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LncRNA-MEG3 inhibits cell proliferation of endometrial carcinoma by repressing Notch signaling
Biomedicine & Pharmacotherapy 82 (2016) 589–594
Available online at
ScienceDirect www.sciencedirect.com
LncRNA-MEG3 inhibits cell proliferation of endometrial carcinoma by repressing Notch signaling Qingyun Guoa , Zhida Qiana , Dingding Yanb , Li Lib , Lili Huanga,* a b
Women’s Hospital, College of Medicine, Zhejiang University, Hangzhou 310006, Zhejiang, China Zhejiang Cancer Hospital, Hangzhou 310022, Zhejiang, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 January 2016 Received in revised form 23 February 2016 Accepted 23 February 2016
Background: The long non-coding RNA MEG3 has shown functional role as a tumor suppressor in many cancer types, excluding endometrial carcinoma (EC). Thus, this study tried to reveal the MEG3 dysregulation in EC samples and potential functional mechanism due to its regulation on Notch signaling pathway. Methods: The expression profiles of MEG3 and two Notch signaling molecules, Notch1 and Hes1, were detected in both EC tissues and cell lines through real time PCR and western blot analysis. Lentiviral vector carrying whole MEG3 transcript or shRNA targeting MEG3 (shMEG3) was transfected for MEG3 dysfunction studies, and cell proliferation was analyzed through MTT and colony-formation assays. Xenograft models were also established by subcutaneous implantation and tumor growth was compared under MEG3 dysregulation. Results: Significant downregulation of MEG3 was observed in EC samples compared to control, while the protein levels of Notch1 and Hes1 were both upregulated. Cell proliferation was obviously inhibited by MEG3 overexpression, while opposite improved result was obtained in MEG3 knockout cells. Interestingly, MEG3-induced changes could be reversed by Notch1 regulators. Moreover, MEG3 overexpressing tumors showed strongly repressed growth in vivo, along with Notch signaling inhibition. Conclusion: Downregulated MEG3 exhibited an anti-proliferative role in EC by repressing Notch signaling pathway. ã 2016 Elsevier Masson SAS. All rights reserved.
Keywords: Endometrial carcinoma Cell proliferation MEG3 Notch signaling
1. Introduction Endometrial carcinoma (EC) is one of the most common gynecologic malignant tumors, with great increase in new cases that is second only to cervical cancer [1]. Generally, EC can be classified into two sub-types, namely estrogen-dependent endometrioid carcinoma (type I) and estrogen-independent nonendometrioid carcinoma (type II) [2]. Type I ECs, accounting for 70–80% cases, are typically well differentiated tumors emerging from hyperplastic endometrial tissues, while Type II ECs derived from atrophic endometrial tissues are primary serous tumors and occur more often in older women at the post-menopausal state [3]. Type I ECs can be diagnosed at an early stage showing favorable prognoses, whereas Type II is often diagnosed at an advanced stage with poor outcomes. Although the five year survival rate of EC patients is up to 74–91% [4], adjuvant treatment option for these
* Corresponding author at: Women’s Hospital, College of Medicine, Zhejiang University, No. 1 Xueshi Road, Hangzhou 310006, Zhejiang, China. E-mail address: [email protected] (L. Huang). http://dx.doi.org/10.1016/j.biopha.2016.02.049 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
with advanced and recurrent tumors is still limited, partly due to unclear mechanism of EC development. Maternally expressed gene 3 (MEG3), also known as gene trap locus 2 (GTL2), is an imprinted gene, encoded by the MEG3 transcript of the DLK1/MEG3 locus on human chromosome, or Meg3 on mouse chromosome [5]. MEG3 can express in many normal tissues, while the loss of MEG3 expression can be found in some tumors and tumor cell lines, indicating the role of MEG3 as a potential tumor suppressor [6]. Since then, the multiple mechanisms of MEG3 downregulation have been explored in cancer development and progression [7], due to its regulation on p53, RB and Notch activity, as well as their signaling pathways. And, increasing functional roles of MEG3 have been demonstrated in various cancer types, including meningioma [8], cervical carcinoma [9], lung cancer [10], gastric cancer [11], and hepatocellular cancer [12]. However, the expression profiles of MEG3 in EC tissues or cell lines have not been explored yet, as well as related functional mechanism. Notch signaling is a highly conserved pathway in developmental biology [13], and shows important regulation on cell proliferation, differentiation and death that associated with tumorigenesis
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[14]. Dysregulated Notch pathway has been documented in multiple types of cancers, including EC [15,16]. Recent studies have also revealed the mechanism of EC development under the Notch signaling control [17,18]. As mentioned above, MEG3 also exert potential regulation on Notch signaling, since Notch pathway is reported to be enriched in Meg3-null embryos [19]. Therefore, in the present study, the potential MEG3/Notch axis was speculated to function in EC development, wherein MEG3 might function by modulating Notch signaling pathways to suppress tumorigenesis.
Hes1 or b-actin, at 4 C for overnight. All primary antibodies were purchased from Epitomics (Burlingame, CA, USA), and used at a dilution of 1:2000 following the manufacturer’s suggestions. Peroxidase-conjugated secondary antibodies provided by Cell Signaling Technology (Danvers, MA, USA) were added to detect the bound primary antibodies. Immunoblot images were visualized via chemiluminescence (Beyotime), and analyzed using ImageQuant LAS 4000 System (GE Healthcare, Little Chalfont, UK). 2.5. Plasmids and cell transfection
2. Materials and methods 2.1. Tumor tissues This study was approved by the local Medical Ethical Committee of Women’s Hospital, School of Medicine, Zhejiang University. Total thirty-paired specimens of tumor tissues and adjacent normal endometrial samples were obtained from EC patients, who received surgical treatment from 2012 to 2014. All patients were free from neoadjuvant therapy or endocrine therapy before the surgery and had signed informed consent to allow molecular research on obtained specimens.
