Down-regulation of long non-coding RNA MALAT1 inhibits granulosa cell proliferation in endometriosis by up-regulating P21 via activation of the ERK/MAPK pathway.

Abstract

Is there a specific mechanism underlying the association between lung adenocarcinoma transcript 1 (MALAT1) and endometriosis-related infertility? The down-regulation of MALAT1 in endometriosis granulosa cells (GCs) may have an adverse effect on the growth and development of oocytes by inhibiting GC proliferation, due to cell cycle-dependent mechanisms that enhance P21 expression through activation of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway. The association between endometriosis and infertility is well supported throughout the literature, and endometriosis per se and its surgical treatment have an adverse effect on the ovarian reserve and on oocyte development. MALAT1, one of the most extensively expressed and evolutionarily conserved transcripts, has been implicated to play a role in human development and many diseases. However, little is known about the role of MALAT1 long non-coding RNA (lncRNA) in endometriosis and its associated infertility. We measured MALAT1 lncRNA expression levels in GCs from 52 endometriosis patients and 52 controls. Also, MALAT1 was knocked down in a human GC tumor-derived cell line, KGN, to investigate the role of MALAT1 and its molecular mechanism in cell proliferation. GCs were collected from women with or without endometriosis undergoing IVF or ICSI treatment. All endometriosis patients were diagnosed by laparoscopy or laparotomy, and control patients were limited to male factor or tubal disease and had a normal ovarian reserve. Quantitative real-time PCR (qRT-PCR) was used to measure the differential expression levels of MALAT1 lncRNA between endometriosis patients and controls. The receiver operating characteristic (ROC) curve was drawn to evaluate the diagnostic values of MALAT1 in endometriosis. In the KGN cell line, MALAT1 was knocked down with locked nucleic acid GapmeRs. Cell counting kit-8 assays, ethynyl-2-deoxyuridine assays and flow cytometry were used to study the role of MALAT1 in cell proliferation and cell-cycle progression, and western blotting was performed to detect the potential underlying mechanism. We first found that MALAT1 lncRNA was significantly down-regulated in endometriosis GCs and was associated with the antral follicle count (R = 0.376, P < 0.001 versus control). In addition, MALAT1 lncRNA levels were significantly lower in the GCs of infertile women with advanced stages of endometriosis (P = 0.01 versus control). The ROC curves illustrated strong separation between all the endometriosis patients and the control group (AUC: 0.705; 95% CI: 0.606-0.804; P < 0.001), Stage I-II and control group (AUC: 0.651; 95% CI: 0.536-0.767; P = 0.016), and Stage III-IV and control group (AUC: 0.827; 95% CI: 0.718-0.936; P < 0.001). MALAT1 lncRNA was primarily localized in the nuclei of GCs. We found a negative correlation between MALAT1 lncRNA and P21 mRNA in the GCs from patients (R = -0.628; P < 0.001). MALAT1 knockdown in KGN cells inhibited cell proliferation and cell-cycle progression. In addition, MALAT1 knockdown induced an increase in both the mRNA and protein levels of P21, and of P53, phosphorylated ERK1/2 (p-ERK1/2) and phosphorylated c-Jun N-terminal protein kinase (p-JNK) protein levels, as well as causing a decrease in cyclin dependent kinase 2 (CDK2), cyclin D1 and p-P38 MAPK protein levels. Furthermore, inhibition of the ERK/MAPK pathway with U0126, the up-regulation of p-ERK1/2, P21 and P53, and the down-regulation of CDK2 and cyclin D1 by the knockdown of MALAT1 were all attenuated by MALAT1 knockdown. Therefore, MALAT1 may regulate GC proliferation through P21/P53-dependent control of the cell cycle, and the ERK/MAPK pathway participates in this process. None. The hormonal treatment used in IVF and surgical removal of endometriotic lesions may have altered MALAT1 expression in GCs. The ovarian granulosa-like tumor cell line, KGN, was used for further functional and mechanistic studies due to the difficulties in obtaining human GCs in sizable amounts and maintaining primary cultures. Our finding represents the first example of an lncRNA-based mechanism in endometriosis GCs. Women with endometriosis show altered MALAT1 expression levels in GCs that may impair fertility by regulating the function of GCs. Therefore, analysis of MALAT1 and its molecular mechanisms of action provide new insights into the pathogenesis of endometriosis and its associated infertility. This work was supported by the National Natural Science Foundation of China (grant number: 81671524) and the National key research and development program of China (grant number: 2017YFC1001100). The authors declare there is no conflict of interest.