http://informahealthcare.com/aan ISSN: 1939-6368 (print), 1939-6376 (electronic) Syst Biol Reprod Med, Early Online: 1–6 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/19396368.2014.977499
RESEARCH COMMUNICATION
A single nucleotide polymorphism in the MTOR gene is associated with recurrent spontaneous abortion in the Chinese female population Huifen Xiang1,2, Shengnan Liu3,4, Chen Zong1,2, Zelian Li1,2, Yunyun Liu1,2, Xu Ma3,4,5*, and Yunxia Cao1,2* 1
Reproductive Medicine Center, 2Institute of Reproductive Genetics, The First Affiliated Hospital, Anhui Medical University, Hefei, China, Graduate School of Peking Union Medical College, 4National Research Institute for Family Planning, and 5World Health Organization Collaborating Center for Research in Human Reproduction, Beijing, China
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Abstract
Keywords
Recurrent spontaneous abortion (RSA) is a multi-factor disease. The mammalian target of the the rapamycin (MTOR) gene has been reported to be involved in mouse embryo development and regulates the proliferation of embryonic stem cells. Our study explored the relationship between the single nucleotide polymorphism (SNP) rs17027478 in the promoter region of MTOR gene and the development of RSA. A total of 306 patients with RSA and 127 healthy females as the controls were recruited in the case-control study. The predesigned TaqMan SNP Genotyping Assay was adopted to analyze the association between rs17027478 and the development of RSA. Quantitative real-time reverse transcription polymerase chain reaction and luciferase reporter assays were conducted to analyze the function of the variant. It was found that a significant association exists between the variant and the risk of RSA among the patients who experienced no less than three spontaneous abortions (p ¼ 0.043). However, the significant difference disappeared among the total samples (p ¼ 0.524). Furthermore, we observed lower MTOR mRNA levels in the blood of RSA patients compared with healthy females (p ¼ 0.020). The luciferase reporter assay showed that the rs17027478A allele significantly reduced the luciferase activity (p ¼ 0.029). The results demonstrated that the variant rs17027478 in the promoter region of MTOR might be a good candidate responsible for the pathogenesis of RSA.
Chinese population, embryos, MTOR, recurrent spontaneous abortion, single nucleotide polymorphism History Received 28 March 2014 Revised 13 August 2014 Accepted 18 August 2014 Published online 2 April 2015
Abbreviations: RSA: recurrent spontaneous abortion; MTOR: mammalian target of rapamycin; SNP: single nucleotide polymorphism; qRT-PCR: quantitative real-time polymerase chain reaction; URSA: unexplained recurrent spontaneous abortion; mTORC1: mTOR complex 1; ESC: embryonic stem cells; HKE-293: human embryonic kidney 293 cells; HWE: Hardy-Weinberg equilibrium; ANOVA: one-way analysis of variance
Introduction Recurrent spontaneous abortion (RSA) refers to two or more consecutive failed clinical pregnancies prior to the 20th week of gestation [Pfeifer et al. 2013]. It has been reported that approximately 15% of clinical pregnancies end up with miscarriage and 0.4–2% of the diagnosed pregnancies suffered from RSA [McNamee et al. 2012]. Human pregnancy is strictly controlled by multiple regulatory mechanisms and any disturbance might lead to fetal loss [Knabl et al. 2013]. The pathogenesis of RSA is complicated. Several causes responsible for RSA have been identified, including parental
*Address correspondence to Xu Ma, Center for Genetics, National Research Institute for Family Planning, 12 Dahuisi Road, Haidian, Beijing 100081, China. Tel: 00861062179059. E-mail: [email protected] or to Yunxia Cao, Professor, Reproductive Medicine Center, The First Affiliated Hospital of Anhui Medical University, 218 Jixi Road, Hefei, Anhui 230022, China. Tel: 00865512922180. E-mail: caoyunxia6@ 126.com
chromosomal anomalies, anatomical abnormalities, endocrine abnormalities, immune dysfunction, thrombophilic disorders, antiphospholipid syndrome, and infections [Rai and Regan 2006]. However, these only account for 50% of the pathogenesis in patients with RSA [Plouffe et al. 1992]. The etiology for the remaining cases is not understood and these are referred to as unexplained recurrent spontaneous abortion (URSA) [Wilczynski et al. 2012]. The safety and efficacy of current therapies are inadequate for preventing RSA, partly due to the poor understanding of the complex pathogenesis and etiology of RSA. Therefore, further studies should be conducted. Research on the genetic architecture of pregnancy losses has provided a novel perspective which can be applied to identify genes involved in this disease and to investigate the molecular mechanisms of RSA. The expression of the tumor protein p53 (TP53) gene and the tumor necrosis factor (ligand) superfamily, member 10 (TNFSF10/TRAIL) gene in placenta are probably involved in the etiology of RSA by controlling the apoptotic signaling
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pathways [Rull et al. 2013b; Shang et al. 2013]. For example, polymorphisms in the histidine-rich glycoprotein (HRG) gene, androgen receptor (AR) gene, chorionic gonadotropin beta5 (CGB5) gene, tumor necrosis factor (TNF-alpha) gene, transforming growth factor, beta 1 (TGFB1) gene, and fas ligand (FAS-L) gene are significantly associated with the risk of RSA, suggesting that RSA has a solid genetic basis and that the pathogenesis is associated with various genes [Alkhuriji et al. 2013; Banzato et al. 2013; Jahaninejad et al. 2013; Lindgren et al. 2013; Magdoud et al. 2013; Rull et al. 2013a]. The mammalian target of rapamycin (MTOR), also known as FRAP1, is a member of the phosphatidylinostitol kinaserelated kinase (PIKK) superfamily which contains a lipid kinase homology domain and functions as a serine/threonine kinase [Laplante and Sabatini 2009]. It is conserved throughout evolution [Laplante and Sabatini 2009]. MTOR plays a critical role in cell growth, survival, proliferation, and angiogenesis [Jiang and Liu 2009]. It integrates extracellular signals (growth factors and steroid hormones) with intracellular amino acid availability and energy status to control translation rates and additional metabolic processes [Mparmpakas et al. 2010]. MTOR is known to encode two biochemically and functionally distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which have distinct downstream