Lentiviral constructs carrying whole MEG3 transcript (LV-MEG3), or shRNA targeting MEG3 (LV-shMEG3), and an empty negative control vector (LV-GFP) were obtained from GenePharma (Shanghai, China). Cells were pre-grown to 30% confluency and then transfected with different lentiviral constructs at a final concentration of 2 mg/mL by using Lipo2000 (Invitrogen) in accordance with the manufacturer’s instructions. After incubation at 37 C for 48 h, fresh medium was added, and cells were collected after another 72-h-incubation to verify the stable overexpression or knockdown of MEG3 through real time PCR analysis. 2.6. MTT assay
2.2. Cell culture and treatment The human endometrial cell line, endometrial stromal cell (ESC), and two human endometrial cancer cell lines, HEC-1A and KLE, were all purchased from the American Type Culture Collection (ATCC, Manassas, VA), and grown in Dulbecco modified Eagle medium (DMEM)/F12 (HyClone, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) at 37 C with a humidified atmosphere of 5% CO2. For the activation or blockage of Notch signaling, cells were incubated with recombinant Jagged-1/Fc (R&D Systems, Minneapolis, USA) at 500 mg/L org-secretase inhibitor (GSI) (MRK-003; Merck Research Laboratories) at 1 mM respectively. 2.3. Quantitative real time PCR Total RNA was isolated from paraffin-embedded tissues or cultured cells by using Trizol Reagent (Invitrogen, CA, USA) following the manufacturer’s instructions. About 5 mg of total RNA was converted to cDNA by M-MLV reverse transcriptase (Invitrogen), supplement with Oligo (dT18) RT primers. Samples for real-time PCR analysis were prepared with SYBR Premix Ex Taq (Takara, Japan) and specific primers as follows: MEG3-F: 50 -CCTCTCCATGCTGAGCTGCT-30 , MEG3-R: 50 -TGTTGGTGGGATCCAGGAAA-30 ; b-actin-F: 50 -AGCGAGCATCCCCCAAAGTT-30 , 0 b-actin-R: 5 -GGGCACGAAGGCTCATCATT-30 . All reactions were performed on the Bio-Rad CFX96 real-time PCR System (Bio-Rad, Foster City, CA, USA) with each sample assayed in triplicate in each of three independent experiments. The level of MEG3 expression in each sample was normalized to the respective b-actin expression level. 2.4. Western blotting Protein lysates from tissues or cells were prepared by using a RIPA kit (Beyotime, Shanghai, China) according to the manufacturer’s protocols. And, an enhanced BCA Protein Assay kit (Beyotime) was used to determine protein concentrations. Samples containing equal amounts of protein were mixed with loading buffer, separated by SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). These membranes were treated with blocking buffer containing 5% non-fat dry milk, and then probed with primary antibodies, against Notch1,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, Sigma) was used here for the measurement of cell viability. Briefly, cells were inoculated at 3 103 cells/well in 96-well plates and 20 ml MTT at 5 mg/ml, or culture medium as a control group, was added to each well for subsequent 4-h-incubation at 37 C. Then, the absorbance of different cell cultures was recorded at 490 nm by using a microplate reader. Result from pure culture medium wells without cell inoculation was used as blank control. Each sample was assayed in quintuplicate through at least three independent experiments. 2.7. Colony-formation assays Different cell lines were digested with trypsin for the preparation of single-cell suspensions. Then, approximately 150 cells/well were seeded into 6-well plates, and incubated for 14 days. After fixed with methanol for 30 min at room temperature, formed colonies were stained with 0.5% crystal violet for 1 h, and photographed by Nikon camera (Nikon, Japan). 2.8. Tumor growth in nude mice Eight-week-old athymic male nude mice (Nu/Nu) were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China) and housed in the College of Medicine of Zhejiang University. All animal studies were also approved by the local Ethics Committee. KLE cells with stable expression of LV-MEG3 or LV-GFP were pre-cultured and resuspended in serum-free DMEM/F12. Eight to ten mice/group were used for the establishment of xenograft models through subcutaneously injection of prepared tumor cells (106 cells per mouse). Three weeks after the inoculation, tumor volume (in mm3) was estimated weekly by measuring the length and width. And, data from the third to seventh week was used to draw the tumor growth curve. 2.9. Statistical analysis All data results were present as means SD. SPSS 13.0 software was used to calculate differences among groups by one-way analysis of variance (ANOVA) or Student’s two-tailed t test. P < 0.05 was considered significant.
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3. Results 3.1. MEG3 downregulation and Notch1 upregulation were observed in EC tissues or cell lines Paraffin-embedded tissues were prepared from EC patients and analyzed by real time PCR or western blotting to detect the expression of targets like MEG3, Notch1 and Hes1. The mean average of MEG3 expression level in EC tissues was 0.38 after compared to that in normal ones (Fig. 1A), showing a significant decrease (P < 0.01). Image of bands from three different EC or normal samples represented that the protein expression of Notch1 or Hes1 was strongly upregulated, with a respective increase of 3.0-fold or 2.2-fold, in EC tissues as compared with normal ones, after normalized to the internal control, b-actin (Fig. 1B; P < 0.01). Accordingly, these targets expression was also measured in two EC cell lines, HEC-1A or KLE, and one normal endometrial cell line, ESC. Similarly, MEG3 expression showed a decrease level to 0.58-fold or 0.38-fold in HEC-1A or KLE cells respectively, as compared to that in ESC (Fig. 1C; P < 0.01). And, the expression level of Notch1 or Hes1 protein was also significantly increased by 1.6-fold or 2.0-fold in HEC-1A cells, or 2.6-fold or 2.7-fold in KLE cells, respectively (Fig. 1D; P < 0.01). The downregulation of MEG3 and upregulation of Notch1 and Hes1 might not only imply the potential function of MEG3 in EC but also indicate the connection between MEG3 function and Notch1-mediated pathways. 3.2. MEG3-induced inhibition on cell proliferation was reversed by Notch1 activator Previous study has implicated the involvement of Notch pathway in regulating the cell proliferation of EC cells [20]. Since MEG3 expression showed potential correlation with Notch1 and its downstream gene Hes1, its regulation on cell proliferation was analyzed through gain- and loss-of-function studies. Firstly,
