miR-451a Modulates Endometriotic Lesion and Cellular Proliferation through c-Myc-RPLP1 Regulation: A Novel Pathway in Endometriosis Pathogenesis

Abstract

Endometriosis is a chronic, debilitating inflammatory disease. It is characterized by ectopic formation of estrogen-sensitive endometrial glands and stroma mostly on the surface of the ovaries, fallopian tubes, and pelvic cavity. Major complaints of endometriosis patients are pelvic pain, dysmenorrhea and infertility, which decrease the quality of life of these women. Non-steroidal anti-inflammatory medications and oral contraceptives are the first line of treatment to alleviate the symptoms and regress the lesions. The diagnosis of the disease is a hurdle; there are no specific markers for endometriosis, and the only approach to confirm the disease is by laparoscopy. The reason for limited treatment and diagnosis options is the lack of complete understanding of endometriosis pathophysiology. Retrograde menstruation is one of the most accepted theories, however, considering that retrograde menstruation is a normal process in most women of reproductive age, the causes of the establishment and survival of endometriotic tissue in these patients is not clear. Several studies suggested that microRNAs may play a significant role in endometriosis development. MicroRNAs are small non-coding RNAs that stimulate or inhibit the transcription of specific gene targets. Mis-expression of microRNAs has been established in endometriosis and is linked to its cellular phenotypes. miRNA-451 (now referred to as miR-451a), is one of the most differential expressed miRNAs in endometriotic lesions from women with endometriosis. It is well-established that miR-451a modulates cell survival, but its potential role in endometriosis is poorly understood. We have previously demonstrated that miR-451a regulates expression of factors essential for endometriotic lesion survival. The objective of this project was to further explore the mechanisms and mediators through which miR-451a may modulate endometriotic lesion and cell survival. To accomplish our goal, we over-expressed miR-451a in the immortalized endometriotic epithelial 12Z cell line and screened differentially expressed proteins by 2-DiGE analysis. As a result, RPLP1, Ribosomal Protein Large P1, was found to be decreased in 12Z cells which over-expressed miR-451a. RPLP1 is a component of 60S ribosomal subunit which aids in the translation elongation process and is speculated to drive cell proliferation. Thus, the overall hypothesis of this research project is that RPLP1is a target of miR-451a and its expression is related to the proliferation and survival of endometriotic ectopic lesions. To test this, we utilized matched human eutopic and ectopic endometrium (endometriotic lesions). We found that in endometriotic lesion samples, the majority 52/77 samples) expressed elevated levels of RPLP1 mRNA compared to matched eutopic tissues. RPLP1 protein was also increased in ectopic lesion tissue compared to matched eutopic endometrium. In addition, a positive correlation was observed between RPLP1 mRNA expression and that of the proliferative marker, cyclin E1 (CCNE1). To examine the function of RPLP1 in 12Z cells, we knocked down RPLP1 expression using siRNA technology and observed a significant decrease in the expression of CCNE1, and a concurrent increase in PTEN (a pro-apoptotic marker). In addition, RPLP1 knockdown by shRNA demonstrated an approximate 90% reduction in cell survival of 12Z cells compared to those infected with non-targeting shRNA. To explore the mechanism of miR-451a in modulating RPLP1 expression, we conducted a literature review and bio-informatic prediction tools search. This revealed that c-Myc was a putative direct target of miR-451a which may potentially regulate RPLP1 expression. C-Myc is a transcriptional factor that regulates the expression of many genes involved in cell proliferation and tumorigenesis. To investigate the possible regulation of c-Myc by miR-451a, we first examined the expression of c-Myc in miR-451a transfected 12Z cells and found significant downregulation on the protein and mRNA level. Furthermore, knockdown of c-Myc by siRNA transfection resulted in a significant reduction of RPLP1 transcript and protein in 12Z cells. To examine the relationship between c-Myc and RPLP1 in endometriotic lesion tissue, we first examined the expression of c-Myc transcript in human eutopic and ectopic endometrial samples. We found that the mean fold change of c-Myc expression was four-fold higher in ectopic lesions compared to eutopic tissues. Expression of c-Myc and RPLP1 mRNA were positively correlated. Collectively, these data confirm our hypothesis that RPLP1 is a player in mediating endometriotic cell/lesion survival. In addition, our data revealed a novel pathway which involve that miR-451a directly target c-Myc, which in turn may control the expression of RPLP1. Outcomes from this research may lead to more specific approaches to treating endometriosis which may avoid use of anti-estrogen therapies and their unwanted side effects