Specific RNA m6A modification sites in bone marrow mesenchymal stem cells from the jawbone marrow of type 2 diabetes patients with dental implant failure

Abstract

The failure rate of dental implantation in patients with well-controlled type 2 diabetes mellitus (T2DM) is higher than that in non-diabetic patients. This due, in part, to the impaired function of bone marrow mesenchymal stem cells (BMSCs) from the jawbone marrow of T2DM patients (DM-BMSCs), limiting implant osseointegration. RNA N6-methyladenine (m6A) is important for BMSC function and diabetes regulation. However, it remains unclear how to best regulate m6A modifications in DM-BMSCs to enhance function. Based on the “m6A site methylation stoichiometry” of m6A single nucleotide arrays, we identified 834 differential m6A-methylated genes in DM-BMSCs compared with normal-BMSCs (N-BMSCs), including 43 and 790 m6A hypermethylated and hypomethylated genes, respectively, and 1 gene containing hyper- and hypomethylated m6A sites. Differential m6A hypermethylated sites were primarily distributed in the coding sequence, while hypomethylated sites were mainly in the 3′-untranslated region. The largest and smallest proportions of m6A-methylated genes were on chromosome 1 and 21, respectively. MazF-PCR and real-time RT-PCR results for the validation of erythrocyte membrane protein band 4.1 like 3, activity-dependent neuroprotector homeobox (ADNP), growth differentiation factor 11 (GDF11), and regulator of G protein signalling 2 agree with m6A single nucleotide array results; ADNP and GDF11 mRNA expression decreased in DM-BMSCs. Furthermore, gene ontology and Kyoto Encyclopedia of Genes and Genomes analyses suggested that most of these genes were enriched in metabolic processes. This study reveals the differential m6A sites of DM-BMSCs compared with N-BMSCs and identifies candidate target genes to enhance BMSC function and improve implantation success in T2DM patients.

References

[1] American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care, 2012, 35(Suppl 1): S64-S71.

[2] Galicia-Garcia U, . Pathophysiology of type 2 diabetes mellitus. Int J. Mol. Sci., 2020, 21: E6275.

[3] Graves DT, Ding Z, Yang Y. The impact of diabetes on periodontal diseases. Periodontol 2000, 2020, 82: 214-224.

[4] Tang D, . Is hyperglycemia the only risk factor for implant in type 2 diabetics during the healing period?. Oral. Dis., 2021, 27: 1551-1563.

[5] Zhang J, . Genipin and insulin combined treatment improves implant osseointegration in type 2 diabetic rats. J. Orthop. Surg. Res., 2021, 16: 59.

[6] Sundar G, . Impact of well-controlled type 2 diabetes mellitus on implant stability and bone biomarkers. Int J. Oral. Maxillofac. Implants, 2019, 34: 1441-1449.

[7] Akintoye SO, . Skeletal site-specific characterization of orofacial and iliac crest human bone marrow stromal cells in same individuals. Bone, 2006, 38: 758-768.

[8] Chen Z, . Tuning chemistry and topography of nanoengineered surfaces to manipulate immune response for bone regeneration applications. ACS Nano, 2017, 11: 4494-4506.

[9] Sun R, . Effects of metformin on the osteogenesis of alveolar BMSCs from diabetic patients and implant osseointegration in rats. Oral. Dis., 2022, 28: 1170-1180.

[10] Li J, Liu CY, Jiang YF, Wei XZ, Li JU. Proliferation and differentiation of human osteoblasts from a type 2 diabetic patient in vitro. Genet. Mol. Res., 2015, 14: 11292-11299.

[11] Hu Y, Zhao X. Role of m6A in osteoporosis, arthritis and osteosarcoma (Review). Exp. Ther. Med., 2021, 22: 926.

[12] Jiang H, . Transcriptome-wide high-throughput m6A sequencing of differential m6A methylation patterns in the human rheumatoid arthritis fibroblast-like synoviocytes cell line MH7A. J. Inflamm. Res., 2021, 14: 575-586.