targets and cellular effects. The mTORC1 complex is able to promote phosphorylation of p70 ribosomal s6 kinase (p70s6k) and Eif4E (4E-BP1) which are critical translation regulators and regulate cellular growth and proliferation [Krymskaya and Goncharova 2009]. A recent study demonstrated that mTORC1 plays a critical role in blastocyst activation and mouse embryo implantation [Gonzalez et al. 2012]. Previous studies have demonstrated that not only in early mouse embryos but also in embryonic stem cells (ESC), MTOR has a central effect on cell proliferation and growth. Gangloff and colleagues reported that MTOR(/) embryos were subject to embryonic developmental arrest at an earlier stage due to the failure of embryonic stem cells to establish and defect of inner cell mass proliferation [Gangloff et al. 2004]. Another study found that both cell size and proliferation were controlled by MTOR in embryonic stem cells and early mouse embryos by disrupting the kinase domain of mouse MTOR, and the homozygous mutant embryos died shortly after implantation because neither embryonic nor extraembryonic tissue proliferated properly [Murakami et al. 2004]. In addition to these observations, the MTOR gene also has an analogous function in human pregnancy. Previous in vitro experiments demonstrated that MTOR plays an important role in regulating the invasion of human trophoblast cells through serine phosphorylation of signal transducer and activator of transcription 3 (STAT3), suggesting that dysregulation of MTOR may be involved in pregnancy-related pathologies affecting trophoblast invasion [Busch et al. 2009]. Dysregulation of MTOR plays a critical role in the development of abnormal embryogenesis and implantation, and the human MTOR gene may be a good candidate risk factor responsible for the pathogenesis of RSA. In order to determine whether the MTOR gene is involved in RSA and promote further understanding of the etiology of RSA, our study analyzed the single nucleotide polymorphism (SNP)
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rs17027478 which causes a single nucleotide change from a C to an A in the promoter region of MTOR in a cohort of Chinese females.
Results Study population The genotype frequencies of MTOR rs17027478 polymorphism in both case and control groups were in Hardy-Weinberg equilibrium ( 2 ¼ 0.157, P ¼ 0.692; 2 ¼ 0.102, p ¼ 0.749). The genotype frequencies of rs17027478 polymorphism were 90.8%, 8.8%, and 0.3% for the CC, CA, and AA genotypes among the cases, and 94.5%, 5.5%, and 0 among the controls, respectively. The genotype frequencies showed no significant difference between cases and controls ( 2 ¼ 1.706, p ¼ 0.524). There was no significant difference in the frequencies of rs17027478 alleles between cases and controls. It was found that the MTOR rs17027478 A allele was present at a frequency of 4.7% in the case group, while the frequency was 2.8% in the control group ( 2 ¼ 1.771, p ¼ 0.183, OR ¼ 1.75, 95%CI: 0.76–4.06). It is worth mentioning that when the 84 RSA patients who had the history of three or more spontaneous abortions were compared with the controls, the results showed a significant difference. The frequency distribution of rs17027478A allele was significantly higher among cases than controls (7.74% versus 2.76%; 2 ¼ 5.56, p ¼ 0.018, OR ¼ 2.96, 95%CI: 1.16– 7.58). In addition, the genotype frequencies of CC, CA, and AA were 85.71%, 13.10%, and 1.19% among the 84 RSA patients, respectively. Fisher’s Exact Test analysis indicated that the frequency distributions of genotype between the cases and controls were significantly different ( 2 ¼ 5.177, p ¼ 0.043; p ¼ 0.029, OR ¼ 2.86, 95%CI: 1.08–7.59 by dominant mode). Expression and analysis of MTOR mRNA In the study, five heterozygous samples were selected from cases and controls, respectively. Quantitative real-time reverse transcription polymerase chain reaction was conducted to access the MTOR RNA levels in the blood of both groups. As shown in Figure 1, the level of MTOR in the cases was significantly lower than that in the controls (p ¼ 0.020). To examine whether the SNP rs17027478 polymorphism affected the expression of MTOR, two luciferase reporter constructs containing the MTOR promoter region with either the rs17047278 C or A allele were tested. The reporter plasmids were transfected into HEK293 cells. As shown in Figure 2, both wild-type and mutant-type MTOR promoter enhanced the activity of pGL3-Basic reporter, and the mutant-type showed remarkable loss of activity compared with the wildtype (p ¼ 0.029).
Discussion In this study, we demonstrated that the MTOR promoter region polymorphism rs17027478 was associated with the development of RSA among the females who experienced at least three spontaneous abortions. The in vitro experiments indicated that the C to A change in rs17027478 substantially altered the transcriptional activity of the MTOR gene, and the
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DOI: 10.3109/19396368.2014.977499
Figure 1. Expression levels of MTOR mRNA in RSA and control groups. MTOR expression level in blood of patients was significantly lower than the healthy females. The left bar of this figure shows the average MTOR expression level in cases, five heterozygous samples were used. The right bar of the figure shows the average MTOR expression level in control samples, also five heterozygous samples were included. The significance of difference was evaluated by DDCt analysis and student’s t test. *p50.05 versus wild-type.
Figure 2. Effect of the MTOR rs17027478 polymorphism on the expression of MTOR. We cotransfected human embryonic kidney 293 cells (HKE-293) with pGL3-Basic vector or pGL3- MTOR reporter construct (wild-type or mutant) together with Renilla luciferase plasmid. The pGL3- MTOR reporter construct contains either the rs17024748 C allele or the A allele. Cell extracts of HKE-293 cells were collected and firefly and Renilla luciferase activities were measured 48 h after transfection. Data represent mean values from at least three independent experiments. The significance of differences was evaluated by the oneway analysis of variance (ANOVA). *p50.05 versus wild-type.
reporter plasmid containing the A allele presented decreased transcriptional activity. In vivo analysis using the method of quantitative real-time reverse transcription polymerase chain reaction also suggested that the MTOR mRNA level was higher among healthy control women.
