Beyond Repetition: The Role of Gray Zone Alleles in the Upregulation of FMR1-Binding miR-323a-3p and the Modification of BMP/SMAD-Pathway Gene Expression in Human Granulosa Cells
Abstract
1. Introduction
2. Results
2.1. Patient Demographics
2.2. FMR1 Expression Is Independent of Gray Zone Allele Presence
2.3. miR-323a-3p Expression Significantly Increased in GCs of the Gray Zone Group
2.4. FMR1 Levels Were Downregulated in Poor Ovarian Responders
2.5. miR-323a-3p Overexpression Decreased SMAD Levels in COV434 Cells
2.6. BMPR2 and SMADs Were Upregulated in GCs of the Gray Zone Group
3. Discussion
4. Materials and Methods
4.1. Ethics Approval
4.2. Study Population
4.3. Ovarian Stimulation and GC Collection
4.4. Cell Culture
4.5. RNA Extraction and Gene Expression Analysis
4.6. CGG Repeat Length Analysis (Adapted from [103])
4.7. Mimic Treatment
4.8. Statistical Analyses
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
- Pisarska, M.D.; Chan, J.L.; Lawrenson, K.; Gonzalez, T.L.; Wang, E.T. Genetics and Epigenetics of Infertility and Treatments on Outcomes. J. Clin. Endocrinol. Metab. 2019, 104, 1871–1886. [Google Scholar] [CrossRef]
- Webber, L.; Davies, M.; Anderson, R.; Bartlett, J.; Braat, D.; Cartwright, B.; Cifkova, R.; de Muinck Keizer-Schrama, S.; Hogervorst, E.; Janse, F.; et al. ESHRE Guideline: Management of women with premature ovarian insufficiency. Hum. Reprod. 2016, 31, 926–937. [Google Scholar] [CrossRef]
- Tarlatzis, B.C.; Zepiridis, L.; Grimbizis, G.; Bontis, J. Clinical management of low ovarian response to stimulation for IVF: A systematic review. Hum. Reprod. Update 2003, 9, 61–76. [Google Scholar] [CrossRef]
- Sullivan, A.K.; Marcus, M.; Epstein, M.P.; Allen, E.G.; Anido, A.E.; Paquin, J.J.; Yadav-Shah, M.; Sherman, S.L. Association of FMR1 repeat size with ovarian dysfunction. Hum. Reprod. 2005, 20, 402–412. [Google Scholar] [CrossRef]
- Rehnitz, J.; Alcoba, D.D.; Brum, I.S.; Dietrich, J.E.; Youness, B.; Hinderhofer, K.; Messmer, B.; Freis, A.; Strowitzki, T.; Germeyer, A. FMR1 expression in human granulosa cells increases with exon 1 CGG repeat length depending on ovarian reserve. Reprod. Biol. Endocrinol. 2018, 16. [Google Scholar] [CrossRef]
- Rosario, R.; Anderson, R. The molecular mechanisms that underlie fragile X-associated premature ovarian insufficiency: Is it RNA or protein based? Mol. Hum. Reprod. 2020, 26, 727–737. [Google Scholar] [CrossRef]
- Straub, D.; Schmitt, L.M.; Boggs, A.E.; Horn, P.S.; Dominick, K.C.; Gross, C.; Erickson, C.A. A sensitive and reproducible qRT-PCR assay detects physiological relevant trace levels of FMR1 mRNA in individuals with Fragile X syndrome. Sci. Rep. 2023, 13, 3808. [Google Scholar] [CrossRef]
- Lyons, J.I.; Kerr, G.R.; Mueller, P.W. Fragile X Syndrome: Scientific Background and Screening Technologies. J. Mol. Diagn. 2015, 17, 463–471. [Google Scholar] [CrossRef]
- Hall, D.A. In the Gray Zone in the Fragile X Gene: What are the Key Unanswered Clinical and Biological Questions? Tremor Other Hyperkinet Mov. 2014, 4, 208. [Google Scholar] [CrossRef]
- Hagerman, R.; Hagerman, P. Advances in clinical and molecular understanding of the FMR1 premutation and fragile X-associated tremor/ataxia syndrome. Lancet Neurol. 2013, 12, 786–798. [Google Scholar] [CrossRef] [PubMed]