[13] Oerum S, Meynier V, Catala M, Tisné C. A comprehensive review of m6A/m6Am RNA methyltransferase structures. Nucleic Acids Res., 2021, 49: 7239-7255.

[14] Han X, Guo J, Fan Z. Interactions between m6A modification and miRNAs in malignant tumors. Cell Death Dis., 2021, 12

[15] Shen F, . Decreased N(6)-methyladenosine in peripheral blood RNA from diabetic patients is associated with FTO expression rather than ALKBH5. J. Clin. Endocrinol. Metab., 2015, 100: E148-E154.

[16] Liang D, . m6A reader YTHDC1 modulates autophagy by targeting SQSTM1 in diabetic skin. Autophagy, 2022, 18: 1318-1337.

[17] Liu T, . The m6A “reader” YTHDF1 promotes osteogenesis of bone marrow mesenchymal stem cells through translational control of ZNF839. Cell Death Dis., 2021, 12

[18] Li Z, . The N6-methyladenosine demethylase ALKBH5 negatively regulates the osteogenic differentiation of mesenchymal stem cells through PRMT6. Cell Death Dis., 2021, 12

[19] Zhou J, Wei T, He Z. ADSCs enhance VEGFR3-mediated lymphangiogenesis via METTL3-mediated VEGF-C m6A modification to improve wound healing of diabetic foot ulcers. Mol. Med., 2021, 27: 146.

[20] Szklarczyk D, . The STRING database in 2017: Quality-controlled protein-protein association networks, made broadly accessible. Nucleic Acids Res., 2017, 45: D362-D368.

[21] Perry RP, Kelley DE, Friderici K, Rottman F. The methylated constituents of L cell messenger RNA: Evidence for an unusual cluster at the 5′ terminus. Cell, 1975, 4: 387-394.

[22] Zaccara S, Ries RJ, Jaffrey SR. Reading, writing and erasing mRNA methylation. Nat. Rev. Mol. Cell Biol., 2019, 20: 608-624.

[23] Garcia-Campos MA, . Deciphering the ‘m6A code’ via antibody-independent quantitative profiling. Cell, 2019, 178: 731-747.e16.

[24] Zhang Z, . Single-base mapping of m6A by an antibody-independent method. Sci. Adv., 2019, 5: eaax0250.

[25] Ma H, . N6-methyladenosine methyltransferase ZCCHC4 mediates ribosomal RNA methylation. Nat. Chem. Biol., 2019, 15: 88-94.

[26] Zhou J, . Dynamic m(6)A mRNA methylation directs translational control of heat shock response. Nature, 2015, 526: 591-594.

[27] Choi J, . N(6)-methyladenosine in mRNA disrupts tRNA selection and translation-elongation dynamics. Nat. Struct. Mol. Biol., 2016, 23: 110-115.

[28] Huang M, . m6A methylation regulates osteoblastic differentiation and bone remodeling. Front Cell Dev. Biol., 2021, 9: 783322.

[29] Yang L, . Nsun4 and Mettl3 mediated translational reprogramming of Sox9 promotes BMSC chondrogenic differentiation. Commun. Biol., 2022, 5: 495.

[30] Tian C, Huang Y, Li Q, Feng Z, Xu Q. Mettl3 regulates osteogenic differentiation and alternative splicing of Vegfa in bone marrow mesenchymal stem cells. Int J. Mol. Sci., 2019, 20: E551.

[31] Zheng J, . Identification of MDM2, YTHDF2 and DDX21 as potential biomarkers and targets for treatment of type 2 diabetes. Biochem. Biophys. Res. Commun., 2021, 581: 110-117.

[32] Yao Y, . METTL3 inhibits BMSC adipogenic differentiation by targeting the JAK1/STAT5/C/EBPβ pathway via an m6A-YTHDF2-dependent manner. FASEB J., 2019, 33: 7529-7544.