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enforced MEG3 expression was conducted in KLE cells, with relative lower MEG3 expression level than HEC-1A (Fig. 1C), by the transfection of MEG3 overexpressing plasmid, LV-MEG3. Cell proliferation was evaluated by detecting cell viability during a 72h-incubation through MTT method, as well as colony formation ability. Since 24 h post-inoculation, a significant decrease of cell growth was observed in MEG3 overexpressing cells (Fig. 2A), whose colony formation was also less than that of normal cells (Fig. 2B). Notably, the expression of Notch1 or Hes1 was found to be downregulated by MEG3 overexpression (Fig. 2C; P < 0.01), with a respective decrease of 0.25-fold or 0.44-fold. And, the addition of Jagged-1/Fc, one Notch1 activator [21], was proven to reverse MEG3-induced inhibition on cell proliferation, including recovered cell growth and colony formation (Fig. 2A, B). Thus, MEG3 indeed showed regulation on cell proliferation which was mediated by Notch1. 3.3. MEG3 silence promoted cell proliferation, which was then repressed by Notch1 inhibitor Loss-of-function study was also performed in HEC-1A cells, with relative higher MEG3 expression level than KLE (Fig. 1C), by the transfection of lentiviral plasmid with shMEG3, LV-shMEG3. Similar analysis for cell proliferation was conducted, wherein cell growth and colony formation both showed significant increase in MEG3 silencing cells (Fig. 3A, B). Accordingly, Notch1 pathway was detected to be induced by MEG3 silence, while the expression of Notch1 or Hes1 was upregulated by respective 2.5-fold or 2.1-fold (Fig. 3C; P < 0.01). Moreover, GSI, one Notch1 inhibitor [22], was selected to demonstrate Notch1-mediated mechanism in MEG3 silencing cells, while the increase of cell proliferation was observed to be strongly inhibited (Fig. 3A, B). With the integration of results from both studies, MEG3 showed negative regulation on the proliferation of EC cells via Notch1-mediated mechanism.
Fig. 1. Expression of MEG3 in EC tissues or cells, as well as Notch1 and Hes1. Relative MEG3 expression in EC tissues (A) or cell lines (C; HEC-1A and KLE) was detected by real time PCR and compared to that in adjacent normal tissues or normal endometrial cells (ESC) respectively, while results from control ones were set at 1. The expression of Notch1 and Hes1 in tissues (B) or cells (D) was also analyzed by western blotting, with b-actin used as an internal control. **P < 0.01 vs control.
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Fig. 2. Effects of MEG3 overexpression in KLE cells. KLE cells were transfected with LV-GFP (control) or LV-MEG3 for MEG3 overexpression. A. The expression of Notch1 and Hes1 in MEG3 overexpressing cells. B. MTT analysis for cell proliferation with Jagged-1/Fc used as Notch1 activator. C. Detection for colony formation. **P < 0.01 vs cells with LV-GFP transfection; ##P < 0.01 vs cells with LV-MEG3 transfection.
Fig. 3. MEG3 interfering in HEC-1A cells. HEC-1A cells were transfected with LV-GFP (control) or LV-shMEG3 for MEG3 interference. A. The expression of Notch1 and Hes1 in MEG3 overexpressing cells. B. MTT analysis for cell proliferation with GSI used as Notch1 inhibitor. C. Detection for colony formation. **P < 0.01 vs cells with LV-GFP transfection; ##P < 0.01 vs cells with LV-shMEG3 transfection.
3.4. MEG3 overexpression in vivo inhibited tumor growth, with downregulated Notch1 and Hes1 As MEG3 overexpression had been proven to inhibit the proliferation of KLE cells, MEG3-induced effects in vivo were also analyzed, by subcutaneous transplantation of tumor cells with or without MEG3 overexpression into mice and subsequent observation for tumor growth. At three-week post-transplantation, tumor
size was recorded weekly to calculate the tumor volume, and tumor growth curves were illustrated accordingly. As shown in Fig. 4A, tumor growth was significantly inhibited in mice transplanted with MEG3 overexpressing tumors, as compared to control mice carrying normal tumor cells, and the difference became greater following the forth-week to the end. At the seventh-week post-transplantation, all mice were sacrificed for the detection of targets expression, including MEG3, Notch1 and Hes1. MEG3
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Fig. 4. Observations for mice transplanted with KLE tumors. KLE cells transfected with lentiviral vectors (LV-GFP or LV-MEG3) were subcutaneously transplanted into mice. A. Measurement for tumor volume three weeks after the transplantation. *P < 0.05, **P < 0.01 vs mice transplanted with LV-GFP-expressing KLE cells. Result from control group at the beginning was set at 100%. B. Relative MEG3 expression levels in tumor tissues. **P < 0.01 vs tissues from mice transplanted with LV-GFP-expressing KLE cells. C. Protein expression of Notch1 and Hes1 in tumor tissues, with b-actin as control.