- Nolin, S.L.; Glicksman, A.; Tortora, N.; Allen, E.; Macpherson, J.; Mila, M.; Vianna-Morgante, A.M.; Sherman, S.L.; Dobkin, C.; Latham, G.J.; et al. Expansions and contractions of the FMR1 CGG repeat in 5,508 transmissions of normal, intermediate, and premutation alleles. Am. J. Med. Genet. A 2019, 179, 1148–1156. [Google Scholar] [CrossRef]
- Loesch, D.Z.; Bui, Q.M.; Huggins, R.M.; Mitchell, R.J.; Hagerman, R.J.; Tassone, F. Transcript levels of the intermediate size or grey zone fragile X mental retardation 1 alleles are raised, and correlate with the number of CGG repeats. J. Med. Genet. 2007, 44, 200–204. [Google Scholar] [CrossRef]
- Bretherick, K.L.; Fluker, M.R.; Robinson, W.P. FMR1 repeat sizes in the gray zone and high end of the normal range are associated with premature ovarian failure. Hum. Genet. 2005, 117, 376–382. [Google Scholar] [CrossRef]
- Pastore, L.M.; Johnson, J. The FMR1 gene, infertility, and reproductive decision-making: A review. Front. Genet. 2014, 5, 195. [Google Scholar] [CrossRef]
- Bodega, B.; Bione, S.; Dalprà, L.; Toniolo, D.; Ornaghi, F.; Vegetti, W.; Ginelli, E.; Marozzi, A. Influence of intermediate and uninterrupted FMR1 CGG expansions in premature ovarian failure manifestation. Hum. Reprod. 2005, 21, 952–957. [Google Scholar] [CrossRef]
- Garber, K.B.; Visootsak, J.; Warren, S.T. Fragile X syndrome. Eur. J. Hum. Genet. 2008, 16, 666–672. [Google Scholar] [CrossRef]
- Gireud, M.; Sirisaengtaksin, N.; Bean, A.J. Chapter 21—Molecular Mechanisms of Neurological Disease. In From Molecules to Networks, 3rd ed.; Byrne, J.H., Heidelberger, R., Waxham, M.N., Eds.; Academic Press: Boston, MA, USA, 2014; pp. 639–661. [Google Scholar]
- Schuettler, J.; Peng, Z.; Zimmer, J.; Sinn, P.; Von Hagens, C.; Strowitzki, T.; Vogt, P.H. Variable expression of the Fragile X Mental Retardation 1 (FMR1) gene in patients with premature ovarian failure syndrome is not dependent on number of (CGG)n triplets in exon 1. Hum. Reprod. 2011, 26, 1241–1251. [Google Scholar] [CrossRef]
- Willemsen, R.; Levenga, J.; Oostra, B. CGG repeat in the FMR1 gene: Size matters. Clin. Genet. 2011, 80, 214–225. [Google Scholar] [CrossRef]
- Yang, L.; Duan, R.; Chen, D.; Wang, J.; Chen, D.; Jin, P. Fragile X mental retardation protein modulates the fate of germline stem cells in Drosophila. Hum. Mol. Genet. 2007, 16, 1814–1820. [Google Scholar] [CrossRef]
- Costa, A.; Wang, Y.; Dockendorff, T.C.; Erdjument-Bromage, H.; Tempst, P.; Schedl, P.; Jongens, T.A. The Drosophila Fragile X Protein Functions as a Negative Regulator in the orb Autoregulatory Pathway. Dev. Cell 2005, 8, 331–342. [Google Scholar] [CrossRef]
- Epstein, A.M.; Bauer, C.R.; Ho, A.; Bosco, G.; Zarnescu, D.C. Drosophila Fragile X Protein controls cellular proliferation by regulating cbl levels in the ovary. Dev. Biol. 2009, 330, 83–92. [Google Scholar]
- Megosh, H.B.; Cox, D.N.; Campbell, C.; Lin, H. The Role of PIWI and the miRNA Machinery in Drosophila Germline Determination. Curr. Biol. 2006, 16, 1884–1894. [Google Scholar]
- Fitzgerald, J.B.; George, J.; Christenson, L.K. Non-Coding RNA in Ovarian Development and Disease; Springer: Dordrecht, The Netherlands, 2016; pp. 79–93. [Google Scholar]
- Lee, R.C.; Feinbaum, R.L.; Ambros, V. The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14. Cell 1993, 75, 843–854. [Google Scholar]
- Makeyev, E.V.; Maniatis, T. Multilevel regulation of gene expression by microRNAs. Science 2008, 319, 1789–1790. [Google Scholar]