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Embryo implantation and pregnancy are complex physiological processes which depend on the regulation of numerous signaling proteins such as ERKs, phosphatidylinositol 3-kinase (PI3K), PTK2 or PI3K/AKT/mammalian target of rapamycin (mTOR) [Pollheimer and Knofler 2005]. MTOR is a protein kinase that plays a central role in cell growth, proliferation, differentiation, and apoptosis [Fingar and Blenis 2004]. In the cases of compromised nutrition (decreased available amino acids or sugars), growth factor withdrawal, or stress, decreased MTOR activity resulted in the reduction of cell proliferation and tissue growth, and the onset of autophagy [Wullschleger et al. 2006]. Under non-stressed conditions, MTOR phosphorylates p70S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) to regulate translational control [Gingras et al. 2001]. Previous studies have demonstrated that the MTOR gene is essential for early mouse embryo growth and proliferation of embryonic stem cells [Chen et al. 2009; Gangloff et al. 2004; Martin and Sutherland 2001; Murakami, et al. 2004]. Additionally, Regnault and colleagues reported that MTOR can regulate the placenta leucine transport that is critical for the fetal growth [Regnault et al. 2007]. Decreased MTOR activity may affect decidualization of endometrial cells and further induce apoptosis in the mouse model [Chen, et al. 2009]. Our analysis suggested that the MTOR polymorphism rs17027478 has significant association with the development of RSA. But, this association only presented when controls were compared with the RSA patients who had a history of at least three spontaneous abortions. When the patients who experienced only two spontaneous abortions were included, the significant difference disappeared. The phenomenon indicated that RSA females experienced two spontaneous abortions that could be accidental with no genetic significance. There are several possible reasons for this result. First, it should be noted that the MTOR rs17027478 A allele displayed significantly reduced transcriptional activity compared with the C allele, therefore the A/C alternation may possibly have an effect on the expression level of MTOR. Thus, the more the women experienced spontaneous abortions, the more genetic significance it has. Secondly, the observed result may also be partly due to the very low mutation rate of the MTOR polymorphism rs17027478 (0.04; HapMap database, see http://hapmap.ncbi.nlm.nih.gov/). Thus, case-control studies based on a larger population and more functional studies are needed in order to better elucidate the relationship between MTOR rs17027478 A allele and susceptibility to RSA. In our study, we also observed that the level of MTOR mRNA was significantly lower in the blood of RSA patients than in healthy individuals consistent with association. Luciferase reporter assays in our investigation showed that MTOR rs17027478 A allele displayed significantly reduced transcriptional activity compared with the C allele. Thus, we speculate that the polymorphism was a functional SNP that affected the transcriptional regulation of MTOR. Additionally, the results of these functional analyses are in accord with previous studies which used mouse model and human trophoblast cell line [Busch et al. 2009; Chen et al. 2009; Gangloff et al. 2004; Murakami et al. 2004]. Taking these
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results together to consider the functional role of the rs17027478 in modulating the expression of MTOR, it is possible that this polymorphism could influence the individual susceptibility to the development of RSA in the Chinese population. However, there are some limitations in our investigation. First, our research only indicated that rs17027478 was significantly associated with the susceptibility to RSA, but whether the polymorphism will influence the phosphorylation level of MTOR and further alter its downstream signal is still unknown. Further investigations need to be conducted to elucidate specific regulatory mechanism. Second, we only analyzed the MTOR mRNA level in heterozygote individuals due to the low frequency of the A allele. The mRNA level of MTOR among all rs17027478 genotypes should be considered. In conclusion, the results presented in this study suggest that the promoter region of MTOR gene rs17027478 was significantly associated with the risk of RSA in the Chinese population. This polymorphism might be a good candidate responsible for the pathogenesis of RSA.
Materials and Methods Ethics statement The study was approved by the Ethics Review Committee on Family Planning of Anhui Medical University and complied with the 1975 Declaration of Helsinki. Written informed consent was obtained from all participants in this study. Study population A total of 306 unrelated Chinese patients diagnosed with RSA and 127 healthy unrelated Chinese females with at least one previous live birth were recruited in the case-control study from The First Affiliated Hospital of Anhui Medical University during the years 2010–2012. The cases and controls were included according to the criteria as follows. All of the females with RSA included were primary aborters with no previous history of live births. Furthermore, all RSA patients enrolled in the study underwent a routine examination to exclude the known risk factors for RSA, including chromosomal aberrations of partners and the embryos, anatomical abnormalities, thrombophilia, metabolic disorders, and positive lupus anticoagulant and anticardiolipin antibodies. As the control group, 127 healthy women with at least one previous live birth and no spontaneous abortion were included. The women enrolled in the control group had no pregnancy-associated complications.
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genomic DNA, 0.12 mL TaqMan SNP Genotyping Assays, 2.5 mL 2 TaqMan Genotyping Master Mix, and 1.88mL of double distilled water to form the 5 mL reaction mixture. The amplification conditions were as follows: 60 C, 30 sec; 95 C, 10 min; subsequently 95 C, 15 sec and 60 C, 1 min for 40 cycles. We selected 84 samples randomly for DNA sequencing to confirm the genotype. The results of DNA sequencing were 100% in accordance with TaqMan SNP Genotyping Assays. Analysis of MTOR mRNA expression Total RNA of fives heterozygous RSA patients and 5 heterozygous healthy controls was extracted from peripheral blood leukocytes using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). In order to avoid the traces of genomic DNA, the extracted RNA was DNase (Takara, Otsu, Japan) treated. After that, reverse transcription was conducted to synthesize cDNA using a SuperScriptÕ VILOÔ cDNA Synthesis Kit (Invitrogen). To measure the expression levels of MTOR mRNA, quantitative real-time polymerase chain reaction (qRT-PCR) was run on a 7000 real-time PCR system Õ (Applied Biosystems) using SYBR Premix Ex TaqÔ (Takara) and specially designed primers. The primers were 50 -GTGCCTCAGCCTCCCGAGTAT-30 (sense) and 50 -TCAG ACGGTGCCCAGGTCGTG-30 (antisense). A qRT-PCR was performed in the presence of SYBRÕ Green (Takara). RNA concentrations were normalized by GAPDH to calculate relative mRNA level of MTOR. Each sample was run in triplicate and three independent repeated experiments were conducted. Construction of promoter-reporter plasmids The 1098-bp MTOR promoter region sequence, containing either the rs17024748 C allele or the A allele, was amplified by PCR using specifically designed primers from human Genome DNA and inserted into the Sma I- and Bgl II(Takara) digested pGL3-Basic firefly luciferase vector (Promega, Madison, WI, USA) to generate the MTOR reporter construct pGL3-MTOR. The primers were 50 -TCCCCCG GGGGACTCGTGCCTCAGCCTCCCGAGTAT-30 (forward) and 50 -GAAGATCTTCTCAGACGGTGCCCAGGTCGTG-30 (reverse). PCR-mediated site-directed mutagenesis was conducted to generate a human MTOR promoter bearing the identified variations in the luciferase reporter constructs using the appropriate primers and the QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA). The introduced variations were confirmed by DNA sequencing.
