Endocrine Abstracts

Introduction: Osteoporosis is a disease characterized by the loss of bone density and deficits in bone microarchitecture. “Diabetoporosity” is a new term for osteoporosis in diabetes mellitus type 2 (T2DM) patients, where classical tools for the assessment of bone properties like dual x-ray absorptiometry or bone turnover markers are lacking prognostic accuracy for the determination of bone quality and fracture risk.

MicroRNAs (miRNAs) are getting into the focus of research not only for their importance in the development of osteogenic lineage cells, but also as biomarkers for bone related diseases. Sequencing of serum samples of elderly T2DM patients with prospective fractures identified differentially expressed miRNA compared to control serum samples. Here we have removed some of the identified microRNAs by CRISPR/Cas and examine the function of these microRNAs upon osteoblast differentiation of immortalized human mesenchymal stem cells (hMSC-TERTs).

Materials and methods: Serum samples of patients with T2DM who developed fractures within two years of follow-up were compared to serum of T2DM patients without fractures. miRNA sequencing was performed in serum of 10 non-fracture and 6 fracture patients. Immortalized human bone marrow derived mesenchymal stem cells expressing Cas9 (MSC-TERT ^Cas9) were used to delete selected miRNAs. Guide RNAs were designed, cloned into the plasmid phU6 and transfected in MSC-TERT^cas9 cells. Individual cells lacking miRNAs were clonally expanded.

Results: miRNA sequencing analysis of serum samples revealed 16 miRNAs (FDR < 0.05) that correlated with prospective fractures in elderly diabetic patients. miRNAs with average relative counts between 100 and 10000 were chosen for further analysis. 8 of the selected 9 were expressed in MSC-TERTs and considered for functional analyses and deletion. Successful creation of three microRNA deletion strains, namely miR140-KO, miR25/93/106b KO and miR-363/19b-KO, could be confirmed by PCR and sanger-sequencing.

Discussion: The establishment of cells lacking our target miRNAs is the first step for the examination of their importance during osteoblast differentiation of hMSCs. Further experiments will reveal the function of these miRNAs in the context of the differentiation potential of the created cell lines.