- Hong, X.; Luense, L.J.; McGinnis, L.K.; Nothnick, W.B.; Christenson, L.K. Dicer1 is essential for female fertility and normal development of the female reproductive system. Endocrinology 2008, 149, 6207–6212. [Google Scholar]
- Gonzalez, G.; Behringer, R.R. Dicer is required for female reproductive tract development and fertility in the mouse. Mol. Reprod. Dev. 2009, 76, 678–688. [Google Scholar]
- Nagaraja, A.K.; Andreu-Vieyra, C.; Franco, H.L.; Ma, L.; Chen, R.; Han, D.Y.; Zhu, H.; Agno, J.E.; Gunaratne, P.H.; DeMayo, F.J.; et al. Deletion of Dicer in Somatic Cells of the Female Reproductive Tract Causes Sterility. Mol. Endocrinol. 2008, 22, 2336–2352. [Google Scholar]
- Tu, J.; Cheung, A.H.; Chan, C.L.; Chan, W.Y. The Role of microRNAs in Ovarian Granulosa Cells in Health and Disease. Front. Endocrinol. 2019, 10, 174. [Google Scholar]
- Jin, P.; Alisch, R.S.; Warren, S.T. RNA and microRNAs in fragile X mental retardation. Nat. Cell Biol. 2004, 6, 1048–1053. [Google Scholar] [PubMed]
- Gong, X.; Zhang, K.; Wang, Y.; Wang, J.; Cui, Y.; Li, S.; Luo, Y. MicroRNA-130b targets Fmr1 and regulates embryonic neural progenitor cell proliferation and differentiation. Biochem. Biophys. Res. Commun. 2013, 439, 493–500. [Google Scholar]
- Lin, S.L. microRNAs and Fragile X Syndrome. Adv. Exp. Med. Biol. 2015, 888, 107–121. [Google Scholar]
- Men, Y.; Zhai, Y.; Wu, L.; Liu, L.; Zhang, W.; Jiang, W.; Bi, N.; Song, Y.; Hui, Z.; Wang, L. MiR-323a-3p acts as a tumor suppressor by suppressing FMR1 and predicts better esophageal squamous cell carcinoma outcome. Cancer Cell Int. 2022, 22, 140. [Google Scholar] [PubMed]
- Yi, Y.H.; Sun, X.S.; Qin, J.M.; Zhao, Q.H.; Liao, W.P.; Long, Y.S. Experimental identification of microRNA targets on the 3′ untranslated region of human FMR1 gene. J. Neurosci. Methods 2010, 190, 34–38. [Google Scholar] [CrossRef]
- Li, J.; Xu, X.; Meng, S.; Liang, Z.; Wang, X.; Xu, M.; Wang, S.; Li, S.; Zhu, Y.; Xie, B.; et al. MET/SMAD3/SNAIL circuit mediated by miR-323a-3p is involved in regulating epithelial–mesenchymal transition progression in bladder cancer. Cell Death Dis. 2017, 8, e3010. [Google Scholar] [PubMed]
- Chen, H.; Gao, S.; Cheng, C. MiR-323a-3p suppressed the glycolysis of osteosarcoma via targeting LDHA. Hum. Cell 2018, 31, 300–309. [Google Scholar] [CrossRef] [PubMed]
- Blank, U.; Karlsson, S. The role of Smad signaling in hematopoiesis and translational hematology. Leukemia 2011, 25, 1379–1388. [Google Scholar] [CrossRef]
- Patiño, L.C.; Silgado, D.; Laissue, P. A potential functional association between mutant BMPR2 and primary ovarian insufficiency. Syst. Biol. Reprod. Med. 2017, 63, 145–149. [Google Scholar] [CrossRef]
- Kaivo-Oja, N.; Bondestam, J.; Kämäräinen, M.; Koskimies, J.; Vitt, U.; Cranfield, M.; Vuojolainen, K.; Kallio, J.P.; Olkkonen, V.M.; Hayashi, M.; et al. Growth Differentiation Factor-9 Induces Smad2 Activation and Inhibin B Production in Cultured Human Granulosa-Luteal Cells. J. Clin. Endocrinol. Metab. 2003, 88, 755–762. [Google Scholar] [CrossRef]
- Li, Q.; Pangas, S.A.; Jorgez, C.J.; Graff, J.M.; Weinstein, M.; Matzuk, M.M. Redundant roles of SMAD2 and SMAD3 in ovarian granulosa cells in vivo. Mol. Cell Biol. 2008, 28, 7001–7011. [Google Scholar] [CrossRef]