expression showed an increase of approximately 8.6-fold in MEG3 overexpressing tumors (Fig. 4B; P < 0.01), while the protein levels of Notch1 and Hes1 were both greatly inhibited (Fig. 4C), showing a decrease of 0.42-fold and 0.36-fold respectively (P < 0.01). Therefore, MEG3 also showed great inhibition on tumor growth in vivo, involving downregulated Notch1 and Hes1. 4. Discussion Long non-coding RNAs (lncRNAs) are a group of non-coding RNAs with more than 200 nucleotides. Nowadays, increasing lncRNAs have been identified in different cell types, where they might function as important regulators in both physiology and pathology [23]. As for tumorigenesis, some well-studied lncRNAs have been reported to be dysregulated in EC tissues as compared with paired normal ones, indicating their participation in EC progression through multiple mechanisms [24]. For example, the expression of lncRNAs HOTAIR, H19 and SRA can be directly affected by changes in the levels of sex hormones, along with the development of EC. In this study, it was demonstrated for the first time that lncRNA MEG3 was also down-regulated in EC samples compared to control samples. Further functional studies revealed the negative regulation of MEG3 on both EC cell proliferation and tumor growth, suggesting its role as a tumor suppressor in EC tumorigenesis. Moreover, concomitant upregulation of Notch1 and Hes1, two Notch signaling molecules, was observed in both EC tissues and cell lines, suggesting the potential mechanism of MEG3/Notch axis in EC development. Notch signaling pathway has been documented to participate in different tumorigenesis [14], and show great promise as
therapeutic target in various cancer types [25]. In general, Notch signaling initiates through ligand–receptor interactions, while the intramembranous proteolytic cleavage of Notch1 receptor contributes to the release of Notch intracellular domain (NICD) in an active form. Subsequently, NICD can function as a transcriptional activator after translocating to the nucleus, showing positive regulation on target genes, including Hairyenhancer of split 1 (Hes1) [26]. Hes1 is one HES family members that can act as a transcriptional repressor by negatively regulating genes required for cell proliferation and differentiation [27], and has been widely used as an indicator of Notch activation [28]. Notably, Notch activation can exert either pro-proliferative or antiproliferative effect on cell proliferation in tissue type and cellular context dependent manner by modulating multiple factors [14]. Studies focusing on the expression profiles of Notch signaling molecules have suggested an inhibition of Notch pathway in human EC tissues, supporting the notion of Notch pathway as tumor suppressor in early EC development [15,16]. However, Notch pathway also shows contribution to the promoted cell proliferation of EC cells [18]. Moreover, the repression of Notch signaling, with decreased Notch1 expression and reduced Hes1 protein level, can exert an anti-proliferative effect on cell cycle progression and apoptosis of Ishikawa EC cells [20]. Similarly, our results showed the accompanied activation of Notch signaling in EC cells or tumors with improved proliferation or growth under MEG3-mediated mechanisms. Therefore, it was speculated that Notch activation in EC development might also depend on tumor stage or cellular context, exhibiting an adaptive role under multiple regulatory mechanisms. Additionally, the negative regulation of MEG3 on EC cell proliferation was also proven to be reversed by the addition of
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Notch1-targeted agent, Jagged-1/Fc as Notch1 activator or GSI as Notch1 inhibitor, implying the key role of Notch1 in MEG3-induced anti-proliferative effect. In conclusion, lncRNA MEG3 was demonstrated to be downregulated in both EC tissues and cell lines, wherein it functioned as a tumor suppressor by negatively regulating the cell proliferation via Notch1-mediated activation of Notch signaling. Further in vivo experiments indicated a promise application of MEG3 overexpression in repressing EC tumor growth. Due to the complicated cross talk of Notch signaling with various cancer progression regulators, more deep studies are still needed to explore Notch1mediated mechanism under MEG3 dysfunction, for exploring therapeutic value of MEG3-targeted methods in EC treatment or prognosis. Conflicts of interest None declared. Acknowledgement This study was supported by Zhejiang Provincial Medical Science and Technology Plan (No: 2016KYA035). References [1] P. Morice, et al., Endometrial cancer, Lancet 387 (10023) (2016) 1094–1108. [2] J.V. Bokhman, Two pathogenetic types of endometrial carcinoma, Gynecol. Oncol. 15 (1) (1983) 10–17. [3] C.G.A.R. Network, Integrated genomic characterization of endometrial carcinoma, Nature 497 (7447) (2013) 67–73. [4] R. Murali, R.A. Soslow, B. Weigelt, Classification of endometrial carcinoma: more than two types, Lancet Oncol. 15 (7) (2014) e268–78. [5] L. Benetatos, G. Vartholomatos, E. Hatzimichael, MEG3 imprinted gene contribution in tumorigenesis, Int. J. Cancer 129 (4) (2011) 773–779. [6] Y. Zhou, X. Zhang, A. Klibanski, MEG3 noncoding RNA: a tumor suppressor, J. Mol. Endocrinol. 48 (3) (2012) R45–R53. [7] Z. Gong, et al., Long non-coding RNAs in cancer, Sci. China Life Sci. 55 (12) (2012) 1120–1124. [8] V. Balik, et al., MEG3: a novel long noncoding potentially tumour-suppressing RNA in meningiomas, J. Neurooncol. 112 (1) (2013) 1–8. [9] J. Zhang, et al., Long noncoding RNA MEG3 is downregulated in cervical cancer and affects cell proliferation and apoptosis by regulating miR-21, Cancer Biol. Ther. 17 (1) (2015) 104–113.
[10] K.H. Lu, et al., Long non-coding RNA MEG3 inhibits NSCLC cells proliferation and induces apoptosis by affecting p53 expression, BMC Cancer 13 (2013) 461. [11] W. Peng, et al., Long non-coding RNA MEG3 functions as a competing endogenous RNA to regulate gastric cancer progression, J. Exp. Clin. Cancer Res. 34 (2015) 79. [12] J. Zhu, et al., Long noncoding RNA MEG3 interacts with p53 protein and regulates partial p53 target genes in hepatoma cells, PLoS One 10 (10) (2015) e139790. [13] S. Artavanis-Tsakonas, M.D. Rand, R.J. Lake, Notch signaling: cell fate control and signal integration in development, Science 284 (5415) (1999) 770–776. [14] R.A. Previs, et al., Molecular pathways: translational and therapeutic implications of the Notch signaling pathway in cancer, Clin. Cancer Res. 21 (5) (2015) 955–961. [15] V. Jonusiene, et al., Down-regulated expression of Notch signaling molecules in human endometrial cancer, Med Oncol. 30 (1) (2013) 438. [16] A. Sasnauskiene, et al., NOTCH1: NOTCH3, NOTCH4, and JAG2 protein levels in human endometrial cancer, Medicina (Kaunas) 50 (1) (2014) 14–18. [17] Y. Wei, et al., Nuclear estrogen receptor-mediated Notch signaling and GPR30mediated PI3K/AKT signaling in the regulation of endometrial cancer cell proliferation, Oncol. Rep. 27 (2) (2012) 504–510. [18] M. Qiu, et al., FOXA1 promotes tumor cell proliferation through AR involving the Notch pathway in endometrial cancer, BMC Cancer 14 (1) (2014) 78. [19] F.E. Gordon, et al., Increased expression of angiogenic genes in the brains of mouse meg3-null embryos, Endocrinology 151 (6) (2010) 2443–2452. [20] M. Gotte, et al., The adult stem cell marker Musashi-1 modulates endometrial carcinoma cell cycle progression and apoptosis via Notch-1 and p21WAF1/ CIP1, Int. J. Cancer 129 (8) (2011) 2042–2049. [21] B.L. Beckstead, D.M. Santosa, C.M. Giachelli, Mimicking cell–cell interactions at the biomaterial–cell interface for control of stem cell differentiation, J. Biomed. Mater. Res. Part A 79 (1) (2006) 94–103. [22] S.S. Rao, et al., Inhibition of NOTCH signaling by gamma secretase inhibitor engages the RB pathway and elicits cell cycle exit in T-cell acute lymphoblastic leukemia cells, Cancer Res. 69 (7) (2009) 3060–3068. [23] S. Geisler, J. Coller, RNA in unexpected places: long non-coding RNA functions in diverse cellular contexts, Nat. Rev. Mol. Cell Biol. 14 (11) (2013) 699–712. [24] M.A. Smolle, et al., Long non-coding RNAs in endometrial carcinoma, Int. J. Mol. Sci. 16 (11) (2015) 26463–26472. [25] N. Takebe, D. Nguyen, S.X. Yang, Targeting notch signaling pathway in cancer: clinical development advances and challenges, Pharmacol. Ther. 141 (2) (2014) 140–149. [26] B. De Strooper, et al., A presenilin-1-dependent gamma-secretase-like protease mediates release of Notch intracellular domain, Nature 398 (6727) (1999) 518–522. [27] S.M. Kwon, et al., The molecular basis of Notch signaling: a brief overview, Adv. Exp. Med. Biol. 727 (2012) 1–14. [28] Y. Sun, et al., Differential Notch1 and Notch2 expression and frequent activation of Notch signaling in gastric cancers, Arch. Pathol. Lab. Med. 135 (4) (2011) 451–458.
