MiR-182 Regulates Osteogenic Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells And Contributes To Diabetic Osteoporosis

Background:Diabetic Osteoporosis (DOP) is a severe chronic complication of diabetes in terms of bone. It is a kind of secondary osteoporosis. Patients who suffer from it may have lower bone mass and damaged microstructure of bone tissue. Meanwhile, their bones are more fragile and prone to fracture. With the aging of population as well as the growth in the living standard, the cases of DOP occur more often year by year. Epidemiological survey shows that among type Ⅰ and type Ⅱ diabetes, the incidence of osteoporosis is 20-72%. DOP seriously hazard health and lives of people.The skeletal system bears good plasticity and regeneration capacity. Under normal circumstances, the processes of osteogenesis and bone absorption are alternately happening within the body in a complex but orderly way, which makes the bone in the state of dynamic rebuilding. While in DOP patients, the balance between the two processes are broke, due to the function impairment of BMSCs.MicroRNA (miRNA), a class of uncoding small RNA newly discovered in recent years, interfere with translation of specific target mRNAs and play an important role in a variety of regulation of gene expression in eukaryotic cells, through which way exerting important influence on many physiological and pathological processes. Studies have found that some changes of microRNA expression profile will lead to distinct results of stem cells fuction. Therefore, to better understand the properties of stem cells and then use them to prevent or treat diseases, we need to figure out the mechanism of how microRNA regulates the self-renewal and differentiation of stem cells. So far, as to DOP, the mechanism of how microRNA acts on stem cells to form osteoblasts is unknown, the regulating methods by target gene is also unclear.Objective:We set up the DOP rat model to isolate and culture BMSCs. then find the changes of their biological properties. We use microRNA deep sequencing to seek for differently expressed microRNA of the two types of BMSCs. and then, based on biological information, make analysis and prediction of how microRNA regulates gene and affectes the osteogenic differentiation of BMSCs. By doing these experiments, we may discover the possible roles of microRNA in the onset of DOP.Methods(1) The biological properties of BMSCs originating from diabetic osteoporosis rats (diabetes-BMSCs) and healthy rats (wistar-BMSCs) were compared. The diabetic rat model was constructed via intraperitoneal injection of streptozotocin (STZ), and the osteoporotic status of rats was assessed by serum biochemical tests, micro-computed tomography (micro-CT) scanning, and morphological analysis of bone tissue. Diabetes-BMSCs and wistar-BMSCs were isolated and cultured using the whole bone marrow adherence method. The biological properties of the two types of BMSCs were then compared through surface molecule identification by flow cytometry, MTT assays, colony formation assays, and induction of osteogenic and adipogenic differentiation.(2) microRNAs that were differentially expressed in diabetes-BMSCs and wistar-BMSCs were screened. microRNA deep sequencing was performed in diabetes-BMSCs and wistar-BMSCs. The sequencing results were validated by quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) to select for differentially expressed microRNAs during osteogenic and adipogenic differentiation and in different tissues.(3) The effects of miR-182 on the proliferative and osteogenic differentiation capacities of BMSCs were analyzed. The expression level of miR-182 in BMSCs was regulated using cell transfection, and changes in proliferative and osteogenic differentiation capacities of BMSCs with over-expressed or inhibited miR-182 were determined by MTT assays, induction of osteogenic differentiation, and Alizarin Red S staining.(4) The regulatory mechanisms of miR-182 in BMSCs during the osteogenic differentiation process were analyzed. Smadl was predicted as a candidate target gene of miR-182 using the bioinformatic approach, and a dual-luciferase reporter gene vector was constructed to verify the target gene. qRT-PCR and western blot were performed to determine the endogenous expression of the target gene (Smadl) and an osteogenesis-related gene (Runx2) during the osteogenic differentiation of BMSCs in the context of miR-182 over-expression.Results(1) At 8 weeks after induction of the diabetic rat model by intraperitoneal injection of STZ, body weights, serum osteocalcin levels, bone mineral densities (BMDs) of the humerus and lumbar spine, and bone-to-tissue volume (BV/TV) ratios were significantly reduced compared with those in the normal group. In contrast, trabeculargaps had increased. BMSCs of both origins expressed high levels of CD44, CD90, and CD 105, but did not express CD34 or CD45. The proliferative capacities of diabetes-BMSCs were significantly lower than those of wistar-BMSCs, and both groups exhibited strong colony-forming ability. Diabetes-BMSCs had a significantly lower osteogenic differentiation capacity but a significantly higher adipogenic differentiation capacity than wistar-BMSCs.(2) A total of 10 microRNAs that were differentially expressed in diabetes-BMSCs and wistar-BMSCs were selected via microRNA deep sequencing. This analysis showed that the expression of miR-182 was significantly elevated in diabetes-BMSCs and the bone tissues of rats with diabetic osteoporosis. The expression of miR-182 in diabetes-BMSCs and wistar-BMSCs was reduced during osteogenic differentiation and increased during adipogenic differentiation. Therefore, miR-182 was selected for subsequent experiments.(3) miR-182 had no significant effect on the proliferative capacity of BMSCs. The osteogenic differentiation capacity of BMSCs was significantly reduced when miR-182 was over-expressed and slightly increased when miR-182 was inhibited.(4) Smadl was predicted to be a candidate target gene of miR-182 using a bioinformatic approach. Additionally, dual-luciferase assays also verified that miR-182 could bind specifically to the 3’-untranslated region (3’-UTR) of Smadl mRNA. Up regulation of miR-182 expression led to down regulation of Smadl and Runx2 expression, whereas down regulation of miR-182 expression led to up regulation of Smadl expression. Up regulation of miR-182 expression in BMSCs could inhibit the expression of Smadl and Runx2 (a key osteogenesis-related gene) during osteogenic differentiation, thereby reducing the osteogenic differentiation capacity of BMSCs.Conclusion(1) In diabetes-induced osteoporosis, BMSCs retained the basic abilities and properties of stem cells. However, their proliferative and osteogenic differentiation capacities were reduced, whereas their adipogenic differentiation capacity was enhanced.(2) In diabetes-induced osteoporosis, the expression of miR-182 was specifically increased in BMSCs.(3) miR-182 negatively regulated the expression of its target gene (Smad1) and affected the osteogenic differentiation of BMSCs by reducing the expression of Smadl and Runx2.