Activation Of Mmu-miR-142-5p Expression Supports Osteoblast Function During Fracture Healing In Mice

Chapter One mmu-miR-142-5p expression patterns in callus during fracture healing in miceObjectivesTo establish a bone fracture model in mice, and investigate mmu-miR-142-5p expression patterns in mice during fracture healing.MethodsA unilateral open femoral shaft transverse osteotomy with locking screw fixation procedure was performed on male,10to11-week-old C57BL/6mice. The characteristics of bone fracture healing of these mice were analyzed by X-ray, callus sections HE staining. The middle of femoral separated from mice which underwent no surgery were used as baseline control. Total RNA of callus and baseline control were extracted with Trizol reagent. The expression levels of osteocalcin, an osteoblast differentiation marker and mmu-miR-142-5p were detected by real-time quantitative RT-PCR. Time points were set by day7,14,21and28postoperatively.Results1. A unilateral open femoral shaft transverse osteotomy with locking screw fixation model for fracture healing study was successfully established in mice.2. Both intramembranous bone formation and endochondral calcification contributed to callus formation, but mainly through the later process. The healing process was completed within4weeks in this model. Bony callus nearly formed around day21postoperatively while osteogenesis was reaching a peak.3. The expression levels of osteocalcin mRNA in callus gradually increased during fracture healing in these mice, reaching a peak (4.15times of baseline value) around day21postoperatively, and then declined slightly. The functional status of osteoblasts represented through osteocalcin expression was consistent with radiological and histological analysis.4. The expression levels of mmu-miR-142-5p in callus gradually increased during fracture healing in these mice, and reached a peak (12.58times of baseline value) around day21postoperatively, and then declined slightly. Expression levels of mmu-miR-142-5p were synchronized with that of osteocalcin mRNA.ConclusionsActivation of mmu-miR-142-5p expression in callus during fracture healing might be associated with osteoblasts activity. Chapter Two The action of mmu-miR-142-5p on osteoblast functionObjectivesTo investigate the role of mmu-miR-142-5p on osteoblast mineralization, and explore the mechanism of mmu-miR-142-5p involved in osteoblast mineralization.MethodsMC3T3-E1cells, representatives of pre-osteoblasts, were induced to differentiate and mineralize in the presence of β-glycerophosphate (β-GP) and ascorbic acid. To establish the mmu-miR-142-5p gain-of-function or loss-of-function in vitro model, MC3T3-E1cells were consecutively and directly transfected with agomiR-142-5p, agomiR-NC, antagomiR-142-5p and antagomiR-NC respectively at day5,11,17of mineralization induction. Total cellular RNA was extracted by Trizol reagent48hours after first transfection, and the expression of alkaline phosphates (ALP), osteocalcin and mmu-miR-142-5p were detected by real-time quantitative RT-PCR. Meanwhile, the culture supernatant of MC3T3-E1cells in each group was collected for ELISA to detect protein levels of ALP and OC secreted into the medium. Matrix mineralization in each model was assessed by Alizarin Red S staining and quantitative at day21of mineralization induction, which is4day after the third transfection. Various miRNA target prediction software tools including TargetScan were used to predict the target genes of mmu-miR-142-5p. WWP1, a putative target of mmu-miR-142-5p involved in osteoblast differentiation and mineralization was initially confirmed by utilizing real time RT-PCR and Western blotting.Results1. Exogenous nucleotides agomir-142-5p or antagomir-142-5p transfection could be used to modulate mmu-miR-142-5p abundance and activity in MC3T3-E1cells.2. Compared with the controls, MC3T3-E1cells transfeced with agomiR-142-5p enhanced ALP and OC expression in both mRNA and secreted protein levels, and promoted the formation of mineralized nodules shown by increased Alizarin Red quantitative. Compared with the control, MC3T3-E1cells transfeced with antagomiR-12-5p reduced ALP and OC expression in both mRNA and sectreted protein levels, and blocked the formation of mineralized nodules presented by decreased Alizarin Red quantitative.3. WWP1was predicted as the target gene of mmu-miR-142-5p involved in osteoblast mineralization.4. Compared with the controls, MC3T3-E1cells transfected with agomiR-142-5p reduced endogenous WWP1protein levels without affecting its mRNA levels. Compared with the control, MC3T3-E1cells transfected with antagomiR-12-5p upregulated endogenous WWP1protein levels without affecting its mRNA levels. Conclusionsmmu-miR-142-5p supports osteoblast function and mineralization through downregulating WWP1expression in a post-transcriptional level.