Study On The Role Of CircRNA＿31307 In Promoting Bone Formation By Exercise

Objective:Osteoporosis is a chronic bone disease characterized by systemic bone mass reduction and bone microstructure degradation,with increased bone fragility and high risk of fracture,the mechanism is the dynamic balance between osteoblast-mediated bone formation process and osteoclast-mediated bone resorption process is destroyed,the rate of bone resorption is greater than that of bone formation,resulting in bone loss.Postmenopausal osteoporosis accounts for a large proportion in the elderly population with osteoporosis,at present,exercise is the most economical and effective choice in the prevention and treatment of osteoporosis.Appropriate exercise has a positive effect on the process of osteogenesis through signal pathway or cytokines,which can be used as an effective means to prevent and treat osteoporosis,a large number of cell level studies have shown that appropriate mechanical stress can promote the osteogenic differentiation of cells.CircRNA,as one of the non-coding RNAs studied in recent years,has been proved to play an important role in regulating bone balance and improving osteoporosis,but its specific mechanism needs to be further explored.This study analyzed and screened the differentially expressed circRNA in bone of osteoporosis mice after exercise intervention,selected important circRNA that may participate in osteogenesis regulation for verification,analyzed their possible targets,and explored the potential regulatory pathways of circRNA participating in mechanical stimulation to promote bone formation.Methods:A total of 32 female C57BL/6 mice were raised to 12 weeks old,randomly divided into sham-operated group（Sham）,sham-operated plus exercise group（Sham+E）,ovariectomized group（OVX）,ovariectomized plus exercise group（OVX+E）,each group of 8.Mice in the OVX group and OVX+E group were ovariectomized,Sham group and Sham+E group mice cut the same size of fat around the ovary at the same position.After two weeks of molding,the mice in the Sham+E group and OVX+E group were subjected to a nine-week treadmill exercise intervention,the first week was pre-adaptation,and the last eight weeks were formal training.All mice were sacrificed at 48h after the end of the exercise intervention,the left femur was subjected to three-dimensional tomography and three-dimensional image reconstruction;decalcification,paraffin sectioning and HE staining were performed after the right femur was fixed for femoral morphology observation;the total RNA was extracted by grinding from the left tibia for circRNA chip hybridization,the key circRNA was screened for target gene prediction and pathway enrichment;the total RNA was extracted from the right tibia to pre-screen the circRNA and verify its miRNA target genes and osteogenic markers expression.Primary osteoblasts were extracted from the skulls of C57BL/6 mice within 3 days,and the osteoblasts were cultured for subculture,the osteoblasts were induced to differentiate into osteoblasts after 3 consecutive generations,which were 1,3,5,and 7 days respectively,and the alkaline phosphatase staining was performed,observing the effect of osteogenesis differentiation.Primary osteoblasts were extracted and cultured to the third generation,the cells were divided into stretch group（Stretch）and control group（Control）,the Stretch group cells were carried out for mechanical traction intervention of 3 consecutive days,4h per day,with a traction strength of 6%and a frequency of 0.5 Hz,the Control cells were cultured under the same conditions.At the end of the stretch intervention,the total protein and RNA of the cells were extracted immediately,the Western Blot experiment was carried out to detect the protein expression of osteogenesis markers in cells,and the expression levels of circRNA and downstream miRNA and osteogenesis markers in the cell were determined by qRT-PCR,possible regulatory mechanisms were explored.All experimental data are expressed as mean ± standard deviation（x± SD）,data processing is performed using IBM SPSS Statistics 26.0 software,one-way analysis of variance or independent sample T test,and Bonferroni method for post-testing.Statistical differences are considered to be statistically significant in p<0.05,all charts are made using GraphPad Prism 9.0 software.Results:1.Three dimensional tomography and reconstruction analysis were performed on the left femur of four groups of mice,it was found that the number of bone trabeculae in the OVX group compared with the Sham group mice was significantly reduced,the degree of separation was increased,the number of bone trabeculae in the two groups of exercise intervention group mice was increased,and the bone trabeculae volume fraction of the OVX group mice was significantly lower than that of the Sham group mice,and the bone trabecular volume fraction of the mice in the OVX+E group was significantly increased than the OVX group.After the HE staining of the femur distal,it can be observed that the structure of the mouse bone trabeculae in the Sham+E group is more clear and complete,and the number of bone trabeculae in the OVX group is significantly reduced,while the OVX+E group is slightly improved.2.CircRNA chip analysis was performed on osteoporosis mice,the five circRNAs with the highest differential expression multiple among each group were screened out,the circRNAs significantly up-regulated in OVX+E group than OVX group were mmu_circRNA₃3006,mmu_circRNA₄2484,mmu_circRNA₁9387,mmu_circRNA₃0190,mmu_circRNA₃1307.Then,according to the pathway enrichment and target gene prediction,the target circRNA of the study is circRNA₃1307,and its possible miRNAs are miR-484 and miR-7017-3p,which jointly target the key osteogenic marker Runx2.3.The circRNA₃1307,miR-484,miR-7017-3p,and osteogenic markers Runx2,Osterix,and ATF4 expressions in four groups of mice were detected by qRT-PCR,and the relative expression of circRNA₃1307 after ovariectomy was significantly reduced and increased after exercise intervention.The expression of miR-484 and miR-7017-3p were significantly increase after ovariectomy,and the expression of osteogenic markers decreased significantly after ovariectomy and increased after exercise intervention.4.ALP staining showed that the activity of ALP was the strongest and the degree of differentiation was the highest on the 7th day,and after 3 days of mechanical stretch intervention,the expression of circRNA₃1307 in cells increased significantly.The PCR results showed that after appropriate mechanical stretch intervention,the expression of osteogenic markers Runx2,Osterix and ATF4 in primary osteoblasts was significantly increased,while the expression of miRNA target genes miR-484 and miR-7017₃p affected by circRNA₃1307 was significantly reduced.Compared with Control cells,the protein expression of the osteogenic markers Runx2,Osterix,ATF4 in Stretch group cells was increased to varying degrees.Conclusion:1.After the osteoporosis model was established in mice,the bone microstructure changed,the volume fraction,number and thickness of trabecular bone decreased significantly,and the separation degree of trabecular bone increased significantly,ovariectomy resulted in osteopenia and microstructure degradation in mice,after exercise intervention,the above indexes were significantly improved,exercise promoted bone formation in osteoporotic mice.2.After osteoporosis molding and exercise intervention,the circRNA expression of the bone tissue of mice changed differently,and the expression of circRNA₃1307 in mice in the OVX+E group was significantly increased,and it was possible to target miR-484 and miR-7017-3p through osteogenic differentiation related pathways and act on osteogenic marker Runx2 to promote osteogenic differentiation.3.After suitable mechanical stretch intervention,the expression of circRNA₃1307 in primary osteoblasts was upregulated,the expression of its miRNA targets miR-484 and miR-7017-3p was downregulated,the relative expression of mRNA and protein of osteogenic markers Runx2,Osterix and ATF4 increased significantly,the regulation axis of circRNA-miRNA-mRNA may exist in the process of osteogenesis.