DNA extraction
Cell culture and transient transfection
Genomic DNA of each participant was obtained from peripheral blood leukocytes using the QIAamp DNA Blood Mini kit (QIAGEN, Hilden, Germany).
The human embryonic kidney 293 cells (HKE-293) were cultured in prepared maintenance medium of Iscove’s modified Dulbecco’s medium containing an additional 10% fetal bovine serum, 100 mg/ml streptomycin, and 100 mg/ml penicillin. HKE-293 cells were transfected using Lipofectamine 2000 (Invitrogen, New York, NY, USA). Transfections were performed in 48-well tissue culture plates seeded at 0.5 105cells/well 24 h prior to transfection
Genotype Predesigned TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA) was applied to perform genotypic analysis of MTOR (rs17027478). We mixed 30 ng
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DOI: 10.3109/19396368.2014.977499
at 60 to 70% confluence. A total of 500 ng reporter construct pGL3-MTOR (wild-type or mutant) or 500 ng pGL3-Basic vector without insert was cotransfected together with 5 ng Renilla luciferase plasmid. After 48 h, cells were lysed with the lysis buffer (Promega) and assayed for luciferase activity using the Dual-Glo Luciferase Assay System (Promaga) according to the manufacturer’s instructions. The results were normalized against those from the Renilla internal control plasmid. Independent experiments were done in triplicate for each plasmid construct. Cells were washed in PBS and lysed in passive lysis buffer (Promega) 48 h after transfection. Luciferase activity was measured by the Dual-Glo Luciferase Assay System (Promega) following the manufacturer’s instruction, which normalized the transfection efficiency to paired Renilla luciferase activity. Transient transfection was conducted in triplicate for each plasmid construct and repeated at least three times. Statistical analysis SNP rs17027478 allele frequencies, genotype distribution, and Hardy-Weinberg equilibrium (HWE) were compared between RSA patients and healthy females by chi-square tests using the Statistic Package for the Social Science (SPSS) version 16.0 software. Kolmogorov-Smirnov test was used to determine whether the data collected from luciferase assays and qRT-PCR were normally distributed. Since all the data were normally distributed (p40.05), the statistical significance of unpaired samples in luciferase assays was determined by the one-way analysis of variance (ANOVA) and the expression levels of MTOR mRNA between cases and controls were tested by DDCt analysis and compared by Student’s t test.
Declaration of interest The authors report no declaration of interest.
Author contributions The authors’ contributions are as follows: Drafted the manuscript and carried out the molecular genetic studies: HX, SL; Functional analysis: CZ; Samples collection: ZL, YL; Conceived of the study and participated in its design and coordination: XM, YC. All authors approved the final manuscript.
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Chen, X., He, J., Ding, Y., Zeng, L., Gao, R., Cheng, S., et al. (2009) The role of MTOR in mouse uterus during embryo implantation. Reproduction 138:351–6. Fingar, D.C., Blenis, J. (2004) Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression. Oncogene 23:3151–71. Gangloff, Y.G., Mueller, M., Dann, S.G., Svoboda, P., Sticker, M., Spetz, J.F., et al. (2004) Disruption of the mouse mTOR gene leads to early postimplantation lethality and prohibits embryonic stem cell development. Mol Cell Biol 24:9508–16. Gingras, A.C., Raught, B., and Sonenberg, N. (2001) Control of translation by the target of rapamycin proteins. Prog Mol Subcell Biol 27:143–74. Gonzalez, I.M., Martin, P.M., Burdsal, C., Sloan, J.L., Mager, S., Harris, T., et al. (2012) Leucine and arginine regulate trophoblast motility through mTOR-dependent and independent pathways in the preimplantation mouse embryo. Dev Biol 361:286–300. Jahaninejad, T., Ghasemi, N., Kalantar, S.M., Sheikhha, M.H., and Pashaiefar, H. (2013) StuI polymorphism on the androgen receptor gene is associated with recurrent spontaneous abortion. J Assist Reprod Genet 30:437–40. Jiang, B.H. and Liu, L.Z. (2009) PI3K/PTEN signaling in angiogenesis and tumorigenesis. Adv Cancer Res 102:19–65. Knabl, J., Pestka, A., Huttenbrenner, R., Plosch, T., Ensenauer, R., Welbergen, L., et al. (2013) The liver x receptor in correlation with other nuclear receptors in spontaneous and recurrent abortions. PPAR Res 2013:575604. Krymskaya, V.P., Goncharova, E.A. (2009) PI3K/mTORC1 activation in hamartoma syndromes: therapeutic prospects. Cell Cycle 8: 403–13. Laplante, M. and Sabatini, D.M. (2009) mTOR signaling at