- Kaivo-oja, N.; Jeffery, L.A.; Ritvos, O.; Mottershead, D.G. Smad signalling in the ovary. Reprod. Biol. Endocrinol. 2006, 4, 21. [Google Scholar] [CrossRef]
- Nguyen, X.P.; Vilkaite, A.; Bender, U.; Dietrich, J.E.; Hinderhofer, K.; Strowitzki, T.; Rehnitz, J. Regulation of Bone Morphogenetic Protein Receptor Type II Expression by FMR1/Fragile X Mental Retardation Protein in Human Granulosa Cells in the Context of Poor Ovarian Response. Int. J. Mol. Sci. 2024, 25, 10643. [Google Scholar] [CrossRef] [PubMed]
- Massagué, J.; Blain, S.W.; Lo, R.S. TGFβ Signaling in Growth Control, Cancer, and Heritable Disorders. Cell 2000, 103, 295–309. [Google Scholar] [PubMed]
- Bertoldo, M.J.; Cheung, M.Y.; Sia, Z.K.; Agapiou, D.; Corley, S.M.; Wilkins, M.R.; Richani, D.; Harrison, C.A.; Gilchrist, R.B. Non-canonical cyclic AMP SMAD1/5/8 signalling in human granulosa cells. Mol. Cell. Endocrinol. 2019, 490, 37–46. [Google Scholar]
- Moore, R.K.; Otsuka, F.; Shimasaki, S. Molecular basis of bone morphogenetic protein-15 signaling in granulosa cells. J. Biol. Chem. 2003, 278, 304–310. [Google Scholar]
- Otsuka, F.; Moore, R.K.; Shimasaki, S. Biological Function and Cellular Mechanism of Bone Morphogenetic Protein-6 in the Ovary*. J. Biol. Chem. 2001, 276, 32889–32895. [Google Scholar] [PubMed]
- Yao, W.; Wang, S.; Du, X.; Lin, C.; Zhang, J.; Pan, Z.; Li, Q. SMAD4 Inhibits Granulosa Cell Apoptosis via the miR-183-96-182 Cluster and FoxO1 Axis. Reprod. Sci. 2022, 29, 1577–1585. [Google Scholar] [CrossRef]
- Yu, M.; Liu, J. MicroRNA-30d-5p promotes ovarian granulosa cell apoptosis by targeting Smad2. Exp. Ther. Med. 2020, 19, 53–60. [Google Scholar] [CrossRef]
- Li, Y.; Xiang, Y.; Song, Y.; Wan, L.; Yu, G.; Tan, L. Dysregulated miR-142, -33b and -423 in granulosa cells target TGFBR1 and SMAD7: A possible role in polycystic ovary syndrome. Mol. Hum. Reprod. 2019, 25, 638–646. [Google Scholar]
- Ferraretti, A.; La Marca, A.; Fauser BC, J.M.; Tarlatzis, B.; Nargund, G.; Gianaroli, L.; ESHRE working group on Poor Ovarian Response Definition. ESHRE consensus on the definition of ‘poor response’ to ovarian stimulation for in vitro fertilization: The Bologna criteria. Hum. Reprod. 2011, 26, 1616–1624. [Google Scholar] [CrossRef]
- Johnson, A.L. Ovarian follicle selection and granulosa cell differentiation. Poult. Sci. 2015, 94, 781–785. [Google Scholar]
- Hsueh, A.J.W.; Kawamura, K.; Cheng, Y.; Fauser, B.C.J.M. Intraovarian Control of Early Folliculogenesis. Endocr. Rev. 2015, 36, 1–24. [Google Scholar]
- Pastore, L.M.; McMurry, T.L.; Williams, C.D.; Baker, V.L.; Young, S.L. AMH in women with diminished ovarian reserve: Potential differences by FMR1 CGG repeat level. J. Assist. Reprod. Genet. 2014, 31, 1295–1301. [Google Scholar] [PubMed]
- Lledo, B.; Guerrero, J.; Ortiz, J.A.; Morales, R.; Ten, J.; Llacer, J.; Gimenez, J.; Bernabeu, R. Intermediate and normal sized CGG repeat on the FMR1 gene does not negatively affect donor ovarian response. Hum. Reprod. 2011, 27, 609–614. [Google Scholar]
- Grynnerup, A.G.; Lindhard, A.; Sørensen, S. The role of anti-Müllerian hormone in female fertility and infertility—An overview. Acta Obstet. Gynecol. Scand. 2012, 91, 1252–1260. [Google Scholar] [PubMed]
- Maalouf, S.W.; Liu, W.S.; Pate, J.L. MicroRNA in ovarian function. Cell Tissue Res. 2016, 363, 7–18. [Google Scholar] [PubMed]
