNAs (lncRNAs) modulate endometriosis. The current study investigated the mechanisms and effects of SNHG4 on endometriosis. The qRT-PCR was conducted to examine the miR-148a-3p and SNHG4 expressions in endometriosis tissues. The 5-ethynyl-2′-deoxyuridine incorporation assay and 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide assay were used to measure the rate of cell proliferation. The association between miR-148a-3p, SNHG4 and c-Met was confirmed via bioinformatical approach and luciferase reporter gene assay. Also, the function of SNHG4 on the growth of endometriotic lesions was investigated in vivo. The SNHG4 expression was considerably upregulated in endometriosis tissues, whereas the level of miR-148a-3p expression was reduced. In addition, SNHG4 can be considered as ceRNAs that bind miR-148a-3p and rise the proliferation activity of HESCs by downregulating miR-148a-3p. Furthermore, silencing SNHG4 could downregulate the c-Met level by enhancing miR-148a-3p expression, and finally inhibiting endometriosis development in vivo. LncRNA SNHG4 promotes the increased growth of endometrial tissue outside the uterine cavity via regulating c-Met mediated by miR-148a-3p, which may be used as diagnostic biomarker as well as molecular target in the treatment of endometriosis.

Introduction

Endometriosis is a frequently occurring gynecological disorder that is associated with the occurrence and outgrowth of endometrial-like tissue on the exterior of the uterine cavity (Giudice, 2010). The disease affects more than 10% of women during their reproductive period and reaches its peak incidence between 25 and 35 years old (Giudice, 2010; Vercellini et al., 2014). This disease has multiple symptoms including dysmenorrhea, infertility, pelvic mass, and malignant transformation, and it can dramatically decrease the quality of life (Falcone and Flyckt, 2018). Laparoscopy, which is a form of invasive surgery, is the gold standard tool for the endometriosis diagnosis, and there are no noninvasive reliable diagnostic biomarkers to confirm endometriosis (Peiris et al., 2018). Therefore, it is particularly necessary to investigate the mechanisms of endometriosis and to identify a noninvasive reliable therapeutic biomarker for endometriosis.

Recently, lncRNAs (long noncoding RNAs) have been shown to regulate cell proliferation, apoptosis, and differentiation (Batista and Chang, 2013; Kung et al., 2013). Dysregulation of lncRNAs is implicated in the initiation, progression and physiological processes of various diseases, such as endometriosis (Ahn et al., 2017; Bhan et al., 2017). A prior study suggested that H19 lncRNA could alter the growth of stromal cells via an insulin-like growth factor, which signaled in the endometrium of endometriosis patients (Ghazal et al., 2015). Another study showed than lncRNA MALAT1 was downregulated by miR-200c which inhibited the endometrial stromal cell proliferation and migration (Liang et al., 2017). Conversely, SNHG4 (small nucleolar RNA host gene 4) is a new lncRNA belonging to the SNHGs family and has a critical function in the evolution of many tumors, including cervical cancer, osteosarcoma, and lung cancer (Xu et al., 2018; Dasgupta et al., 2018; Tang et al., 2019). However, to the best of our knowledge, the possible mechanisms of SNHG4 in the progression of endometriosis remain unclear.

Previous studies indicated that lncRNAs could suppress miRNAs’ expression resulting in the inhibition of gene silencing complexes formation (Zhang et al., 2018). The miR-148a-3p dysregulation is implicated in many human diseases, including cervical cancer, pancreatic cancer, and gastric cancer (Dasgupta et al., 2018; Li et al., 2017; Liffers et al., 2011). Although MiR-148a-3p and SNHG4 have complementary binding sequences (Dasgupta et al., 2018), their mutual function in endometriosis is still obscure. Besides, the molecular mechanisms underlying this relationship are not well-documented.

