Mechanisms Of MiRNA-92a-3p Regulates Osteoblast Differentiation In Patients With Fracture And Traumatic Brain Injury

Part ?: Quicker callus formation and higher plasma MIRNA-92A-3P concentrations in fracture patients with traumatic brain injuryObjective: The rate of callus formation at the fracture site of patients with traumatic brain injury is faster than that of patients with simple fracture.This clinical phenomenon has long been widely concerned by first-line doctors,but its cause is still unclear.However,with the continuous improvement of surgical techniques and instruments,the rate of fracture nonunion of femur and tibia has not been significantly reduced but remains high.By exploring the causes of rapid callus formation in patients with traumatic brain injury and fracture,it is expected to explain the causes of this clinical phenomenon,and then provide theoretical basis for the or the treatment of fracture nonunion.Methods: Collect union hospital affiliated to Tongji medical college,Huazhong university of science and technology,orthopaedic trauma 08,2018-October 2018 of 30 cases of hospitalized patients with venous plasma,according to the fracture combined traumatic brain injuries,simple fracture,remove the internal fixation of patients in three groups,in the hospital 1 day,3 days,7 days venous blood collection,collection of 10 cases in each group,the RT-PCR detection has-micro RNA-92a-3p expression level.Male C57 BL mice aged 8 weeks were selected and divided into 3 groups: fracture complicated with brain trauma,simple fracture,and control group.The traumatic brain injury model was constructed by means of the vertical beating instrument.The femoral fracture model was constructed by cutting off the right femur.The femur was only exposed and sutured in the control group.At 3,7,and 14 days after the operation,epicanthus vein blood was collected for RT-PCR detection of the expression level of miRNA-92a-3p.At 14 days after the model was established,21 days after the model was established,the femur of the mice was harvested for micro-CT evaluation of callus formation.Results: The plasma RT-PCR results of clinical patients showed that the expression level of miRNA-92a-3p was increased at 1,3 and 7 days after admission in the fracture combined with traumatic brain injury group compared with the fracture group and the internal fixation group,and the difference was statistically significant(P<0.05);The expression level of miRNA-92a-3p was detected by plasma RT-PCR 3,7,and 14 days after the animal model was made,and the difference was statistically significant between the fracture combined with traumatic brain injury group and the single fracture group(P<0.05).Conclusions: Callus formation was faster in patients with fracture complicated with brain injury than in patients with simple fracture,and the plasma expression level of miRNA-92a-3p was increased.Part ?:The effect of MIRNA-92A-3P on osteogenic precursor cells and its MechanismsObjective: To explore whether miRNA-92a-3p mediated callus formation to promote fracture healing through affecting osteogenic precursor cells in vitro trial.Methods: Firstly,RT-PCR and western-blot analysis were used to assess the expression level of osteoblast differentiation genes and proteins in MC3T3-E1 48 hours after transfection of agomiRNA-92a-3p,antagomiRNA-92a-3p,agomiRNA-negative control(agomiRNA-NC),antagomiRNA-negative control(antagomiRNA-NC),at 200 um using Lipofectiectamine 3000.And then alkaline phosphatase staining and alizarin red staining were performed 14 days after transfection to observe osteoblast differentiation and matrix mineralization.Secondly,the effect of miRNA-92a-3p on IBSP was examined via luciferase reporter constructs.Further,the RT-PCR and western-blot analysis were used to assess the expression of IBSP after transfecting cell as above,and then the effect of IBSP on expression of osteoblast differentiation genes and proteins were examined through RT-PCR and Western-blot analysis.Finally,in order to further clarify the relationship between IBSP and PI3K/AKT signaling pathway,PI3 K,phosphorylated PI3K(p-PI3K),AKT,and phosphorylated AKT(p-AKT)levels were assessed by western-blot analysis in cells transfected with siRNA-IBST,siRNA-PI3 K ? siRNA-AKT,and subsequently the effected of PI3 K and AKT on expression level of osteoblast differentiation genes and proteins were examined and alkaline phophatase staining was performed.Results: Compared with control,the miRNA-92a-3p group showed that the expression of osteoblast differentiation genes and proteins were up-regulated,and osteoblast differentiation and matrix mineralization were markedly increased.However,the expression level of IBSP genes and proteins decreased,which made the expression level of PI3 K /AKT up-regulated.Conclusions: miRNA-92a-3p could promote osteogenic precursor cells activation and matrix mineralization by inhibiting PI3K/AKT signaling mediated by IBSP.Part ?:The effect of MIRNA-92A-3P on fracture healing in miceObjective: To assess the effect of miRNA-92a-3p on callus formation after the mice were received local injection of miRNA-92a-3p at the fracture site.Methods: Four mices per group were received local injection of agomiRNA-92a-3p,antagomiRNA-92a-3p and control,and local injection was performed at three time points(1 day,3 days,7 days)after femur fracture.CT examination was performed in 21 days after femur fracture to assess the BMD,BV,TV,and BV/TV of each group.Mice were sacrificed after m CT examination,and fracture end were harvested for HE-stained sections to observe the bone callus volume.Further,bone calluses at the fractured position were collected for western blot and PCR analyses which detected the expression level of Col1a1?ALP?OCN and Runx2 in 21 days after femur fracture.Results: Mice treated with agomiRNA-92a-3p exhibited a greater callus volume relative to control and mice with antagomiRNA-92a-3p,and they were also present a lack of any visible boundary between the hardened callus and the cortical bone,with clear evidence of remodeling.However,mice treated with antagomiRNA-92a-3p exhibited a lower callus volume and visible boundary between the hardened callus and the cortical bone.In addition,agomiRNA-92a-3p mice exhibited significantly higher BMD,BV,TV,BV/TV than did the animals in the other two groups.Furthermore,up-regulated expression of osteoblast differentiation genes and proteins were observed in miRNA-92a-3p group.Conclusions: Local injection of miRNA-92a-3p at the fracture site can promote fracture healing by up-regulated expression of osteoblast differentiation genes.