Min Zhou1,2, Jing Fu1, Li Xiao1, Shiyuan Yang1, Yong Song1, Xianghui Zhang1, Xue Feng1, Huaqin Sun3, Wenming Xu3, Wei Huang4. 1. Department of Obstetrics and Gynecology, West China Second University Hospital of Sichuan University, Chengdu, Sichuan 610041, PR China. 2. Department of Obstetrics and Gynecology, Hospital of University of Electronic Science and Technology of China and Sichuan Provincial People's Hospital, Chengdu, Sichuan 610072, PR China. 3. Department of Obstetrics and Gynecology Key Laboratory of Obstetric Gynecologic, and Pediatric Diseases and Birth Defects of Ministry of Education, West China Second University Hospital of Sichuan University, Chengdu, Sichuan 610041, P R China. 4. Department of Obstetrics and Gynecology, West China Second University Hospital of Sichuan University, Chengdu, Sichuan 610041, PR China weihuang64@163.com.
Abstract
STUDY QUESTION: Do microRNAs (miRNAs) contribute to aberrant progesterone receptor (PGR) expression in the eutopic endometrium of women with endometriosis? SUMMARY ANSWER: miR-196a upregulates MEK/ERK signalling, mediating a downregulation of PGR expression in the eutopic endometrium of women with minimal or mild endometriosis. WHAT IS KNOWN ALREADY: Implantation failure is strongly suggested as an underlying cause for the observed infertility in minimal or mild endometriosis. Progesterone resistance, which is mainly caused by aberrantly expressed progesterone receptor in the eutopic endometrium, is considered as a key factor of decreased endometrial receptivity; thus far, epigenetics, but not miRNA, has been shown to affect PGR expression in the endometrium. STUDY DESIGN SIZE, DURATION: Microarray analysis was used to analyse the eutopic endometrium. The differential expression of miR-196a was validated. Bioinformatics analysis predicted that miR-196a targets the 3'-untranslated region (UTR) of the PGR. The relationship between the miR-196a level and PGR expression was studied and the role of the MEK/ERK signal pathway was investigated. PARTICIPANTS/MATERIALS, SETTING, METHODS: Total RNA was extracted from eutopic endometrium samples in three infertile women with mild/minimal endometriosis and three disease-free control subjects. The miRNA and mRNA expression levels were analysed by microarray analysis. The miR-196a expression was validated by qRT-PCR [endometriosis (n = 22) and control (n = 20)], while functional analysis utilised in vitro transfection of endometrial stromal cells (ESCs), induction of decidualization of ESCs, and luciferase reporter assays in 293 T cell lines. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 66 dysregulated miRNAs and 357 dysregulated mRNAs were screened by microarray analysis. miR-196a and P-MEK/P-ERK were both found to be significantly upregulated in the eutopic endometrium in patients with mild/minimal endometriosis. PGR and PGR-B mRNA were inhibited by miR-196a overexpression and upregulated by miR-196a inhibition. Luciferase reporter failed to confirm the target regulation of miR-196a on PGR. Transfection of ESCs with a miR-196a mimic led to an increase in the P-MEK/P-ERK protein levels, decrease in the PGR protein levels, and atypical decidualization. Following miR-196a inhibition, the P-MEK/P-ERK protein was downregulated and the PGR protein was upregulated. Inhibition of P-MEK/P-ERK also increased PGR expression. LARGE SCALE DATA: Data are presented in Supplementary Tables SI and SII. LIMITATIONS REASONS FOR CAUTION: This study focused on the role of miR-196a, and therefore does not involve other miRNAs; hence, it is possible that other miRNAs may also be responsible for progestin resistance in endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: Our data revealed altered miRNA expression and activated MEK/ERK signalling in the eutopic endometrium in minimal or mild endometriosis. We showed that the miR-196a level is associated with reduced expression of PGR isoforms through MEK/ERK, suggesting that miR-196a and MEK/ERK are both potential biomarkers of endometriosis. These results provide a novel approach to target the mechanisms behind progesterone resistance in endometriosis. STUDY FUNDING/COMPETING INTERESTS: This research was supported by the National Natural Science Foundation of China (No. 81370693). The authors have no conflicts of interest.
STUDY QUESTION: Do microRNAs (miRNAs) contribute to aberrant progesterone receptor (PGR) expression in the eutopic endometrium of women with endometriosis? SUMMARY ANSWER: miR-196a upregulates MEK/ERK signalling, mediating a downregulation of PGR expression in the eutopic endometrium of women with minimal or mild endometriosis. WHAT IS KNOWN ALREADY: Implantation failure is strongly suggested as an underlying cause for the observed infertility in minimal or mild endometriosis. Progesterone resistance, which is mainly caused by aberrantly expressed progesterone receptor in the eutopic endometrium, is considered as a key factor of decreased endometrial receptivity; thus far, epigenetics, but not miRNA, has been shown to affect PGR expression in the endometrium. STUDY DESIGN SIZE, DURATION: Microarray analysis was used to analyse the eutopic endometrium. The differential expression of miR-196a was validated. Bioinformatics analysis predicted that miR-196a targets the 3'-untranslated region (UTR) of the PGR. The relationship between the miR-196a level and PGR expression was studied and the role of the MEK/ERK signal pathway was investigated. PARTICIPANTS/MATERIALS, SETTING, METHODS: Total RNA was extracted from eutopic endometrium samples in three infertilewomen with mild/minimal endometriosis and three disease-free control subjects. The miRNA and mRNA expression levels were analysed by microarray analysis. The miR-196a expression was validated by qRT-PCR [endometriosis (n = 22) and control (n = 20)], while functional analysis utilised in vitro transfection of endometrial stromal cells (ESCs), induction of decidualization of ESCs, and luciferase reporter assays in 293 T cell lines. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 66 dysregulated miRNAs and 357 dysregulated mRNAs were screened by microarray analysis. miR-196a and P-MEK/P-ERK were both found to be significantly upregulated in the eutopic endometrium in patients with mild/minimal endometriosis. PGR and PGR-B mRNA were inhibited by miR-196a overexpression and upregulated by miR-196a inhibition. Luciferase reporter failed to confirm the target regulation of miR-196a on PGR. Transfection of ESCs with a miR-196a mimic led to an increase in the P-MEK/P-ERK protein levels, decrease in the PGR protein levels, and atypical decidualization. Following miR-196a inhibition, the P-MEK/P-ERK protein was downregulated and the PGR protein was upregulated. Inhibition of P-MEK/P-ERK also increased PGR expression. LARGE SCALE DATA: Data are presented in Supplementary Tables SI and SII. LIMITATIONS REASONS FOR CAUTION: This study focused on the role of miR-196a, and therefore does not involve other miRNAs; hence, it is possible that other miRNAs may also be responsible for progestin resistance in endometriosis. WIDER IMPLICATIONS OF THE FINDINGS: Our data revealed altered miRNA expression and activated MEK/ERK signalling in the eutopic endometrium in minimal or mild endometriosis. We showed that the miR-196a level is associated with reduced expression of PGR isoforms through MEK/ERK, suggesting that miR-196a and MEK/ERK are both potential biomarkers of endometriosis. These results provide a novel approach to target the mechanisms behind progesterone resistance in endometriosis. STUDY FUNDING/COMPETING INTERESTS: This research was supported by the National Natural Science Foundation of China (No. 81370693). The authors have no conflicts of interest.