METTL3-mediated long non-coding RNA MIR99AHG methylation targets miR-4660 to promote bone marrow mesenchymal stem cell osteogenic differentiation

ABSTRACT Whether long non-coding RNA Mir-99a-Let-7c Cluster Host Gene (LncRNA MIR99AHG) is involved in osteoporosis (OP) remains vague, so we hereby center on its implication. Old C57BL/6J mice were injected with the silencing lentivirus of MIR99AHG and subjected to microCT analysis and immunohistochemistry on osteogenic cells. The osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) with or without transfection was determined by alkaline phosphatase (ALP) and Alizarin Red S staining. Total N(6)-methyladenosine (m6A) on the bone marrow mesenchymal stem cells (BMSCs) was quantified. The potential methylation site and the complementary binding sites with candidate microRNA (miR) were predicted via bioinformatic analyses, with the latter being confirmed via dual-luciferase reporter, RNA immunoprecipitation and RNA pull-down assays. Quantitative real-time PCR and Western blot were used for quantification assays. MIR99AHG was decreased during the osteogenic differentiation of BMSCs, where increased Osterix (OSX), Collagen, Type I, Alpha 1 (Col1A1), Osteocalcin (OCN) and RUNX Family Transcription Factor 2 (RUNX2) as well as more color-stained areas were found. Also, silencing MIR99AHG relieved the OP in mice and reduced the loss of osteogenic cells. M6A methylation in undifferentiated BMSCs was low and MIR99AHG overexpression abolished the effects of overexpressed METTL3 on promoting osteogenic differentiation. MiR-4660, which was downregulated in BMSCs without differentiation but increased during osteogenic differentiation, could bind with MIR99AHG. Furthermore, miR-4660 promoted osteogenic differentiation and reversed the effects of overexpressed MIR99AHG. The present study demonstrated that METTL3-mediated LncRNA MIR99AHG methylation enhanced the osteogenic differentiation of BMSCs via targeting miR-4660.

[1]  T. Rasmussen,et al.  lncRNA involvement in cancer stem cell function and epithelial-mesenchymal transitions. , 2020, Seminars in cancer biology.

[2]  A. Arthur,et al.  Clinical Application of Bone Marrow Mesenchymal Stem/Stromal Cells to Repair Skeletal Tissue , 2020, International journal of molecular sciences.

[3]  G. Ma,et al.  N6-methyladenosine methyltransferase plays a role in hypoxic preconditioning partially through the interaction with lncRNA H19. , 2020, Acta biochimica et biophysica Sinica.

[4]  Qiwen Li,et al.  Alpha-ketoglutarate ameliorates age-related osteoporosis via regulating histone methylations , 2020, Nature Communications.

[5]  K. Xia,et al.  Autophagy receptor OPTN (optineurin) regulates mesenchymal stem cell fate and bone-fat balance during aging by clearing FABP3 , 2020, Autophagy.

[6]  A. Oryan,et al.  Effects of bisphosphonates on osteoporosis: Focus on zoledronate. , 2020, Life sciences.

[7]  Jiwei Zheng,et al.  SNHG5/miR-582-5p/RUNX3 feedback loop regulates osteogenic differentiation and apoptosis of bone marrow mesenchymal stem cells. , 2020, Journal of cellular physiology.

[8]  W. Xie,et al.  ALKBH1 Promotes Lung Cancer by Regulating m6A RNA Demethylation. , 2020, Biochemical pharmacology.

[9]  Yang Wang,et al.  MiR‐140‐5p promotes osteogenic differentiation of mouse embryonic bone marrow mesenchymal stem cells and post‐fracture healing of mice , 2020, Cell biochemistry and function.

[10]  P. Zhou,et al.  Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses , 2020, Stem cell research & therapy.

[11]  T. Shao,et al.  Long Non-coding RNA AGAP2-AS1 Silencing Inhibits PDLIM5 Expression Impeding Prostate Cancer Progression via Up-Regulation of MicroRNA-195-5p , 2020, Frontiers in Genetics.

[12]  J. Ghosh,et al.  Osteogenic differentiation potential of porcine bone marrow mesenchymal stem cell subpopulations selected in different basal media , 2020, Biology Open.

[13]  Wenyi Wei,et al.  Phosphorylation‐dependent osterix degradation negatively regulates osteoblast differentiation , 2020, FASEB journal : official publication of the Federation of American Societies for Experimental Biology.