a glance. J Cell Sci 122:3589–94. Lindgren, K.E., Karehed, K., Karypidis, H., Hosseini, F., Bremme, K., Landgren, B.M., et al. (2013) Histidine-rich glycoprotein gene polymorphism in patients with recurrent miscarriage. Acta Obstet Gynecol Scand 92:974–7. Magdoud, K., Granados Herbepin, V., Messaoudi, S., Hizem, S., Bouafia, N., Almawi, W.Y., et al. (2013) Genetic variation in TGFB1 gene and risk of idiopathic recurrent pregnancy loss. Mol Hum Reprod 19:438–43. Martin, P.M., Sutherland, A.E. (2001) Exogenous amino acids regulate trophectoderm differentiation in the mouse blastocyst through an mTOR-dependent pathway. Dev Biol 240:182–93. McNamee, K., Dawood, F., and Farquharson, R. (2012) Recurrent miscarriage and thrombophilia: an update. Curr Opin Obstet Gynecol 24:229–34. Mparmpakas, D., Zachariades, E., Foster, H., Kara, A., Harvey, A., Goumenou, A., et al. (2010) Expression of mTOR and downstream signalling components in the JEG-3 and BeWo human placental choriocarcinoma cell lines. Int J Mol Med 25:65–9. Murakami, M., Ichisaka, T., Maeda, M., Oshiro, N., Hara, K., Edenhofer, F., et al. (2004) mTOR is essential for growth and proliferation in early mouse embryos and embryonic stem cells. Mol Cell Biol 24:6710–18. Pfeifer, S., Goldberg, J., Lobo, R., Thomas, M., Widra, E., Licht, M., et al. (2013) Definitions of infertility and recurrent pregnancy loss: a committee opinion. Fertil Steril 99:63. Plouffe Jr., L., White, E.W., Tho, S.P., Sweet, C.S., Layman, L.C., Whitman, G.F., et al. (1992) Etiologic factors of recurrent abortion and subsequent reproductive performance of couples: have we made any progress in the past 10 years? Am J Obstet Gynecol 167:313–20; discussion 320–1. Pollheimer, J. and Knofler, M. (2005) Signalling pathways regulating the invasive differentiation of human trophoblasts: a review. Placenta 26:S21–30. Rai, R., Regan, L. (2006) Recurrent miscarriage. Lancet 368:601–11. Regnault, T.R., Kudo, Y., Glazier, J., Roos, S., Lewis, R.M., and Jansson, T. (2007) Heterodimeric amino acid transporters in the placenta – a workshop report. Placenta 28:S103–6. Rull, K., Christiansen, O.B., Nagirnaja, L., Steffensen, R., Margus, T., and Laan, M. (2013a) A modest but significant effect of CGB5 gene promoter polymorphisms in modulating the risk of recurrent miscarriage. Fertil Steril 99:1930–1936.e6. Rull, K., Tomberg, K., Koks, S., Mannik, J., Mols, M., Sirotkina, M., et al. (2013b) Increased placental expression and maternal serum
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RESEARCH COMMUNICATION
A single nucleotide polymorphism in the MTOR gene is associated with recurrent spontaneous abortion in the Chinese female population Huifen Xiang1,2, Shengnan Liu3,4, Chen Zong1,2, Zelian Li1,2, Yunyun Liu1,2, Xu Ma3,4,5*, and Yunxia Cao1,2* 1
Reproductive Medicine Center, 2Institute of Reproductive Genetics, The First Affiliated Hospital, Anhui Medical University, Hefei, China, Graduate School of Peking Union Medical College, 4National Research Institute for Family Planning, and 5World Health Organization Collaborating Center for Research in Human Reproduction, Beijing, China
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Abstract
Keywords
Recurrent spontaneous abortion (RSA) is a multi-factor disease. The mammalian target of the the rapamycin (MTOR) gene has been reported to be involved in mouse embryo development and regulates the proliferation of embryonic stem cells. Our study explored the relationship between the single nucleotide polymorphism (SNP) rs17027478 in the promoter region of MTOR gene and the development of RSA. A total of 306 patients with RSA and 127 healthy females as the controls were recruited in the case-control study. The predesigned TaqMan SNP Genotyping Assay was adopted to analyze the association between rs17027478 and the development of RSA. Quantitative real-time reverse transcription polymerase chain reaction and luciferase reporter assays were conducted to analyze the function of the variant. It was found that a significant association exists between the variant and the risk of RSA among the patients who experienced no less than three spontaneous abortions (p ¼ 0.043). However, the significant difference disappeared among the total samples (p ¼ 0.524). Furthermore, we observed lower MTOR mRNA levels in the blood of RSA patients compared with healthy females (p ¼ 0.020). The luciferase reporter assay showed that the rs17027478A allele significantly reduced the luciferase activity (p ¼ 0.029). The results demonstrated that the variant rs17027478 in the promoter region of MTOR might be a good candidate responsible for the pathogenesis of RSA.