Available online at
ScienceDirect www.sciencedirect.com
LncRNA-MEG3 inhibits cell proliferation of endometrial carcinoma by repressing Notch signaling Qingyun Guoa , Zhida Qiana , Dingding Yanb , Li Lib , Lili Huanga,* a b
Women’s Hospital, College of Medicine, Zhejiang University, Hangzhou 310006, Zhejiang, China Zhejiang Cancer Hospital, Hangzhou 310022, Zhejiang, China
A R T I C L E I N F O
A B S T R A C T
Article history: Received 28 January 2016 Received in revised form 23 February 2016 Accepted 23 February 2016
Background: The long non-coding RNA MEG3 has shown functional role as a tumor suppressor in many cancer types, excluding endometrial carcinoma (EC). Thus, this study tried to reveal the MEG3 dysregulation in EC samples and potential functional mechanism due to its regulation on Notch signaling pathway. Methods: The expression profiles of MEG3 and two Notch signaling molecules, Notch1 and Hes1, were detected in both EC tissues and cell lines through real time PCR and western blot analysis. Lentiviral vector carrying whole MEG3 transcript or shRNA targeting MEG3 (shMEG3) was transfected for MEG3 dysfunction studies, and cell proliferation was analyzed through MTT and colony-formation assays. Xenograft models were also established by subcutaneous implantation and tumor growth was compared under MEG3 dysregulation. Results: Significant downregulation of MEG3 was observed in EC samples compared to control, while the protein levels of Notch1 and Hes1 were both upregulated. Cell proliferation was obviously inhibited by MEG3 overexpression, while opposite improved result was obtained in MEG3 knockout cells. Interestingly, MEG3-induced changes could be reversed by Notch1 regulators. Moreover, MEG3 overexpressing tumors showed strongly repressed growth in vivo, along with Notch signaling inhibition. Conclusion: Downregulated MEG3 exhibited an anti-proliferative role in EC by repressing Notch signaling pathway. ã 2016 Elsevier Masson SAS. All rights reserved.
Keywords: Endometrial carcinoma Cell proliferation MEG3 Notch signaling
1. Introduction Endometrial carcinoma (EC) is one of the most common gynecologic malignant tumors, with great increase in new cases that is second only to cervical cancer [1]. Generally, EC can be classified into two sub-types, namely estrogen-dependent endometrioid carcinoma (type I) and estrogen-independent nonendometrioid carcinoma (type II) [2]. Type I ECs, accounting for 70–80% cases, are typically well differentiated tumors emerging from hyperplastic endometrial tissues, while Type II ECs derived from atrophic endometrial tissues are primary serous tumors and occur more often in older women at the post-menopausal state [3]. Type I ECs can be diagnosed at an early stage showing favorable prognoses, whereas Type II is often diagnosed at an advanced stage with poor outcomes. Although the five year survival rate of EC patients is up to 74–91% [4], adjuvant treatment option for these
* Corresponding author at: Women’s Hospital, College of Medicine, Zhejiang University, No. 1 Xueshi Road, Hangzhou 310006, Zhejiang, China. E-mail address: [email protected] (L. Huang). http://dx.doi.org/10.1016/j.biopha.2016.02.049 0753-3322/ã 2016 Elsevier Masson SAS. All rights reserved.
with advanced and recurrent tumors is still limited, partly due to unclear mechanism of EC development. Maternally expressed gene 3 (MEG3), also known as gene trap locus 2 (GTL2), is an imprinted gene, encoded by the MEG3 transcript of the DLK1/MEG3 locus on human chromosome, or Meg3 on mouse chromosome [5]. MEG3 can express in many normal tissues, while the loss of MEG3 expression can be found in some tumors and tumor cell lines, indicating the role of MEG3 as a potential tumor suppressor [6]. Since then, the multiple mechanisms of MEG3 downregulation have been explored in cancer development and progression [7], due to its regulation on p53, RB and Notch activity, as well as their signaling pathways. And, increasing functional roles of MEG3 have been demonstrated in various cancer types, including meningioma [8], cervical carcinoma [9], lung cancer [10], gastric cancer [11], and hepatocellular cancer [12]. However, the expression profiles of MEG3 in EC tissues or cell lines have not been explored yet, as well as related functional mechanism. Notch signaling is a highly conserved pathway in developmental biology [13], and shows important regulation on cell proliferation, differentiation and death that associated with tumorigenesis
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[14]. Dysregulated Notch pathway has been documented in multiple types of cancers, including EC [15,16]. Recent studies have also revealed the mechanism of EC development under the Notch signaling control [17,18]. As mentioned above, MEG3 also exert potential regulation on Notch signaling, since Notch pathway is reported to be enriched in Meg3-null embryos [19]. Therefore, in the present study, the potential MEG3/Notch axis was speculated to function in EC development, wherein MEG3 might function by modulating Notch signaling pathways to suppress tumorigenesis.
