The Effect Of HIF-1α Activation On Fracture Repair And Hypoxia-Induced MiR-429 On Regulating Osteoblastic Differentiation Of MC3T3-E1 Cells

ObjectivesWith the development of the aging of the population, the incidence of fracture is increasing. However, Elderly patients with functional decline, reduced angiogenesis impaired fracture healing. Nonunion and delayed union become a common complication of fracture. Fracture healing is a complex process, vascular injury and hematoma after fracture leads to the tissues and cells have to repair bone in hypoxia. HIF-1α is the most direct factor under hypoxia to regulat lots of downstream target genes, they can control the angiogenesis, energy metabolism, cell proliferation and differentiation. So we observed the effect HIF-1α activation on fracture repair in vivo.At the same time, there are kinds of microRNA to control precisely in hypoxia. They play a very important role in maintaining the activity of cells in the hypoxic environment. Thus, we focus on miR-429, to explore its mechanism on regulating osteoblastic differentiation of MC3T3-E1 cells.Methods 1. A total of 48 SD rats were divided into two groups and treated with Co Cl2 orsaline, respectively. Rats with tibial fractures were sacrificed at 7, 28 and 42days subsequent to fracture. Then the bone structure, bone strength, hypoxiarelated factors and osteogenic marker luciferase assessed by Radiograph,biomechanical analysis,RT-PCR, Western blot, and immunohistochemistry. 2. To mimic the effects of hypoxia, 200 μM CoCl2 was added to the medium, thenwe detected the cell proliferation and apoptosis, and observe the expression ofmiR-429 in MC3T3-E1 cells through the qRT-PCR. MiR-429 overexpressionlentivirus, verified by qRT-PCR was prepared in HEK293 T cells and theninfected MC3T3-E1 cells. We evaluated the effect of miR-429 overexpressionon osteogenic differentiation by ALP assay, alizarin red staining. 3. Bioinformatic analysis was performed with TargetScan, miRBase onlinesoftware, ZFPM2 was considered as a putative target of miR-429, we detectedthe mRNA and protein level of ZFPM2 after mi R-429 overexpression. Thenluciferase reporter assays were performed in MC3T3-E1 cells to test whetherZFPM2 was a direct target of miR-429. 4. MC3T3-E1 cells were transfected with ZFPM2 small interfering RNA(si RNA),the effects of ZFPM2 knockdown was confirmed by Western blot. Then weevaluated the effect of siZFPM2 on osteogenic differentiation by ALP assay,alizarin red staining and Western blot. 5. To determine whether mi R-429 could trigger fracture repair in vivo, as observedin cultured cells, Lentivirus was injected into the subcutaneous region of localfracture, and radiographic examination was taken after 28 days. Then wecollected the fracture zone to assess the bone structure, mi R-429、ZFPM2andosteogenic marker by Radiograph, RT-PCR, Western blot. Results 1. The bone structure, bone strength, hypoxia related factors(HIF-1α, VEGF) andosteogenic markers(ALP, Runx2, OC)were higher in CoCl2 treated group thancontrol group. 2. The expression of mi R-429 was upregulated by CoCl2 in MC3T3-E1 cells, withthe peak value at 4 hours. CoCl2 treatment had no significant effect on cellproliferation and apoptosis. Overexpression of miR-429 greatly increased inmineralized nodule formation and ALP activity compared to the control virus.ALP, OC, RUNX2 expression levels were also significantly increased withoverexpression of miR-429. 3. ZFPM2 expression at both the mRNA and protein levels was reduced upon miR-429 overexpression in MC3T3-E1 cells. The real-time PCR assay showedthat the mRNA level of ZFPM2 was decreased and inversely correlated withmiR-429 expression in MC3T3-E1 cells treated with CoCl2. MiR-429 markedlyreduced firefly luciferase activity of reporter plasmids containing the wild-typeZFPM2 3’ UTR or ZFPM2-MUT2. 4. Knockdown of ZFPM2 in MC3T3-E1 cells increased the ALP activity compared to the negative control. Mineralization was significantly higher at 21 days in the ZFPM2 knockdown cells compared to the NC cells. ZFPM2 silencing stronglyinduced the expression of ALP, OC and RUNX2 and resulted in a decrease ofGATA4. 5. MiR-429 was found to remarkably enhance new bone formation during the course of fracture repair compared to control vector. The expression of miR-429, Runx2, OC was significantly increased in mice infected with the miR-429 lentivirus and resulted in a decrease of ZFPM2. Conclusions 1. The results of the present study demonstrated that CoCl2 enhances fracture healing. It was demonstrated that CoCl2 induces bone and cartilage formation, increases tissue vascularization and may activate the HIF-1α pathway. 2. MiR-429 was induced by CoCl2 in MC3T3-E1 cells, and it promoted preosteoblastic cell differentiation. We additionally identified ZFPM2 as a direct target of miR-429 involved in osteoblast differentiation.