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Medroxyprogesterone Acetate Causes the Alterations of Endoplasmic

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Medroxyprogesterone Acetate Causes the Alterations of Endoplasmic Cao et al. BMC Medical Genomics (2019) 12:163 https://doi.org/10.1186/s12920-019-0601-9 RESEARCH ARTICLE Open Access Medroxyprogesterone acetate causes the alterations of endoplasmic reticulum related mRNAs and lncRNAs in endometrial cancer cells Wenjiao Cao1†, Wuyuan Gao1†, Panchan Zheng1, Xiao Sun1 and Lihua Wang1,2,3,4* Abstract Background: Progestin is effective to promote endometrial cancer (EC) cells apoptosis, however, continuous progestin administration causes low level of progestin receptor B (PRB), further resulting in progestin resistance. Here, we performed microarray analysis on Ishikawa cells (PRB+) treated with medroxyprogesterone acetate (MPA) to explore the molecular mechanism underlying the inhibitory influence of MPA on PRB+ EC cells. Methods: Microarray analysis was performed by using Ishikawa cells (PRB+) treated with MPA. Differentially expressed mRNA and long noncoding RNAs (lncRNAs) were identified. Furthermore, the functions of these mRNAs and lncRNAs were predicted by functional enrichment analysis. QRT-PCR was further performed to verify the microarray data. Results: A total of 358 differentially expressed genes and 292 lncRNAs were identified in Ishikawa cells (PRB+) treated with MPA. QRT-PCR verified these data. Functional enrichment analysis identified endoplasmic reticulum (ER) stress as the key pathway involved in the inhibitory effect of MPA on EC cells. And the ER stress apoptotic molecule CHOP and ER stress related molecule HERPUD1 were both highly expressed in Ishikawa cells (PRB+) treated with MPA. Co-expression analysis showed lnc-CETP-3 was highly correlated with CHOP and HERPUD1, suggesting it might participate in ER stress pathway-related EC cell apoptosis caused by MPA. In addition, compared with untreated cells, lnc-CETP-3, CHOP and HERPUD1 were significantly up-regulated in Ishikawa cells (PRB+) treated with MPA, whereas they have no statistical significance in KLE cells (PRB-). Conclusions: MPA may activate ER stress by progesterone-PRB pathway to up-regulate CHOP expression, which may be one of the molecular mechanisms underlying the inhibitory effect of MPA on EC cells with PRB+. Lnc-CETP-3 might be involved in this process. These findings may provide therapeutic targets for EC patients with PRB-, and resistance-related targets to increase the sensitivity of MPA on EC cells. Keywords: Endometrial cancer, Medroxyprogesterone acetate, Endoplasmic reticulum stress, CHOP, Lnc-CETP-3 * Correspondence: [email protected] †Wenjiao Cao and Wuyuan Gao contributed equally to this work. 1Department of Obstetrics and Gynecology, the International Peace Maternity & Child Health Hospital of China Welfare Institute (IPMCH), Shanghai Jiaotong University, No.910, Hengshan Road, Xuhui District, Shanghai 200030, China 2The International Peace Maternity and Child Health Hospital, School of Medicine, Shanghai Jiao Tong University, Shanghai, China Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Cao et al. BMC Medical Genomics (2019) 12:163 Page 2 of 11 Background mediated progesterone receptor B (PRB) and dopa- Endometrial carcinoma (EC) is the most common malig- mine receptor D2 (DRD2), and decreases the ratio of nant tumor of the female reproductive tract resulting in p-AKT/AKT in PI3K/AKT signal pathway, which fur- 70,000 deaths worldwide annually [1]. Medroxyproges- ther inhibits EC cells proliferation and promotes cell terone acetate (MPA), a steroidal progestin, has been apoptosis [14]. used as a conservative treatment for young patients with Long noncoding RNAs (lncRNAs) encoded by a vast less clinical stage I, grade I EC for a long time, which is de- explores region of the human genome, have been reported sirable in patients hoping to preserve fertility [2]. The to be biomarkers and therapeutic targets for cancer, since overall response rate of progestin treatment for EC they play a critical in promoting and maintaining tumor ini- ranges from 15 to 40% [3]. When treated with MAP, 10 tiation and progression [15]. LncRNAs interact with pro- mg daily for 12–14 days each month, 80–90% patients teins, RNA, and lipids, suggesting that they are essential have regressed hyperplasia without atypia to normal mediators of cancer signaling pathways. In addition, the endometrium [4]. However, most patients with EC de- dysregulation of lncRNAs is associated with the stage and velop progestin-resistance