[33] Du R, Bai Y, Li L. Biological networks in gestational diabetes mellitus: insights into the mechanism of crosstalk between long non-coding RNA and N6-methyladenine modification. BMC Pregnancy Childbirth, 2022, 22

[34] An Y, Duan H. The role of m6A RNA methylation in cancer metabolism. Mol. Cancer, 2022, 21

[35] Louvanto K, . Methylation of viral and host genes and severity of cervical lesions associated with human papillomavirus type 16. Int J. Cancer, 2015, 136: E638-E645.

[36] Lorincz AT, . Methylation of HPV and a tumor suppressor gene reveals anal cancer and precursor lesions. Oncotarget, 2017, 8: 50510-50520.

[37] Giuliano AR, . Methylation of HPV 16 and EPB41L3 in oral gargles: Associations with oropharyngeal cancer detection and tumor characteristics. Int J. Cancer, 2020, 146: 1018-1030.

[38] Xie Y, . ADNP prompts the cisplatin-resistance of bladder cancer via TGF-β-mediated epithelial-mesenchymal transition (EMT) pathway. J. Cancer, 2021, 12: 5114-5124.

[39] Mollinedo P, . Cellular and animal models of skin alterations in the autism-related ADNP syndrome. Sci. Rep., 2019, 9

[40] Idan-Feldman A, . Davunetide (NAP) as a preventative treatment for central nervous system complications in a diabetes rat model. Neurobiol. Dis., 2011, 44: 327-339.

[41] Leker RR, . NAP, a femtomolar-acting peptide, protects the brain against ischemic injury by reducing apoptotic death. Stroke, 2002, 33: 1085-1092.

[42] D’Amico AG, . Nap interferes with hypoxia-inducible factors and VEGF expression in retina of diabetic rats. J. Mol. Neurosci., 2017, 61: 256-266.

[43] D’Amico AG, . NAP counteracts hyperglycemia/hypoxia induced retinal pigment epithelial barrier breakdown through modulation of HIFs and VEGF expression. J. Cell Physiol., 2018, 233: 1120-1128.

[44] Sethy NK, Sharma NK, Das M, Bhargava K. Protein profiling reveals antioxidant and signaling activities of NAP (davunetide) in rodent hippocampus exposed to hypobaric hypoxia. J. Mol. Neurosci., 2014, 54: 414-429.

[45] Maugeri G, . Activity-dependent neuroprotective protein (ADNP)-derived peptide (NAP) counteracts UV-B radiation-induced ROS Formation in corneal epithelium. Antioxidants, 2022, 11: 128.

[46] Escher U, . Anti-inflammatory effects of the octapeptide NAP in human microbiota-associated mice suffering from subacute ileitis. Eur. J. Microbiol Immunol., 2018, 8: 34-40.

[47] Nakashima M, Toyono T, Akamine A, Joyner A. Expression of growth/differentiation factor 11, a new member of the BMP/TGFbeta superfamily during mouse embryogenesis. Mech. Dev., 1999, 80: 185-189.

[48] Qi X, . Endogenous GDF11 regulates odontogenic differentiation of dental pulp stem cells. J. Cell Mol. Med, 2020, 24: 11457-11464.

[49] Nakashima M, . Induction of reparative dentin formation by ultrasound-mediated gene delivery of growth/differentiation factor 11. Hum. Gene Ther., 2003, 14: 591-597.

[50] Nakashima M, . Stimulation of reparative dentin formation by ex vivo gene therapy using dental pulp stem cells electrotransfected with growth/differentiation factor 11 (Gdf11). Hum. Gene Ther., 2004, 15: 1045-1053.

[51] Nakashima M, Mizunuma K, Murakami T, Akamine A. Induction of dental pulp stem cell differentiation into odontoblasts by electroporation-mediated gene delivery of growth/differentiation factor 11 (Gdf11). Gene Ther., 2002, 9: 814-818.