[14]  Qiang Yang,et al.  m6A transferase METTL3‐induced lncRNA ABHD11‐AS1 promotes the Warburg effect of non‐small‐cell lung cancer , 2020, Journal of cellular physiology.

[15]  Qiangfang Zhao,et al.  Long noncoding RNA MIR99AHG promotes gastric cancer progression by inducing EMT and inhibiting apoptosis via miR577/FOXP1 axis , 2020, Cancer cell international.

[16]  Changjun Li,et al.  Regulation of Bone marrow mesenchymal stem cell fate by long non-coding RNA. , 2020, Bone.

[17]  Huiyong Shen,et al.  Aberrantly Expressed lncRNAs and mRNAs of Osteogenically Differentiated Mesenchymal Stem Cells in Ossification of the Posterior Longitudinal Ligament , 2020, Frontiers in Genetics.

[18]  Xiaoyu Chen,et al.  Novel insights into the interplay between m6A modification and noncoding RNAs in cancer , 2020, Molecular Cancer.

[19]  Guoqiang Wang,et al.  miR-483-3p promotes the osteogenesis of human osteoblasts by targeting Dikkopf 2 (DKK2) and the Wnt signaling pathway , 2020, International journal of molecular medicine.

[20]  L. Jia,et al.  Long non-coding RNA MIR22HG promotes osteogenic differentiation of bone marrow mesenchymal stem cells via PTEN/ AKT pathway , 2020, Cell Death & Disease.

[21]  W. Geng,et al.  Emerging role of RNA methyltransferase METTL3 in gastrointestinal cancer , 2020, Journal of Hematology & Oncology.

[22]  G. Toro,et al.  Pharmacological Therapy of Osteoporosis: What’s New? , 2020, Clinical interventions in aging.

[23]  H. Zhong,et al.  N6-methyladenine RNA modification (m6A): an emerging regulator of metabolic diseases. , 2020, Current drug targets.

[24]  D. Shoback,et al.  New Frontiers in Osteoporosis Therapy. , 2020, Annual review of medicine.

[25]  Guowei Li,et al.  Regulatory Role of RNA N6-Methyladenosine Modification in Bone Biology and Osteoporosis , 2020, Frontiers in Endocrinology.

[26]  A. Qian,et al.  Senile Osteoporosis: The Involvement of Differentiation and Senescence of Bone Marrow Stromal Cells , 2020, International journal of molecular sciences.

[27]  Hui Shen,et al.  A road map for understanding molecular and genetic determinants of osteoporosis , 2019, Nature Reviews Endocrinology.

[28]  L. Su,et al.  KR-12-a6 promotes the osteogenic differentiation of human bone marrow mesenchymal stem cells via BMP/SMAD signaling , 2019, Molecular medicine reports.

[29]  Qiang Xu,et al.  miR-204 inhibits the osteogenic differentiation of mesenchymal stem cells by targeting bone morphogenetic protein 2 , 2019, Molecular medicine reports.

[30]  Y. Miao,et al.  The RNA m6A methyltransferase METTL3 promotes pancreatic cancer cell proliferation and invasion. , 2019, Pathology, research and practice.

[31]  Junxiao Yang,et al.  LncRNA MALAT1 shuttled by bone marrow-derived mesenchymal stem cells-secreted exosomes alleviates osteoporosis through mediating microRNA-34c/SATB2 axis , 2019, Aging.

[32]  Yuzhu Zhu,et al.  Long non-coding RNA GAS5 promotes osteogenic differentiation of bone marrow mesenchymal stem cells by regulating the miR-135a-5p/FOXO1 pathway , 2019, Molecular and Cellular Endocrinology.

[33]  Hongbo He,et al.  MicroRNA‐130a controls bone marrow mesenchymal stem cell differentiation towards the osteoblastic and adipogenic fate , 2019, Cell proliferation.

[34]  Y. Teng,et al.  Genome-Wide Identification of a Novel Eight-lncRNA Signature to Improve Prognostic Prediction in Head and Neck Squamous Cell Carcinoma , 2019, Front. Oncol..

[35]  Zhenming Hu,et al.  MiR-128 inhibits the osteogenic differentiation in osteoporosis by down-regulating SIRT6 expression , 2019, Bioscience reports.

[36]  B. Zhu,et al.  MiRNA-27a-3p promotes osteogenic differentiation of human mesenchymal stem cells through targeting ATF3. , 2019, European review for medical and pharmacological sciences.