Chinese population, embryos, MTOR, recurrent spontaneous abortion, single nucleotide polymorphism History Received 28 March 2014 Revised 13 August 2014 Accepted 18 August 2014 Published online 2 April 2015
Abbreviations: RSA: recurrent spontaneous abortion; MTOR: mammalian target of rapamycin; SNP: single nucleotide polymorphism; qRT-PCR: quantitative real-time polymerase chain reaction; URSA: unexplained recurrent spontaneous abortion; mTORC1: mTOR complex 1; ESC: embryonic stem cells; HKE-293: human embryonic kidney 293 cells; HWE: Hardy-Weinberg equilibrium; ANOVA: one-way analysis of variance
Introduction Recurrent spontaneous abortion (RSA) refers to two or more consecutive failed clinical pregnancies prior to the 20th week of gestation [Pfeifer et al. 2013]. It has been reported that approximately 15% of clinical pregnancies end up with miscarriage and 0.4–2% of the diagnosed pregnancies suffered from RSA [McNamee et al. 2012]. Human pregnancy is strictly controlled by multiple regulatory mechanisms and any disturbance might lead to fetal loss [Knabl et al. 2013]. The pathogenesis of RSA is complicated. Several causes responsible for RSA have been identified, including parental
*Address correspondence to Xu Ma, Center for Genetics, National Research Institute for Family Planning, 12 Dahuisi Road, Haidian, Beijing 100081, China. Tel: 00861062179059. E-mail: [email protected] or to Yunxia Cao, Professor, Reproductive Medicine Center, The First Affiliated Hospital of Anhui Medical University, 218 Jixi Road, Hefei, Anhui 230022, China. Tel: 00865512922180. E-mail: caoyunxia6@ 126.com
chromosomal anomalies, anatomical abnormalities, endocrine abnormalities, immune dysfunction, thrombophilic disorders, antiphospholipid syndrome, and infections [Rai and Regan 2006]. However, these only account for 50% of the pathogenesis in patients with RSA [Plouffe et al. 1992]. The etiology for the remaining cases is not understood and these are referred to as unexplained recurrent spontaneous abortion (URSA) [Wilczynski et al. 2012]. The safety and efficacy of current therapies are inadequate for preventing RSA, partly due to the poor understanding of the complex pathogenesis and etiology of RSA. Therefore, further studies should be conducted. Research on the genetic architecture of pregnancy losses has provided a novel perspective which can be applied to identify genes involved in this disease and to investigate the molecular mechanisms of RSA. The expression of the tumor protein p53 (TP53) gene and the tumor necrosis factor (ligand) superfamily, member 10 (TNFSF10/TRAIL) gene in placenta are probably involved in the etiology of RSA by controlling the apoptotic signaling
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pathways [Rull et al. 2013b; Shang et al. 2013]. For example, polymorphisms in the histidine-rich glycoprotein (HRG) gene, androgen receptor (AR) gene, chorionic gonadotropin beta5 (CGB5) gene, tumor necrosis factor (TNF-alpha) gene, transforming growth factor, beta 1 (TGFB1) gene, and fas ligand (FAS-L) gene are significantly associated with the risk of RSA, suggesting that RSA has a solid genetic basis and that the pathogenesis is associated with various genes [Alkhuriji et al. 2013; Banzato et al. 2013; Jahaninejad et al. 2013; Lindgren et al. 2013; Magdoud et al. 2013; Rull et al. 2013a]. The mammalian target of rapamycin (MTOR), also known as FRAP1, is a member of the phosphatidylinostitol kinaserelated kinase (PIKK) superfamily which contains a lipid kinase homology domain and functions as a serine/threonine kinase [Laplante and Sabatini 2009]. It is conserved throughout evolution [Laplante and Sabatini 2009]. MTOR plays a critical role in cell growth, survival, proliferation, and angiogenesis [Jiang and Liu 2009]. It integrates extracellular signals (growth factors and steroid hormones) with intracellular amino acid availability and energy status to control translation rates and additional metabolic processes [Mparmpakas et al. 2010]. MTOR is known to encode two biochemically and functionally distinct protein complexes, mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2), which have distinct downstream targets and cellular effects. The mTORC1 complex is able to promote phosphorylation of p70 ribosomal s6 kinase (p70s6k) and Eif4E (4E-BP1) which are critical translation regulators and regulate cellular growth and proliferation [Krymskaya and Goncharova 2009]. A recent study demonstrated that mTORC1 plays a critical role in blastocyst activation and mouse embryo implantation [Gonzalez et al. 2012]. Previous studies have demonstrated that not only in early mouse embryos but also in embryonic stem cells (ESC), MTOR has a central effect on cell proliferation and growth. Gangloff and colleagues reported that MTOR(/) embryos were subject to embryonic developmental arrest at an earlier stage due to the failure of embryonic stem cells to establish and defect of inner cell mass proliferation [Gangloff et al. 2004]. Another study found that both cell size and proliferation were controlled by MTOR in embryonic stem cells and early mouse embryos by disrupting the kinase domain of mouse MTOR, and the homozygous mutant embryos died shortly after implantation because neither embryonic nor extraembryonic tissue proliferated properly [Murakami et al. 2004]. In addition to these observations, the MTOR gene also has an analogous function in human pregnancy. Previous in vitro experiments demonstrated that MTOR plays an important role in regulating the invasion of human trophoblast cells through serine phosphorylation of signal transducer and activator of transcription 3 (STAT3), suggesting that dysregulation of MTOR may be involved in pregnancy-related pathologies affecting trophoblast invasion [Busch et al. 2009]. Dysregulation of MTOR plays a critical role in the development of abnormal embryogenesis and implantation, and the human MTOR gene may be a good candidate risk factor responsible for the pathogenesis of RSA. In order to determine whether the MTOR gene is involved in RSA and promote further understanding of the etiology of RSA, our study analyzed the single nucleotide polymorphism (SNP)
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rs17027478 which causes a single nucleotide change from a C to an A in the promoter region of MTOR in a cohort of Chinese females.
Results Study population The genotype frequencies of MTOR rs17027478 polymorphism in both case and control groups were in Hardy-Weinberg equilibrium ( 2 ¼ 0.157, P ¼ 0.692; 2 ¼ 0.102, p ¼ 0.749). The genotype frequencies of rs17027478 polymorphism were 90.8%, 8.8%, and 0.3% for the CC, CA, and AA genotypes among the cases, and 94.5%, 5.5%, and 0 among the controls, respectively. The genotype frequencies showed no significant difference between cases and controls ( 2 ¼ 1.706, p ¼ 0.524). There was no significant difference in the frequencies of rs17027478 alleles between cases and controls. It was found that the MTOR rs17027478 A allele was present at a frequency of 4.7% in the case group, while the frequency was 2.8% in the control group ( 2 ¼ 1.771, p ¼ 0.183, OR ¼ 1.75, 95%CI: 0.76–4.06). It is worth mentioning that when the 84 RSA patients who had the history of three or more spontaneous abortions were compared with the controls, the results showed a significant difference. The frequency distribution of rs17027478A allele was significantly higher among cases than controls (7.74% versus 2.76%; 2 ¼ 5.56, p ¼ 0.018, OR ¼ 2.96, 95%CI: 1.16– 7.58). In addition, the genotype frequencies of CC, CA, and AA were 85.71%, 13.10%, and 1.19% among the 84 RSA patients, respectively. Fisher’s Exact Test analysis indicated that the frequency distributions of genotype between the cases and controls were significantly different ( 2 ¼ 5.177, p ¼ 0.043; p ¼ 0.029, OR ¼ 2.86, 95%CI: 1.08–7.59 by dominant mode). Expression and analysis of MTOR mRNA In the study, five heterozygous samples were selected from cases and controls, respectively. Quantitative real-time reverse transcription polymerase chain reaction was conducted to access the MTOR RNA levels in the blood of both groups. As shown in Figure 1, the level of MTOR in the cases was significantly lower than that in the controls (p ¼ 0.020). To examine whether the SNP rs17027478 polymorphism affected the expression of MTOR, two luciferase reporter constructs containing the MTOR promoter region with either the rs17047278 C or A allele were tested. The reporter plasmids were transfected into HEK293 cells. As shown in Figure 2, both wild-type and mutant-type MTOR promoter enhanced the activity of pGL3-Basic reporter, and the mutant-type showed remarkable loss of activity compared with the wildtype (p ¼ 0.029).