- Guo, Y.; Sun, J.; Lai, D. Role of microRNAs in premature ovarian insufficiency. Reprod. Biol. Endocrinol. 2017, 15, 38. [Google Scholar]
- Zhang, J.; Xu, Y.; Liu, H.; Pan, Z. MicroRNAs in ovarian follicular atresia and granulosa cell apoptosis. Reprod. Biol. Endocrinol. 2019, 17, 9. [Google Scholar]
- Qin, J.; Sun, Y.; Liu, S.; Zhao, R.; Zhang, Q.; Pang, W. MicroRNA-323-3p promotes myogenesis by targeting Smad2. J. Cell Biochem. 2019, 120, 18751–18761. [Google Scholar]
- Xu, T.; Li, L.; Huang, C.; Li, X.; Peng, Y.; Li, J. MicroRNA-323-3p with clinical potential in rheumatoid arthritis, Alzheimer’s disease and ectopic pregnancy. Expert. Opin. Ther. Targets 2014, 18, 153–158. [Google Scholar] [CrossRef]
- Yang, H.A.; Wang, X.; Ding, F.; Pang, Q. MiRNA-323-5p Promotes U373 Cell Apoptosis by Reducing IGF-1R. Med. Sci. Monit. 2015, 21, 3880–3886. [Google Scholar]
- Yang, L.; Xiong, Y.; Hu, X.F.; Du, Y.H. MicroRNA-323 regulates ischemia/reperfusion injury-induced neuronal cell death by targeting BRI3. Int. J. Clin. Exp. Pathol. 2015, 8, 10725–10733. [Google Scholar] [PubMed]
- Zhao, Y.; Tao, M.; Wei, M.; Du, S.; Wang, H.; Wang, X. Mesenchymal stem cells derived exosomal miR-323-3p promotes proliferation and inhibits apoptosis of cumulus cells in polycystic ovary syndrome (PCOS). Artif. Cells Nanomed. Biotechnol. 2019, 47, 3804–3813. [Google Scholar] [CrossRef]
- Wang, T.; Liu, Y.; Lv, M.; Xing, Q.; Zhang, Z.; He, X.; Xu, Y.; Wei, Z.; Cao, Y. miR-323-3p regulates the steroidogenesis and cell apoptosis in polycystic ovary syndrome (PCOS) by targeting IGF-1. Gene 2019, 683, 87–100. [Google Scholar]
- Rodriguez, C.M.; Wright, S.E.; Kearse, M.G.; Haenfler, J.M.; Flores, B.N.; Liu, Y.; Ifrim, M.F.; Glineburg, M.R.; Krans, A.; Jafar-Nejad, P.; et al. A native function for RAN translation and CGG repeats in regulating fragile X protein synthesis. Nat. Neurosci. 2020, 23, 386–397. [Google Scholar] [PubMed]
- Hagerman, R.J.; Berry-Kravis, E.; Hazlett, H.C.; Bailey, D.B., Jr.; Moine, H.; Kooy, R.F.; Tassone, F.; Gantois, I.; Sonenberg, N.; Mandel, J.L.; et al. Fragile X syndrome. Nat. Rev. Dis. Primers 2017, 3, 17065. [Google Scholar] [CrossRef] [PubMed]
- Ascano, M., Jr.; Mukherjee, N.; Bandaru, P.; Miller, J.B.; Nusbaum, J.D.; Corcoran, D.L.; Langlois, C.; Munschauer, M.; Dewell, S.; Hafner, M.; et al. FMRP targets distinct mRNA sequence elements to regulate protein expression. Nature 2012, 492, 382–386. [Google Scholar]
- Hoffman, G.E.; Le, W.W.; Entezam, A.; Otsuka, N.; Tong, Z.B.; Nelson, L.; Flaws, J.A.; McDonald, J.H.; Jafar, S.; Usdin, K. Ovarian abnormalities in a mouse model of fragile X primary ovarian insufficiency. J. Histochem. Cytochem. 2012, 60, 439–456. [Google Scholar]
- Rehnitz, J.; Youness, B.; Nguyen, X.P.; Dietrich, J.E.; Roesner, S.; Messmer, B.; Strowitzki, T.; Vogt, P.H. FMR1 expression in human granulosa cells and variable ovarian response: Control by epigenetic mechanisms. Mol. Hum. Reprod. 2021, 27. [Google Scholar]
- Rehnitz, J.; Alcoba, D.D.; Brum, I.S.; Hinderhofer, K.; Youness, B.; Strowitzki, T.; Vogt, P.H. FMR1 and AKT/mTOR signalling pathways: Potential functional interactions controlling folliculogenesis in human granulosa cells. Reprod. BioMedicine Online 2017, 35, 485–493. [Google Scholar]
- Boustanai, I.; Raanani, H.; Aizer, A.; Orvieto, R.; Elizur, S.E. Granulosa Cell Dysfunction Is Associated With Diminished Ovarian Response in FMR1 Premutation Carriers. J. Clin. Endocrinol. Metab. 2022, 107, 3000–3009. [Google Scholar] [CrossRef]