[52] Li H, . GDF11 attenuates development of Type 2 diabetes via improvement of islet β-cell function and survival. Diabetes, 2017, 66: 1914-1927.

[53] Walker RG, . Exogenous GDF11, but not GDF8, reduces body weight and improves glucose homeostasis in mice. Sci. Rep., 2020, 10

[54] Onodera K, . Decrease in an anti-ageing factor, growth differentiation factor 11, in chronic obstructive pulmonary disease. Thorax, 2017, 72: 893-904.

[55] Mei W, . GDF11 protects against endothelial injury and reduces atherosclerotic lesion formation in apolipoprotein E-null mice. Mol. Ther., 2016, 24: 1926-1938.

[56] Su HH, . Exogenous GDF11 attenuates non-canonical TGF-β signaling to protect the heart from acute myocardial ischemia-reperfusion injury. Basic Res Cardiol., 2019, 114: 20.

[57] Li W, . GDF11 antagonizes TNF-α-induced inflammation and protects against the development of inflammatory arthritis in mice. FASEB J., 2019, 33: 3317-3329.

[58] Chen X, . Regulatory role of RNA N6-methyladenosine modification in bone biology and osteoporosis. Front. Endocrinol., 2020, 10: 911.

[59] Tsingotjidou A, Nervina JM, Pham L, Bezouglaia O, Tetradis S. Parathyroid hormone induces RGS-2 expression by a cyclic adenosine 3′,5′-monophosphate-mediated pathway in primary neonatal murine osteoblasts. Bone, 2002, 30: 677-684.

[60] Uribe P, . Soluble silica stimulates osteogenic differentiation and gap junction communication in human dental follicle cells. Sci. Rep., 2020, 10

[61] Madrigal A, Tan L, Zhao Y. Expression regulation and functional analysis of RGS2 and RGS4 in adipogenic and osteogenic differentiation of human mesenchymal stem cells. Biol. Res, 2017, 50

[62] Keinan D, . Role of regulator of G protein signaling proteins in bone. Front. Biosci., 2014, 19: 634-648.

[63] Wolosowicz M, Prokopiuk S, Kaminski TW. Recent advances in the treatment of insulin resistance targeting molecular and metabolic pathways: Fighting a losing battle?. Medicina, 2022, 58: 472.

[64] Aguilar-Recarte D, Palomer X, Wahli W, Vázquez-Carrera M. The PPARβ/δ-AMPK connection in the treatment of insulin resistance. Int J. Mol. Sci., 2021, 22: 8555.

[65] Entezari M, . AMPK signaling in diabetes mellitus, insulin resistance and diabetic complications: a pre-clinical and clinical investigation. Biomed. Pharmacother., 2022, 146: 112563.

[66] Hu XF, . Adiponectin improves the osteointegration of titanium implant under diabetic conditions by reversing mitochondrial dysfunction via the AMPK pathway in vivo and in vitro. Acta Biomater., 2017, 61: 233-248.

[67] Li Y, . Selenomethionine protects oxidative-stress-damaged bone-marrow-derived mesenchymal stem cells via an antioxidant effect and the PTEN/PI3K/AKT pathway. Exp. Cell Res., 2021, 408: 112864.

[68] Liang C, . Osteogenic potential of three different autogenous bone particles harvested during implant surgery. Oral. Dis., 2017, 23: 1099-1108.

[69] Liang C, Sun R, Xu Y, Geng W, Li J. Effect of the abnormal expression of BMP-4 in the blood of diabetic patients on the osteogenic differentiation potential of alveolar BMSCs and the rescue effect of metformin: a bioinformatics-based study. BioMed. Res. Int., 2020, 2020: 7626215.

[70] Imanishi M, Tsuji S, Suda A, Futaki S. Detection of N6-methyladenosine based on the methyl-sensitivity of MazF RNA endonuclease. Chem. Commun., 2017, 53: 12930-12933.