Discussion In this study, we demonstrated that the MTOR promoter region polymorphism rs17027478 was associated with the development of RSA among the females who experienced at least three spontaneous abortions. The in vitro experiments indicated that the C to A change in rs17027478 substantially altered the transcriptional activity of the MTOR gene, and the
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DOI: 10.3109/19396368.2014.977499
Figure 1. Expression levels of MTOR mRNA in RSA and control groups. MTOR expression level in blood of patients was significantly lower than the healthy females. The left bar of this figure shows the average MTOR expression level in cases, five heterozygous samples were used. The right bar of the figure shows the average MTOR expression level in control samples, also five heterozygous samples were included. The significance of difference was evaluated by DDCt analysis and student’s t test. *p50.05 versus wild-type.
Figure 2. Effect of the MTOR rs17027478 polymorphism on the expression of MTOR. We cotransfected human embryonic kidney 293 cells (HKE-293) with pGL3-Basic vector or pGL3- MTOR reporter construct (wild-type or mutant) together with Renilla luciferase plasmid. The pGL3- MTOR reporter construct contains either the rs17024748 C allele or the A allele. Cell extracts of HKE-293 cells were collected and firefly and Renilla luciferase activities were measured 48 h after transfection. Data represent mean values from at least three independent experiments. The significance of differences was evaluated by the oneway analysis of variance (ANOVA). *p50.05 versus wild-type.
reporter plasmid containing the A allele presented decreased transcriptional activity. In vivo analysis using the method of quantitative real-time reverse transcription polymerase chain reaction also suggested that the MTOR mRNA level was higher among healthy control women.
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Embryo implantation and pregnancy are complex physiological processes which depend on the regulation of numerous signaling proteins such as ERKs, phosphatidylinositol 3-kinase (PI3K), PTK2 or PI3K/AKT/mammalian target of rapamycin (mTOR) [Pollheimer and Knofler 2005]. MTOR is a protein kinase that plays a central role in cell growth, proliferation, differentiation, and apoptosis [Fingar and Blenis 2004]. In the cases of compromised nutrition (decreased available amino acids or sugars), growth factor withdrawal, or stress, decreased MTOR activity resulted in the reduction of cell proliferation and tissue growth, and the onset of autophagy [Wullschleger et al. 2006]. Under non-stressed conditions, MTOR phosphorylates p70S6 kinase (p70S6K) and eukaryotic translation initiation factor 4E-binding protein 1 (4E-BP1) to regulate translational control [Gingras et al. 2001]. Previous studies have demonstrated that the MTOR gene is essential for early mouse embryo growth and proliferation of embryonic stem cells [Chen et al. 2009; Gangloff et al. 2004; Martin and Sutherland 2001; Murakami, et al. 2004]. Additionally, Regnault and colleagues reported that MTOR can regulate the placenta leucine transport that is critical for the fetal growth [Regnault et al. 2007]. Decreased MTOR activity may affect decidualization of endometrial cells and further induce apoptosis in the mouse model [Chen, et al. 2009]. Our analysis suggested that the MTOR polymorphism rs17027478 has significant association with the development of RSA. But, this association only presented when controls were compared with the RSA patients who had a history of at least three spontaneous abortions. When the patients who experienced only two spontaneous abortions were included, the significant difference disappeared. The phenomenon indicated that RSA females experienced two spontaneous abortions that could be accidental with no genetic significance. There are several possible reasons for this result. First, it should be noted that the MTOR rs17027478 A allele displayed significantly reduced transcriptional activity compared with the C allele, therefore the A/C alternation may possibly have an effect on the expression level of MTOR. Thus, the more the women experienced spontaneous abortions, the more genetic significance it has. Secondly, the observed result may also be partly due to the very low mutation rate of the MTOR polymorphism rs17027478 (0.04; HapMap database, see http://hapmap.ncbi.nlm.nih.gov/). Thus, case-control studies based on a larger population and more functional studies are needed in order to better elucidate the relationship between MTOR rs17027478 A allele and susceptibility to RSA. In our study, we also observed that the level of MTOR mRNA was significantly lower in the blood of RSA patients than in healthy individuals consistent with association. Luciferase reporter assays in our investigation showed that MTOR rs17027478 A allele displayed significantly reduced transcriptional activity compared with the C allele. Thus, we speculate that the polymorphism was a functional SNP that affected the transcriptional regulation of MTOR. Additionally, the results of these functional analyses are in accord with previous studies which used mouse model and human trophoblast cell line [Busch et al. 2009; Chen et al. 2009; Gangloff et al. 2004; Murakami et al. 2004]. Taking these
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results together to consider the functional role of the rs17027478 in modulating the expression of MTOR, it is possible that this polymorphism could influence the individual susceptibility to the development of RSA in the Chinese population. However, there are some limitations in our investigation. First, our research only indicated that rs17027478 was significantly associated with the susceptibility to RSA, but whether the polymorphism will influence the phosphorylation level of MTOR and further alter its downstream signal is still unknown. Further investigations need to be conducted to elucidate specific regulatory mechanism. Second, we only analyzed the MTOR mRNA level in heterozygote individuals due to the low frequency of the A allele. The mRNA level of MTOR among all rs17027478 genotypes should be considered. In conclusion, the results presented in this study suggest that the promoter region of MTOR gene rs17027478 was significantly associated with the risk of RSA in the Chinese population. This polymorphism might be a good candidate responsible for the pathogenesis of RSA.