- Treiber, T.; Treiber, N.; Meister, G. Regulation of microRNA biogenesis and its crosstalk with other cellular pathways. Nat. Rev. Mol. Cell Biol. 2019, 20, 5–20. [Google Scholar] [PubMed]
- Wang, C.; Liu, P.; Wu, H.; Cui, P.; Li, Y.; Liu, Y.; Liu, Z.; Gou, S. MicroRNA-323-3p inhibits cell invasion and metastasis in pancreatic ductal adenocarcinoma via direct suppression of SMAD2 and SMAD3. Oncotarget 2016, 7, 14912–14924. [Google Scholar] [PubMed]
- Che, F.; Du, H.; Wei, J.; Zhang, W.; Cheng, Z.; Tong, Y. MicroRNA-323 suppresses nerve cell toxicity in cerebral infarction via the transforming growth factor-β1/SMAD3 signaling pathway. Int. J. Mol. Med. 2019, 43, 993–1002. [Google Scholar]
- Schmierer, B.; Hill, C.S. TGFbeta-SMAD signal transduction: Molecular specificity and functional flexibility. Nat. Rev. Mol. Cell Biol. 2007, 8, 970–982. [Google Scholar]
- Knight, P.G.; Glister, C. TGF-beta superfamily members and ovarian follicle development. Reproduction 2006, 132, 191–206. [Google Scholar] [CrossRef]
- Kristensen, S.G.; Andersen, K.; Clement, C.A.; Franks, S.; Hardy, K.; Andersen, C.Y. Expression of TGF-beta superfamily growth factors, their receptors, the associated SMADs and antagonists in five isolated size-matched populations of pre-antral follicles from normal human ovaries. Mol. Hum. Reprod. 2014, 20, 293–308. [Google Scholar]
- Rosairo, D.; Kuyznierewicz, I.; Findlay, J.; Drummond, A. Transforming growth factor-beta: Its role in ovarian follicle development. Reproduction 2008, 136, 799–809. [Google Scholar] [PubMed]
- Vasudevan, S.; Tong, Y.; Steitz, J.A. Switching from Repression to Activation: MicroRNAs Can Up-Regulate Translation. Science 2007, 318, 1931–1934. [Google Scholar]
- Xu, X.H.; Song, W.; Li, J.H.; Huang, Z.Q.; Liu, Y.F.; Bao, Q.; Shen, Z.W. Long Non-coding RNA EBLN3P Regulates UHMK1 Expression by Sponging miR-323a-3p and Promotes Colorectal Cancer Progression. Front. Med. 2021, 8, 651600. [Google Scholar]
- Bhavsar, S.P.; Olsen, L.; Løkke, C.; Koster, J.; Flægstad, T.; Einvik, C. Hsa-miR-323a-3p functions as a tumor suppressor and targets STAT3 in neuroblastoma cells. Front. Pediatr. 2023, 11, 1098999. [Google Scholar]
- Koussounadis, A.; Langdon, S.P.; Um, I.H.; Harrison, D.J.; Smith, V.A. Relationship between differentially expressed mRNA and mRNA-protein correlations in a xenograft model system. Sci. Rep. 2015, 5, 10775. [Google Scholar] [CrossRef]
- de Sousa Abreu, R.; Penalva, L.O.; Marcotte, E.M.; Vogel, C. Global signatures of protein and mRNA expression levels. Mol. Biosyst. 2009, 5, 1512–1526. [Google Scholar] [CrossRef] [PubMed]
- Schneider, A.; Winarni, T.I.; Cabal-Herrera, A.M.; Bacalman, S.; Gane, L.; Hagerman, P.; Tassone, F.; Hagerman, R. Elevated FMR1-mRNA and lowered FMRP—A double-hit mechanism for psychiatric features in men with FMR1 premutations. Transl. Psychiatry 2020, 10, 205. [Google Scholar] [CrossRef] [PubMed]
- Xu, P.; Lin, X.; Feng, X.H. Posttranslational Regulation of Smads. Cold Spring Harb. Perspect. Biol. 2016, 8, a022087. [Google Scholar] [CrossRef] [PubMed]
- Zumwalt, M.; Ludwig, A.; Hagerman, P.J.; Dieckmann, T. Secondary Structure and Dynamics of the r(CGG) Repeat in the mRNA of the Fragile X Mental Retardation 1 (FMR1) Gene. RNA Biol. 2007, 4, 93–100. [Google Scholar] [CrossRef]