though they initially respond prognosis of many tumor types, including EC, as well as in- to progestin treatment, which further causes tumor pro- volved in resistance against chemotherapy and targeted gression [2]. A study has reported that 62% of patients therapy [15]. Previous studies have identified several with EC have an initial response to progestin, and 23% lncRNAs correlated with EC progression. LncRNA-GAS5 of initial responders later have developed recurrent dis- promotes EC cell apoptosis by regulating the expression of ease [5]. In addition, > 30% of young patients have no re- miR-103 and PTEN [16]. High expression level of lncRNA sponse to progestin due to de novo or acquired BANCR in type 1 EC tissue promotes cell proliferation, mi- progestin resistance during treatment [6]. However, the gration and invasion by activating ERK/MAPK signaling mechanism underlying progestin resistance remains pathway [17]. LncRNA HOTAIR regulates NPM1 via inter- largely unrevealed. acting with miR-646, and further mediates the estrogem- At present, many studies have reported some pos- induced metastasis of EC cells [18]. High levels of lncRNA sible factors associated with progestin resistance. Low MALAT1 have been reported in EC, which is associated expression level of progestin receptor (PR) caused by with the aberrant activation of the wnt/beta-catenin path- continuous progestin administration is regarded as way leading to the interaction of wnt-effector transcription one of the reasons since it further decreases the sensi- factor TCF4 and MALAT1 promoter region [19]. Further- tivity of cancer cells to progestin [7]. The other mole- more, lncRNA LINC00672 has been demonstrated to in- cules including up-regulation of growth factors or crease the sensitivity of EC xenograft mice to paclitaxel their receptors contribute to progestin resistance. [20]. Though various lncRNAs show a relationship to EC Increased level of epidermal growth factor receptor progression, to our knowledge, the association between (EGFR) reduces Ishikawa EC cells sensitivity to pro- lncRNAs and progestin resistance in EC cells has not been gestin and PR expression, and abnormally activated evaluated. the mitogen-activated protein kinase (MAPK) signal- Here, to explore the molecular mechanism underlying ing pathway [8]. Overexpression of insulin-like growth the inhibitory influence of MPA on PRB+ EC cells, we per- factor II (IGF-II) inhibited PR expression partially formed microarray to analyze the mRNA and lncRNA ex- mediating through activating AMP-activated protein pression profiling between Ishikawa cells (stably expressing kinase (AMPK) and inhibiting the over activated PRB) with MPA treatment and untreated cells. And then, mTOR pathway [9]. In addition, activation of the differentially expressed genes and lncRNAs were identified. PI3K/Akt pathway by progestin without PR mediation TopredictthefunctionsofthesegenesandlncRNAsin also causes progestin resistance to EC cells, and inhi- EC, we further performed functional enrichment analysis. biting this pathway is considered as an effective The results showed that endoplasmic reticulum stress (ER method to reverse resistance [10]. These reports sug- stress) might be involved in the influence of MAP on EC gest that the association with PR does not fully explain progression. We further used CHOP (ER stress apoptotic the mechanism of progestin resistance. Our previous molecule) and HERPUD1 (ER stress related molecule) to studies have found RANK-RANKL (the targeting of performed co-expression analysis, and found lnc-CETP-3 receptor activator of nuclear factor-κB ligand) system was significantly increased, and closely correlated with promotes EC progression via MAPK pathway [11]and CHOP and HERPUD1 expression. The functional enrich- epithelial-mesenchymal transition (EMT) [12], and in- ment analysis based on the co-expression mRNAs of lnc- duces EC metastasis mediated by AKT/β-catenin/Snail CETP-3 showed that lnc-CETP-3 was involved in cell pathwaybothinvitroandinvivo[13]. We further find apoptosis, cell cycle, ER stress, and cancer pathway. To val- that a combination of thioridazine (THIO) and MAP idate the reliability of microarray data, we further evaluated significantly enhances the expression levels of MPA- the expression levels of selected lncRNAs by qRT-PCR. Cao et al. BMC Medical Genomics (2019) 12:163 Page 3 of 11 CHOP, HERPUD1 and lnc-CETP-3 were significantly in- Differentially expressed mRNA and lncRNA analysis creased in PRB+ EC cells, whereas they have no statistical
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