Hes1 or b-actin, at 4 C for overnight. All primary antibodies were purchased from Epitomics (Burlingame, CA, USA), and used at a dilution of 1:2000 following the manufacturer’s suggestions. Peroxidase-conjugated secondary antibodies provided by Cell Signaling Technology (Danvers, MA, USA) were added to detect the bound primary antibodies. Immunoblot images were visualized via chemiluminescence (Beyotime), and analyzed using ImageQuant LAS 4000 System (GE Healthcare, Little Chalfont, UK). 2.5. Plasmids and cell transfection
2. Materials and methods 2.1. Tumor tissues This study was approved by the local Medical Ethical Committee of Women’s Hospital, School of Medicine, Zhejiang University. Total thirty-paired specimens of tumor tissues and adjacent normal endometrial samples were obtained from EC patients, who received surgical treatment from 2012 to 2014. All patients were free from neoadjuvant therapy or endocrine therapy before the surgery and had signed informed consent to allow molecular research on obtained specimens.
Lentiviral constructs carrying whole MEG3 transcript (LV-MEG3), or shRNA targeting MEG3 (LV-shMEG3), and an empty negative control vector (LV-GFP) were obtained from GenePharma (Shanghai, China). Cells were pre-grown to 30% confluency and then transfected with different lentiviral constructs at a final concentration of 2 mg/mL by using Lipo2000 (Invitrogen) in accordance with the manufacturer’s instructions. After incubation at 37 C for 48 h, fresh medium was added, and cells were collected after another 72-h-incubation to verify the stable overexpression or knockdown of MEG3 through real time PCR analysis. 2.6. MTT assay
2.2. Cell culture and treatment The human endometrial cell line, endometrial stromal cell (ESC), and two human endometrial cancer cell lines, HEC-1A and KLE, were all purchased from the American Type Culture Collection (ATCC, Manassas, VA), and grown in Dulbecco modified Eagle medium (DMEM)/F12 (HyClone, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) at 37 C with a humidified atmosphere of 5% CO2. For the activation or blockage of Notch signaling, cells were incubated with recombinant Jagged-1/Fc (R&D Systems, Minneapolis, USA) at 500 mg/L org-secretase inhibitor (GSI) (MRK-003; Merck Research Laboratories) at 1 mM respectively. 2.3. Quantitative real time PCR Total RNA was isolated from paraffin-embedded tissues or cultured cells by using Trizol Reagent (Invitrogen, CA, USA) following the manufacturer’s instructions. About 5 mg of total RNA was converted to cDNA by M-MLV reverse transcriptase (Invitrogen), supplement with Oligo (dT18) RT primers. Samples for real-time PCR analysis were prepared with SYBR Premix Ex Taq (Takara, Japan) and specific primers as follows: MEG3-F: 50 -CCTCTCCATGCTGAGCTGCT-30 , MEG3-R: 50 -TGTTGGTGGGATCCAGGAAA-30 ; b-actin-F: 50 -AGCGAGCATCCCCCAAAGTT-30 , 0 b-actin-R: 5 -GGGCACGAAGGCTCATCATT-30 . All reactions were performed on the Bio-Rad CFX96 real-time PCR System (Bio-Rad, Foster City, CA, USA) with each sample assayed in triplicate in each of three independent experiments. The level of MEG3 expression in each sample was normalized to the respective b-actin expression level. 2.4. Western blotting Protein lysates from tissues or cells were prepared by using a RIPA kit (Beyotime, Shanghai, China) according to the manufacturer’s protocols. And, an enhanced BCA Protein Assay kit (Beyotime) was used to determine protein concentrations. Samples containing equal amounts of protein were mixed with loading buffer, separated by SDS-PAGE and then transferred to PVDF membranes (Millipore, Billerica, MA, USA). These membranes were treated with blocking buffer containing 5% non-fat dry milk, and then probed with primary antibodies, against Notch1,
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT, Sigma) was used here for the measurement of cell viability. Briefly, cells were inoculated at 3 103 cells/well in 96-well plates and 20 ml MTT at 5 mg/ml, or culture medium as a control group, was added to each well for subsequent 4-h-incubation at 37 C. Then, the absorbance of different cell cultures was recorded at 490 nm by using a microplate reader. Result from pure culture medium wells without cell inoculation was used as blank control. Each sample was assayed in quintuplicate through at least three independent experiments. 2.7. Colony-formation assays Different cell lines were digested with trypsin for the preparation of single-cell suspensions. Then, approximately 150 cells/well were seeded into 6-well plates, and incubated for 14 days. After fixed with methanol for 30 min at room temperature, formed colonies were stained with 0.5% crystal violet for 1 h, and photographed by Nikon camera (Nikon, Japan). 2.8. Tumor growth in nude mice Eight-week-old athymic male nude mice (Nu/Nu) were purchased from the Shanghai Laboratory Animal Center of the Chinese Academy of Sciences (Shanghai, China) and housed in the College of Medicine of Zhejiang University. All animal studies were also approved by the local Ethics Committee. KLE cells with stable expression of LV-MEG3 or LV-GFP were pre-cultured and resuspended in serum-free DMEM/F12. Eight to ten mice/group were used for the establishment of xenograft models through subcutaneously injection of prepared tumor cells (106 cells per mouse). Three weeks after the inoculation, tumor volume (in mm3) was estimated weekly by measuring the length and width. And, data from the third to seventh week was used to draw the tumor growth curve. 2.9. Statistical analysis All data results were present as means SD. SPSS 13.0 software was used to calculate differences among groups by one-way analysis of variance (ANOVA) or Student’s two-tailed t test. P < 0.05 was considered significant.