- Handa, V.; Goldwater, D.; Stiles, D.; Cam, M.; Poy, G.; Kumari, D.; Usdin, K. Long CGG-repeat tracts are toxic to human cells: Implications for carriers of Fragile X premutation alleles. FEBS Lett. 2005, 579, 2702–2708. [Google Scholar] [CrossRef]
- Handa, V.; Saha, T.; Usdin, K. The fragile X syndrome repeats form RNA hairpins that do not activate the interferon-inducible protein kinase, PKR, but are cut by Dicer. Nucleic Acids Res. 2003, 31, 6243–6248. [Google Scholar] [CrossRef]
- Hall, D.A.; Nag, S.; Ouyang, B.; Bennett, D.A.; Liu, Y.; Ali, A.; Zhou, L.; Berry-Kravis, E. Fragile X Gray Zone Alleles Are Associated with Signs of Parkinsonism and Earlier Death. Mov. Disord. 2020, 35, 1448–1456. [Google Scholar] [CrossRef]
- Teng, Y.; Zhu, M.; Qiu, Z. G-Quadruplexes in Repeat Expansion Disorders. Int. J. Mol. Sci. 2023, 24, 2375. [Google Scholar] [CrossRef]
- Georgakopoulos-Soares, I.; Chan, C.S.Y.; Ahituv, N.; Hemberg, M. High-throughput techniques enable advances in the roles of DNA and RNA secondary structures in transcriptional and post-transcriptional gene regulation. Genome Biol. 2022, 23, 159. [Google Scholar]
- Rouleau, S.; Glouzon, J.S.; Brumwell, A.; Bisaillon, M.; Perreault, J.P. 3′ UTR G-quadruplexes regulate miRNA binding. Rna 2017, 23, 1172–1179. [Google Scholar] [PubMed]
- Corgiat, E.B.; List, S.M.; Rounds, J.C.; Yu, D.; Chen, P.; Corbett, A.H.; Moberg, K.H. The Nab2 RNA-binding protein patterns dendritic and axonal projections through a planar cell polarity-sensitive mechanism. G3 2022, 12. [Google Scholar]
- Fabian, M.R.; Sonenberg, N.; Filipowicz, W. Regulation of mRNA translation and stability by microRNAs. Annu. Rev. Biochem. 2010, 79, 351–379. [Google Scholar]
- Place, R.F.; Li, L.-C.; Pookot, D.; Noonan, E.J.; Dahiya, R. MicroRNA-373 induces expression of genes with complementary promoter sequences. Proc. Natl. Acad. Sci. USA 2008, 105, 1608–1613. [Google Scholar] [CrossRef] [PubMed]
- Huang, V.; Zheng, J.; Qi, Z.; Wang, J.; Place, R.F.; Yu, J.; Li, H.; Li, L.-C. Ago1 Interacts with RNA Polymerase II and Binds to the Promoters of Actively Transcribed Genes in Human Cancer Cells. PLOS Genet. 2013, 9, e1003821. [Google Scholar]
- Rehnitz, J.; Capp, E.; Messmer, B.; Nguyen, X.P.; Germeyer, A.; Freis, A.; Dietrich, J.E.; Hinderhofer, K.; Strowitzki, T.; Vogt, P.H. FMR1 and AKT/mTOR Signaling in Human Granulosa Cells: Functional Interaction and Impact on Ovarian Response. J. Clin. Med. 2021, 10, 3892. [Google Scholar] [CrossRef]
- Zhang, H.; Vollmer, M.; De Geyter, M.; Litzistorf, Y.; Ladewig, A.; Dürrenberger, M.; Guggenheim, R.; Miny, P.; Holzgreve, W.; De Geyter, C. Characterization of an immortalized human granulosa cell line (COV434). Mol. Hum. Reprod. 2000, 6, 146–153. [Google Scholar]
- Rooda, I.; Hasan, M.M.; Roos, K.; Viil, J.; Andronowska, A.; Smolander, O.P.; Jaakma, Ü.; Salumets, A.; Fazeli, A.; Velthut-Meikas, A. Cellular, Extracellular and Extracellular Vesicular miRNA Profiles of Pre-Ovulatory Follicles Indicate Signaling Disturbances in Polycystic Ovaries. Int. J. Mol. Sci. 2020, 21, 9550. [Google Scholar] [CrossRef] [PubMed]
- Scalici, E.; Traver, S.; Mullet, T.; Molinari, N.; Ferrières, A.; Brunet, C.; Belloc, S.; Hamamah, S. Circulating microRNAs in follicular fluid, powerful tools to explore in vitro fertilization process. Sci. Rep. 2016, 6, 24976. [Google Scholar]
