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
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
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长链非编码 RNA MALAT1 的下调通过激活 ERK/MAPK 通路上调 P21 抑制子宫内膜异位症中的颗粒细胞增殖
DOI：
10.1093/molehr/gay045
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发表时间：
2019-01-01
影响因子：
4
通讯作者：
Zhang, Jun
中科院分区：
文献类型：
--
作者：
Li, Ying;Liu, Yu-dong;Zhang, Jun
研究问题：肺腺癌转录本1(MALAT1)与子宫内膜异位症相关性不孕症的关系是否有特定的机制？总结回答：MALAT1在子宫内膜异位症颗粒细胞(GCs)中的下调可能通过抑制GC的增殖而对卵母细胞的生长和发育产生不利影响，这是由于细胞周期依赖的机制，通过激活细胞外信号调节激酶(ERK)/丝裂原活化蛋白激酶(MAPK)途径增强P21的表达。目前已知：子宫内膜异位症与不孕症之间的联系已被文献很好地支持，子宫内膜异位症本身及其手术治疗对卵巢储备和卵母细胞发育有不利影响。MALAT1是最广泛表达和进化保守的转录本之一，已被认为在人类发育和许多疾病中发挥作用。然而，对MALAT1长非编码RNA(LncRNA)在子宫内膜异位症及其相关不孕症中的作用知之甚少。研究设计、大小、持续时间：我们检测了52例子宫内膜异位症患者和52名对照的GCs中MALAT1非编码RNA的表达水平。此外，为了研究MALAT1在人胃癌细胞系KGN中的作用及其在细胞增殖中的分子机制，我们还对MALAT1基因进行了敲除。所有子宫内膜异位症患者均经腹腔镜术或开腹术确诊，对照组仅限于男性因素或输卵管疾病，卵巢储备正常。采用实时定量聚合酶链式反应(qRT-PCR)检测MALAT1基因在子宫内膜异位症患者和对照组中的表达水平。绘制受试者工作特征(ROC)曲线，评价MALAT1对子宫内膜异位症的诊断价值。在KGN细胞系中，MALAT1被锁定的核酸GapmeRs击倒。应用细胞计数试剂盒-8法、乙炔-2-脱氧尿嘧啶核苷分析和流式细胞术研究MALAT1在细胞增殖和细胞周期进程中的作用，并通过Western blotting检测其可能的机制。MAIN结果和Chance的作用：我们首次发现MALAT1在子宫内膜异位症GCs中显著下调，并与有腔卵泡计数相关(R=0.376，P<；0.001与对照组相比)。此外，子宫内膜异位症晚期不孕症患者癌组织中MALAT1 lncRNA水平显著低于对照组(P=0.01)。所有子宫内膜异位症患者与对照组(AUC：0.705；95%CI：0.606-0.804；P<；0.001)、I-II期与对照组(AUC：0.651；95%CI：0.536-0.767；P=0.016)、III-IV期与对照组(AUC：0.827；95%CI：0.718-0.936；P<；0001)的ROC曲线显示出很强的分离性。MALAT1lncRNA主要定位于GCs的胞核。我们发现患者GCs中MALAT1lncRNA与P21mRNA呈负相关(R=-0.628；P<；0.001)。KGN细胞中MALAT1基因敲除抑制了细胞增殖和细胞周期进程。此外，MALAT1基因敲除后，P21、P53、磷酸化ERK1/2(p-ERK1/2)和磷酸化c-Jun氨基末端蛋白激酶(p-JNK)蛋白水平均升高，细胞周期蛋白依赖性蛋白2(CDK2)、细胞周期蛋白D1和p-P38MAPK蛋白水平降低。此外，U0126对ERK/MAPK通路的抑制、p-ERK1/2、P21和P53的上调以及MALAT1基因敲除后CDK2和Cyclin D1的下调均被MALAT1基因敲除所减弱。因此，MALAT1可能通过依赖于P21/P53对细胞周期的控制来调节GC的增殖，ERK/MAPK通路参与了这一过程。LARGE Scale数据：无。限制，谨慎的理由：在体外受精和手术切除子宫内膜异位病变中使用的激素治疗可能改变了GC中MALAT1的表达。卵巢颗粒样肿瘤细胞系KGN被用于进一步的功能和机制研究，因为难以获得相当数量的人GC并保持原代培养。研究结果的广泛意义：我们的发现是子宫内膜异位症GCs中基于lncRNA机制的第一个例子。患有子宫内膜异位症的妇女表现出GCs中MALAT1表达水平的改变，这可能通过调节GCs的功能而损害生育能力。因此，对MALAT1基因及其分子作用机制的研究为深入了解子宫内膜异位症及其相关不孕症的发病机制提供了新的思路。
STUDY QUESTION: Is there a specific mechanism underlying the association between lung adenocarcinoma transcript 1 (MALAT1) and endometriosis-related infertility?SUMMARY ANSWER: 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.WHAT IS KNOWN ALREADY: 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.STUDY DESIGN, SIZE, DURATION: 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.PARTICIPANTS/MATERIALS, SETTING, METHODS: 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.MAIN RESULTS AND THE ROLE OF CHANCE: 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.LARGE SCALE DATA: None.LIMITATIONS, REASONS FOR CAUTION: 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.WIDER IMPLICATIONS OF THE FINDINGS: 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.