Materials and Methods Ethics statement The study was approved by the Ethics Review Committee on Family Planning of Anhui Medical University and complied with the 1975 Declaration of Helsinki. Written informed consent was obtained from all participants in this study. Study population A total of 306 unrelated Chinese patients diagnosed with RSA and 127 healthy unrelated Chinese females with at least one previous live birth were recruited in the case-control study from The First Affiliated Hospital of Anhui Medical University during the years 2010–2012. The cases and controls were included according to the criteria as follows. All of the females with RSA included were primary aborters with no previous history of live births. Furthermore, all RSA patients enrolled in the study underwent a routine examination to exclude the known risk factors for RSA, including chromosomal aberrations of partners and the embryos, anatomical abnormalities, thrombophilia, metabolic disorders, and positive lupus anticoagulant and anticardiolipin antibodies. As the control group, 127 healthy women with at least one previous live birth and no spontaneous abortion were included. The women enrolled in the control group had no pregnancy-associated complications.
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genomic DNA, 0.12 mL TaqMan SNP Genotyping Assays, 2.5 mL 2 TaqMan Genotyping Master Mix, and 1.88mL of double distilled water to form the 5 mL reaction mixture. The amplification conditions were as follows: 60 C, 30 sec; 95 C, 10 min; subsequently 95 C, 15 sec and 60 C, 1 min for 40 cycles. We selected 84 samples randomly for DNA sequencing to confirm the genotype. The results of DNA sequencing were 100% in accordance with TaqMan SNP Genotyping Assays. Analysis of MTOR mRNA expression Total RNA of fives heterozygous RSA patients and 5 heterozygous healthy controls was extracted from peripheral blood leukocytes using the TRIzol reagent (Invitrogen, Carlsbad, CA, USA). In order to avoid the traces of genomic DNA, the extracted RNA was DNase (Takara, Otsu, Japan) treated. After that, reverse transcription was conducted to synthesize cDNA using a SuperScriptÕ VILOÔ cDNA Synthesis Kit (Invitrogen). To measure the expression levels of MTOR mRNA, quantitative real-time polymerase chain reaction (qRT-PCR) was run on a 7000 real-time PCR system Õ (Applied Biosystems) using SYBR Premix Ex TaqÔ (Takara) and specially designed primers. The primers were 50 -GTGCCTCAGCCTCCCGAGTAT-30 (sense) and 50 -TCAG ACGGTGCCCAGGTCGTG-30 (antisense). A qRT-PCR was performed in the presence of SYBRÕ Green (Takara). RNA concentrations were normalized by GAPDH to calculate relative mRNA level of MTOR. Each sample was run in triplicate and three independent repeated experiments were conducted. Construction of promoter-reporter plasmids The 1098-bp MTOR promoter region sequence, containing either the rs17024748 C allele or the A allele, was amplified by PCR using specifically designed primers from human Genome DNA and inserted into the Sma I- and Bgl II(Takara) digested pGL3-Basic firefly luciferase vector (Promega, Madison, WI, USA) to generate the MTOR reporter construct pGL3-MTOR. The primers were 50 -TCCCCCG GGGGACTCGTGCCTCAGCCTCCCGAGTAT-30 (forward) and 50 -GAAGATCTTCTCAGACGGTGCCCAGGTCGTG-30 (reverse). PCR-mediated site-directed mutagenesis was conducted to generate a human MTOR promoter bearing the identified variations in the luciferase reporter constructs using the appropriate primers and the QuikChange II Site-Directed Mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA). The introduced variations were confirmed by DNA sequencing.
DNA extraction
Cell culture and transient transfection
Genomic DNA of each participant was obtained from peripheral blood leukocytes using the QIAamp DNA Blood Mini kit (QIAGEN, Hilden, Germany).
The human embryonic kidney 293 cells (HKE-293) were cultured in prepared maintenance medium of Iscove’s modified Dulbecco’s medium containing an additional 10% fetal bovine serum, 100 mg/ml streptomycin, and 100 mg/ml penicillin. HKE-293 cells were transfected using Lipofectamine 2000 (Invitrogen, New York, NY, USA). Transfections were performed in 48-well tissue culture plates seeded at 0.5 105cells/well 24 h prior to transfection
Genotype Predesigned TaqMan SNP Genotyping Assays (Applied Biosystems, Foster City, CA, USA) was applied to perform genotypic analysis of MTOR (rs17027478). We mixed 30 ng
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DOI: 10.3109/19396368.2014.977499
at 60 to 70% confluence. A total of 500 ng reporter construct pGL3-MTOR (wild-type or mutant) or 500 ng pGL3-Basic vector without insert was cotransfected together with 5 ng Renilla luciferase plasmid. After 48 h, cells were lysed with the lysis buffer (Promega) and assayed for luciferase activity using the Dual-Glo Luciferase Assay System (Promaga) according to the manufacturer’s instructions. The results were normalized against those from the Renilla internal control plasmid. Independent experiments were done in triplicate for each plasmid construct. Cells were washed in PBS and lysed in passive lysis buffer (Promega) 48 h after transfection. Luciferase activity was measured by the Dual-Glo Luciferase Assay System (Promega) following the manufacturer’s instruction, which normalized the transfection efficiency to paired Renilla luciferase activity. Transient transfection was conducted in triplicate for each plasmid construct and repeated at least three times. Statistical analysis SNP rs17027478 allele frequencies, genotype distribution, and Hardy-Weinberg equilibrium (HWE) were compared between RSA patients and healthy females by chi-square tests using the Statistic Package for the Social Science (SPSS) version 16.0 software. Kolmogorov-Smirnov test was used to determine whether the data collected from luciferase assays and qRT-PCR were normally distributed. Since all the data were normally distributed (p40.05), the statistical significance of unpaired samples in luciferase assays was determined by the one-way analysis of variance (ANOVA) and the expression levels of MTOR mRNA between cases and controls were tested by DDCt analysis and compared by Student’s t test.
Declaration of interest The authors report no declaration of interest.
Author contributions The authors’ contributions are as follows: Drafted the manuscript and carried out the molecular genetic studies: HX, SL; Functional analysis: CZ; Samples collection: ZL, YL; Conceived of the study and participated in its design and coordination: XM, YC. All authors approved the final manuscript.
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