- Livak, K.J.; Schmittgen, T.D. Analysis of Relative Gene Expression Data Using Real-Time Quantitative PCR and the 2−ΔΔCT Method. Methods 2001, 25, 402–408. [Google Scholar]
- Rehnitz, J.; Messmer, B.; Bender, U.; Nguyen, X.P.; Germeyer, A.; Hinderhofer, K.; Strowitzki, T.; Capp, E. Activation of AKT/mammalian target of rapamycin signaling in the peripheral blood of women with premature ovarian insufficiency and its correlation with FMR1 expression. Reprod. Biol. Endocrinol. 2022, 20, 44. [Google Scholar] [CrossRef] [PubMed]
- Zhang, J.; Lang, Y.; Guo, L.; Pei, Y.; Hao, S.; Liang, Z.; Su, G.; Shu, L.; Liu, H.; Huang, C.; et al. MicroRNA-323a-3p Promotes Pressure Overload-Induced Cardiac Fibrosis by Targeting TIMP3. Cell Physiol. Biochem. 2018, 50, 2176–2187. [Google Scholar] [CrossRef] [PubMed]
Demographics | Normal Genotype | Gray Zone Genotype | p-Value | ||
---|---|---|---|---|---|
n | Mean (±SD) | n | Mean (±SD) | ||
Age | 55 | 34.47 (±4.91) | 28 | 35.07 (±4.88) | 0.6467 |
BMI | 54 | 23.93 (±4.63) | 28 | 24.24 (±4.63) | 0.7736 |
FSH, U/L | 48 | 9.07 (±3.68) | 24 | 8.133 (±2.34) | 0.2592 |
LH, U/L | 52 | 5.39 (±2.31) | 27 | 6.367 (±6.37) | 0.1592 |
Estradiol, pg/nL | 51 | 52.26 (±27.64) | 26 | 59.03 (±58.27) | 0.4905 |
AMH, ng/nL | 51 | 2.10 (±2.03) | 28 | 3.095 (±4.02) | 0.1449 |
AFC | 35 | 12.29 (±12.81) | 18 | 10.22 (±9.25) | 0.5475 |
Oocytes retrieved | 55 | 7.77 (±5.74) | 28 | 8.25 (±8.01) | 0.7245 |
Mature (MII) oocytes | 37 | 5.57 (±4.69) | 17 | 6.588 (±5.82) | 0.4949 |
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content. |
© 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Share and Cite
Vilkaite, A.; Nguyen, X.P.; Güzel, C.T.; Gottschlich, L.; Bender, U.; Dietrich, J.E.; Hinderhofer, K.; Strowitzki, T.; Rehnitz, J. Beyond Repetition: The Role of Gray Zone Alleles in the Upregulation of FMR1-Binding miR-323a-3p and the Modification of BMP/SMAD-Pathway Gene Expression in Human Granulosa Cells. Int. J. Mol. Sci. 2025, 26, 3192. https://doi.org/10.3390/ijms26073192
Vilkaite A, Nguyen XP, Güzel CT, Gottschlich L, Bender U, Dietrich JE, Hinderhofer K, Strowitzki T, Rehnitz J. Beyond Repetition: The Role of Gray Zone Alleles in the Upregulation of FMR1-Binding miR-323a-3p and the Modification of BMP/SMAD-Pathway Gene Expression in Human Granulosa Cells. International Journal of Molecular Sciences. 2025; 26(7):3192. https://doi.org/10.3390/ijms26073192
Chicago/Turabian StyleVilkaite, Adriana, Xuan Phuoc Nguyen, Cansu Türkan Güzel, Lucas Gottschlich, Ulrike Bender, Jens E. Dietrich, Katrin Hinderhofer, Thomas Strowitzki, and Julia Rehnitz. 2025. "Beyond Repetition: The Role of Gray Zone Alleles in the Upregulation of FMR1-Binding miR-323a-3p and the Modification of BMP/SMAD-Pathway Gene Expression in Human Granulosa Cells" International Journal of Molecular Sciences 26, no. 7: 3192. https://doi.org/10.3390/ijms26073192
APA StyleVilkaite, A., Nguyen, X. P., Güzel, C. T., Gottschlich, L., Bender, U., Dietrich, J. E., Hinderhofer, K., Strowitzki, T., & Rehnitz, J. (2025). Beyond Repetition: The Role of Gray Zone Alleles in the Upregulation of FMR1-Binding miR-323a-3p and the Modification of BMP/SMAD-Pathway Gene Expression in Human Granulosa Cells. International Journal of Molecular Sciences, 26(7), 3192. https://doi.org/10.3390/ijms26073192