Further, it was revealed that miR-148a-3p could inhibit the development of epithelial ovarian cancer mainly by interacting with c-Met (Wang et al., 2018). C-Met (proto-oncogene that encodes the hepatocyte growth factor receptor) has been implicated in endometriosis (KhoshdelRad et al., 2014). Currently, there is no enough data on the potential cross-talks involving c-Met, SNHG4, and miR-148a-3p on endometriosis. Consequently, the study herein was conducted to elucidate the mechanisms of SNHG4, c-Met, and miR-148a-3p in endometriosis. Such mechanisms may provide important insights into a therapeutic target for endometriosis.

Section snippets

Clinical specimens

In the present study, ectopic endometrium (EC) and eutopic endometrium (EU) were obtained from 25 patients undergoing laparoscopic surgery for ovarian endometriosis diagnosis or treatment. The diagnosis of endometriosis was confirmed by histology. The patients included had mild or moderate (Stage I: n = 2, Stage II: n = 10, Stage III: n = 13) endometriosis. The stages of endometriosis were evaluated according to the American Society of Reproductive Medicine revised classification (Giudice, 2010

The SNHG4 and miR-148a-3p expression levels in EC, EU and EN

The results of qRT-PCR showed that the level of SNHG4 expression was considerably higher in EC tissues than in the EN and EU tissues [Fig. 1A–C] (P < 0.05). In addition, miR-148a-3p level of expression was considerably lower in EC tissues than in EN and EU tissues [Fig. 1B–D] (P < 0.05), and a remarkable inverse correlation was observed between miR-148a-3p and SNHG4 expression levels in EC tissues [Fig. 1E].

Effect of SNHG4 knockdown on the HESCs growth

We transfected si-SNHG4 into HESCs with the aim of evaluating the effects of SNHG4

Discussion

This study revealed that SNHG4 was significantly overexpressed in ectopic endometrium than normal endometrium, while miR-148a-3p expression level was considerably reduced in ectopic endometrium tissues compared to normal endometrium and eutopic endometrium, and a remarkable inverse correlation was observed in miR-148a-3p and SNHG4 expressions in ectopic endometrium. We assume that miR-148a-3p and SNHG4 may be associated with the progression of endometriosis.

LncRNAs act as competing endogenous

Conclusions

LncRNA SNHG4 enhances the growth of endometrial tissue outside the uterine cavity by interacting with c-Met through miR-148a-3p, and this may provide a promising diagnostic marker and therapeutic target for the endometriosis treatment.

Author contributions

JCW and MYM designed the experiments and wrote the manuscript. LYJ, HXW, XB, FYL, LX and ZHY conducted the experiments. LYJ, HXW, and YCH analyzed the data. LD, HXW, XRN and WSY provided clinical specimens. All of the authors gave final approval to the submitted version of the manuscript.

Ethical approval and participation consents

This study was conducted after receiving approval from the Institutional Animal Research Ethics Committee and the Research Ethics Committee of Beijing Obstetrics and Gynecology Hospital, Capital Medical University. Also, written informed consent was received from every patient who took part in the study.

Publication consent

This is not relevant.

Data sharing and data accessibility

All materials and data of this study are available and can be requested from the corresponding author.

Funding

This work was supported by the National Key Research and Development Program of China (No: 2018YFC1003003), the Capital's Funds for Health Improvement and Research (No.2016-1-2111), the Beijing Municipal Science & Technology Commission (No. Z171100001017047), and the Beijing Municipal Administration of Hospitals' Ascent Plan (No. DFL20151301).

CRediT authorship contribution statement

Yanjun Liu: Methodology, Validation, Investigation, Software, Data curation, Formal analysis. Xiaowu Huang: Methodology, Validation, Investigation, Software, Data curation, Formal analysis, Resources. Dan Lu: Resources. Yuelan Feng: Methodology, Validation, Investigation. Ruonan Xu: Resources. Xuan Li: Methodology, Validation, Investigation. Chenghong Yin: Software, Data curation, Formal analysis. Bing Xue: Methodology, Validation, Investigation. Huanying Zhao: Methodology, Validation,

Declaration of competing interest

We declare that no conflicts of interest exist among authors or with the funding body.

Acknowledgements

We thank all participants involved in this study.

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