[37]  Shizhang Liu,et al.  Roles for miRNAs in osteogenic differentiation of bone marrow mesenchymal stem cells , 2019, Stem Cell Research & Therapy.

[38]  Zhike Lu,et al.  m6A mRNA demethylase FTO regulates melanoma tumorigenicity and response to anti-PD-1 blockade , 2019, Nature Communications.

[39]  Ji-Fu Wei,et al.  METTL3 promote tumor proliferation of bladder cancer by accelerating pri-miR221/222 maturation in m6A-dependent manner , 2019, Molecular cancer.

[40]  Mingjun Cai,et al.  The role of resveratrol in bone marrow‐derived mesenchymal stem cells from patients with osteoporosis , 2019, Journal of cellular biochemistry.

[41]  Liang Ming,et al.  The role of m6A RNA methylation in cancer. , 2019, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie.

[42]  Min-Cong He,et al.  Polydatin promotes the osteogenic differentiation of human bone mesenchymal stem cells by activating the BMP2-Wnt/β-catenin signaling pathway. , 2019, Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie.

[43]  X. Tu,et al.  Human MiR-4660 regulates the expression of alanine–glyoxylate aminotransferase and may be a biomarker for idiopathic oxalosis , 2019, Clinical and Experimental Nephrology.

[44]  G. Schmidmaier,et al.  Osteogenic differentiation of mesenchymal stem cells is enhanced in a 45S5-supplemented β-TCP composite scaffold: an in-vitro comparison of Vitoss and Vitoss BA , 2019, PloS one.

[45]  Zhihui Feng,et al.  Mettl3 Regulates Osteogenic Differentiation and Alternative Splicing of Vegfa in Bone Marrow Mesenchymal Stem Cells , 2019, International journal of molecular sciences.

[46]  M. Fayyad-kazan,et al.  Evaluation of the Osteogenic Potential of Different Scaffolds Embedded with Human Stem Cells Originated from Schneiderian Membrane: An In Vitro Study , 2019, BioMed research international.

[47]  Y. Wang,et al.  Mettl3-mediated m6A RNA methylation regulates the fate of bone marrow mesenchymal stem cells and osteoporosis , 2018, Nature Communications.

[48]  Chengjian He,et al.  Mechanisms of Zuogui Pill in Treating Osteoporosis: Perspective from Bone Marrow Mesenchymal Stem Cells , 2018, Evidence-based complementary and alternative medicine : eCAM.

[49]  Gang Chen,et al.  Identification and characterization of human ovary-derived circular RNAs and their potential roles in ovarian aging , 2018, Aging.

[50]  A. Rentmeister,et al.  Emerging approaches for detection of methylation sites in RNA , 2018, Open Biology.

[51]  P. Ma,et al.  Multiple functions of m6A RNA methylation in cancer , 2018, Journal of Hematology & Oncology.

[52]  A. Ruggieri,et al.  m6A RNA methylation, a new hallmark in virus-host interactions. , 2017, The Journal of general virology.

[53]  Xiu-Jie Wang,et al.  Mettl3-mediated m6A regulates spermatogonial differentiation and meiosis initiation , 2017, Cell Research.

[54]  T. Carell,et al.  The chemistries and consequences of DNA and RNA methylation and demethylation , 2017, RNA biology.

[55]  Qiang Wang,et al.  Structural basis of N6-adenosine methylation by the METTL3–METTL14 complex , 2016, Nature.

[56]  S. Emmrich,et al.  LincRNAs MONC and MIR100HG act as oncogenes in acute megakaryoblastic leukemia , 2014, Molecular Cancer.

[57]  Miao Yu,et al.  A METTL3-METTL14 complex mediates mammalian nuclear RNA N6-adenosine methylation , 2013, Nature chemical biology.

[58]  Q. Wang,et al.  Regulation of osteogenic differentiation of rat bone marrow stromal cells on 2D nanorod substrates. , 2010, Biomaterials.

[59]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[60]  Zhike Lu,et al.  m6A mRNA demethylase FTO regulates melanoma tumorigenicity and response to anti-PD-1 blockade , 2019, Nature Communications.

[61]  M. Kassem,et al.  Skeletal (stromal) stem cells: an update on intracellular signaling pathways controlling osteoblast differentiation. , 2015, Bone.