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3. Results 3.1. MEG3 downregulation and Notch1 upregulation were observed in EC tissues or cell lines Paraffin-embedded tissues were prepared from EC patients and analyzed by real time PCR or western blotting to detect the expression of targets like MEG3, Notch1 and Hes1. The mean average of MEG3 expression level in EC tissues was 0.38 after compared to that in normal ones (Fig. 1A), showing a significant decrease (P < 0.01). Image of bands from three different EC or normal samples represented that the protein expression of Notch1 or Hes1 was strongly upregulated, with a respective increase of 3.0-fold or 2.2-fold, in EC tissues as compared with normal ones, after normalized to the internal control, b-actin (Fig. 1B; P < 0.01). Accordingly, these targets expression was also measured in two EC cell lines, HEC-1A or KLE, and one normal endometrial cell line, ESC. Similarly, MEG3 expression showed a decrease level to 0.58-fold or 0.38-fold in HEC-1A or KLE cells respectively, as compared to that in ESC (Fig. 1C; P < 0.01). And, the expression level of Notch1 or Hes1 protein was also significantly increased by 1.6-fold or 2.0-fold in HEC-1A cells, or 2.6-fold or 2.7-fold in KLE cells, respectively (Fig. 1D; P < 0.01). The downregulation of MEG3 and upregulation of Notch1 and Hes1 might not only imply the potential function of MEG3 in EC but also indicate the connection between MEG3 function and Notch1-mediated pathways. 3.2. MEG3-induced inhibition on cell proliferation was reversed by Notch1 activator Previous study has implicated the involvement of Notch pathway in regulating the cell proliferation of EC cells [20]. Since MEG3 expression showed potential correlation with Notch1 and its downstream gene Hes1, its regulation on cell proliferation was analyzed through gain- and loss-of-function studies. Firstly,
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enforced MEG3 expression was conducted in KLE cells, with relative lower MEG3 expression level than HEC-1A (Fig. 1C), by the transfection of MEG3 overexpressing plasmid, LV-MEG3. Cell proliferation was evaluated by detecting cell viability during a 72h-incubation through MTT method, as well as colony formation ability. Since 24 h post-inoculation, a significant decrease of cell growth was observed in MEG3 overexpressing cells (Fig. 2A), whose colony formation was also less than that of normal cells (Fig. 2B). Notably, the expression of Notch1 or Hes1 was found to be downregulated by MEG3 overexpression (Fig. 2C; P < 0.01), with a respective decrease of 0.25-fold or 0.44-fold. And, the addition of Jagged-1/Fc, one Notch1 activator [21], was proven to reverse MEG3-induced inhibition on cell proliferation, including recovered cell growth and colony formation (Fig. 2A, B). Thus, MEG3 indeed showed regulation on cell proliferation which was mediated by Notch1. 3.3. MEG3 silence promoted cell proliferation, which was then repressed by Notch1 inhibitor Loss-of-function study was also performed in HEC-1A cells, with relative higher MEG3 expression level than KLE (Fig. 1C), by the transfection of lentiviral plasmid with shMEG3, LV-shMEG3. Similar analysis for cell proliferation was conducted, wherein cell growth and colony formation both showed significant increase in MEG3 silencing cells (Fig. 3A, B). Accordingly, Notch1 pathway was detected to be induced by MEG3 silence, while the expression of Notch1 or Hes1 was upregulated by respective 2.5-fold or 2.1-fold (Fig. 3C; P < 0.01). Moreover, GSI, one Notch1 inhibitor [22], was selected to demonstrate Notch1-mediated mechanism in MEG3 silencing cells, while the increase of cell proliferation was observed to be strongly inhibited (Fig. 3A, B). With the integration of results from both studies, MEG3 showed negative regulation on the proliferation of EC cells via Notch1-mediated mechanism.
Fig. 1. Expression of MEG3 in EC tissues or cells, as well as Notch1 and Hes1. Relative MEG3 expression in EC tissues (A) or cell lines (C; HEC-1A and KLE) was detected by real time PCR and compared to that in adjacent normal tissues or normal endometrial cells (ESC) respectively, while results from control ones were set at 1. The expression of Notch1 and Hes1 in tissues (B) or cells (D) was also analyzed by western blotting, with b-actin used as an internal control. **P < 0.01 vs control.
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Fig. 2. Effects of MEG3 overexpression in KLE cells. KLE cells were transfected with LV-GFP (control) or LV-MEG3 for MEG3 overexpression. A. The expression of Notch1 and Hes1 in MEG3 overexpressing cells. B. MTT analysis for cell proliferation with Jagged-1/Fc used as Notch1 activator. C. Detection for colony formation. **P < 0.01 vs cells with LV-GFP transfection; ##P < 0.01 vs cells with LV-MEG3 transfection.
Fig. 3. MEG3 interfering in HEC-1A cells. HEC-1A cells were transfected with LV-GFP (control) or LV-shMEG3 for MEG3 interference. A. The expression of Notch1 and Hes1 in MEG3 overexpressing cells. B. MTT analysis for cell proliferation with GSI used as Notch1 inhibitor. C. Detection for colony formation. **P < 0.01 vs cells with LV-GFP transfection; ##P < 0.01 vs cells with LV-shMEG3 transfection.
3.4. MEG3 overexpression in vivo inhibited tumor growth, with downregulated Notch1 and Hes1 As MEG3 overexpression had been proven to inhibit the proliferation of KLE cells, MEG3-induced effects in vivo were also analyzed, by subcutaneous transplantation of tumor cells with or without MEG3 overexpression into mice and subsequent observation for tumor growth. At three-week post-transplantation, tumor
size was recorded weekly to calculate the tumor volume, and tumor growth curves were illustrated accordingly. As shown in Fig. 4A, tumor growth was significantly inhibited in mice transplanted with MEG3 overexpressing tumors, as compared to control mice carrying normal tumor cells, and the difference became greater following the forth-week to the end. At the seventh-week post-transplantation, all mice were sacrificed for the detection of targets expression, including MEG3, Notch1 and Hes1. MEG3
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Fig. 4. Observations for mice transplanted with KLE tumors. KLE cells transfected with lentiviral vectors (LV-GFP or LV-MEG3) were subcutaneously transplanted into mice. A. Measurement for tumor volume three weeks after the transplantation. *P < 0.05, **P < 0.01 vs mice transplanted with LV-GFP-expressing KLE cells. Result from control group at the beginning was set at 100%. B. Relative MEG3 expression levels in tumor tissues. **P < 0.01 vs tissues from mice transplanted with LV-GFP-expressing KLE cells. C. Protein expression of Notch1 and Hes1 in tumor tissues, with b-actin as control.
expression showed an increase of approximately 8.6-fold in MEG3 overexpressing tumors (Fig. 4B; P < 0.01), while the protein levels of Notch1 and Hes1 were both greatly inhibited (Fig. 4C), showing a decrease of 0.42-fold and 0.36-fold respectively (P < 0.01). Therefore, MEG3 also showed great inhibition on tumor growth in vivo, involving downregulated Notch1 and Hes1. 4. Discussion Long non-coding RNAs (lncRNAs) are a group of non-coding RNAs with more than 200 nucleotides. Nowadays, increasing lncRNAs have been identified in different cell types, where they might function as important regulators in both physiology and pathology [23]. As for tumorigenesis, some well-studied lncRNAs have been reported to be dysregulated in EC tissues as compared with paired normal ones, indicating their participation in EC progression through multiple mechanisms [24]. For example, the expression of lncRNAs HOTAIR, H19 and SRA can be directly affected by changes in the levels of sex hormones, along with the development of EC. In this study, it was demonstrated for the first time that lncRNA MEG3 was also down-regulated in EC samples compared to control samples. Further functional studies revealed the negative regulation of MEG3 on both EC cell proliferation and tumor growth, suggesting its role as a tumor suppressor in EC tumorigenesis. Moreover, concomitant upregulation of Notch1 and Hes1, two Notch signaling molecules, was observed in both EC tissues and cell lines, suggesting the potential mechanism of MEG3/Notch axis in EC development. Notch signaling pathway has been documented to participate in different tumorigenesis [14], and show great promise as
therapeutic target in various cancer types [25]. In general, Notch signaling initiates through ligand–receptor interactions, while the intramembranous proteolytic cleavage of Notch1 receptor contributes to the release of Notch intracellular domain (NICD) in an active form. Subsequently, NICD can function as a transcriptional activator after translocating to the nucleus, showing positive regulation on target genes, including Hairyenhancer of split 1 (Hes1) [26]. Hes1 is one HES family members that can act as a transcriptional repressor by negatively regulating genes required for cell proliferation and differentiation [27], and has been widely used as an indicator of Notch activation [28]. Notably, Notch activation can exert either pro-proliferative or antiproliferative effect on cell proliferation in tissue type and cellular context dependent manner by modulating multiple factors [14]. Studies focusing on the expression profiles of Notch signaling molecules have suggested an inhibition of Notch pathway in human EC tissues, supporting the notion of Notch pathway as tumor suppressor in early EC development [15,16]. However, Notch pathway also shows contribution to the promoted cell proliferation of EC cells [18]. Moreover, the repression of Notch signaling, with decreased Notch1 expression and reduced Hes1 protein level, can exert an anti-proliferative effect on cell cycle progression and apoptosis of Ishikawa EC cells [20]. Similarly, our results showed the accompanied activation of Notch signaling in EC cells or tumors with improved proliferation or growth under MEG3-mediated mechanisms. Therefore, it was speculated that Notch activation in EC development might also depend on tumor stage or cellular context, exhibiting an adaptive role under multiple regulatory mechanisms. Additionally, the negative regulation of MEG3 on EC cell proliferation was also proven to be reversed by the addition of
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Notch1-targeted agent, Jagged-1/Fc as Notch1 activator or GSI as Notch1 inhibitor, implying the key role of Notch1 in MEG3-induced anti-proliferative effect. In conclusion, lncRNA MEG3 was demonstrated to be downregulated in both EC tissues and cell lines, wherein it functioned as a tumor suppressor by negatively regulating the cell proliferation via Notch1-mediated activation of Notch signaling. Further in vivo experiments indicated a promise application of MEG3 overexpression in repressing EC tumor growth. Due to the complicated cross talk of Notch signaling with various cancer progression regulators, more deep studies are still needed to explore Notch1mediated mechanism under MEG3 dysfunction, for exploring therapeutic value of MEG3-targeted methods in EC treatment or prognosis. Conflicts of interest None declared. Acknowledgement This study was supported by Zhejiang Provincial Medical Science and Technology Plan (No: 2016KYA035). References [1] P. Morice, et al., Endometrial cancer, Lancet 387 (10023) (2016) 1094–1108. [2] J.V. Bokhman, Two pathogenetic types of endometrial carcinoma, Gynecol. Oncol. 15 (1) (1983) 10–17. [3] C.G.A.R. Network, Integrated genomic characterization of endometrial carcinoma, Nature 497 (7447) (2013) 67–73. [4] R. Murali, R.A. Soslow, B. Weigelt, Classification of endometrial carcinoma: more than two types, Lancet Oncol. 15 (7) (2014) e268–78. [5] L. Benetatos, G. Vartholomatos, E. Hatzimichael, MEG3 imprinted gene contribution in tumorigenesis, Int. J. Cancer 129 (4) (2011) 773–779. [6] Y. Zhou, X. Zhang, A. Klibanski, MEG3 noncoding RNA: a tumor suppressor, J. Mol. Endocrinol. 48 (3) (2012) R45–R53. [7] Z. Gong, et al., Long non-coding RNAs in cancer, Sci. China Life Sci. 55 (12) (2012) 1120–1124. [8] V. Balik, et al., MEG3: a novel long noncoding potentially tumour-suppressing RNA in meningiomas, J. Neurooncol. 112 (1) (2013) 1–8. [9] J. Zhang, et al., Long noncoding RNA MEG3 is downregulated in cervical cancer and affects cell proliferation and apoptosis by regulating miR-21, Cancer Biol. Ther. 17 (1) (2015) 104–113.
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