| Purpose:Relevant research shows that the aging population is the most susceptible group of orthopedic diseases.In all cases,the most common diseases include fractures caused by osteoporosis and osteoporosis.There data indicate that nearly 15% of the people,over the age of 50,suffer from osteoporosis or fracture disease in the world.Every 30 seconds,there will be a fracture patient(Caused by osteoporosis).And if the disease investigation object is the special occupational groups such as astronauts or warriors,the incidence of osteoporosis and fractures will be higher.Therefore,faced with such a high incidence,the related diseases cannot be ignored.In view of the slow healing speed of fracture treatments and the difficulties of osteoporosis treatment;We design related experiments: Screening for small molecule regulators(microRNAs)that regulate the formation of a negative regulatory gene(CKIP-1),confirming the targeting relationship and effects on bone formation at the cellular and animal levels.Cell experiments have confirmed the regulation of differentiation of mesenchymal stem cells(BMSCs);it has been shown to promote bone formation(fracture,osteoporosis)in animal experiments.The related experiments are expected to provide a new theoretical basis for the treatment and rehabilitation of modern orthopedic diseases.Method:Based on the previous experimental basis,several miRNAs that can regulate CKIP-1 were screened.After that,we extracted bone marrow mesenchymal stem cells(BMSCs)from rat femur,and confirmed the let-7i-5p could regulate the osteogenic differentiation of BMSCs by cell infection.In animal experiments,according to the previous study that mir-98-5p target CKIP-1 in BMSCs.The conclusion show that the regulation of osteogenic differentiation is obvious,the gene modification and molecular targeted design of miR-98-5p promoter "agomir-98-5p" can be injected into the bone defect mice and osteoporosis mice,which can be obvious.Promotes miR-98-5p in mouse bone tissue and inhibits expression of the CKIP-1 gene.Attempts to inhibit the expression of CKIP-1 gene in vivo have been used to promote the process of bone formation in mice.Result:1.Screening for potential miRNAs targeting CKIP-1 during osteogenic differentiation of BMSCsThe miRNAs most closely related to the CKIP-1 gene were screened.These candidate genes are: miR-687,miR-1961,let-7i-5p,miR-98-5p,miR-3072-3p.In order to confirm the role of these miRNAs in cell osteogenic differentiation,BMSCs were isolated,purified and identified.Osteogenic differentiation of bone marrow mesenchymal stem cells was promoted by special medium for osteogenic differentiation.The changes of five candidate miRNAs before and after osteogenic differentiation were detected by biomolecular means.The results showed that let-7i-5p and miR-98-5p were more obvious.These results suggest that these two CKIP-1-targeting miRNAs are likely to play an important role in regulating osteogenic differentiation of bone marrow mesenchymal stem cells;regulating miRNA expression or permitting regulation of osteogenic differentiation-related signaling pathways.2.Let-7i-5p can promote osteogenic differentiation of BMSCs by targeting CKIP-1 expression.Further explore the role of let-7i-5p in the osteogenic differentiation of BMSCs.BMSCs cells were transfected with let-7i-5p mimics and miR-NC,respectively.After transfection for two days,the transfection efficiency was detected by RT-PCR and fluorescence identification.It is indicated that rat BMSCs have significantly promoted the expression of let-7i-5p.After 48 hours of normal culture,the mRNA expression levels of the marker genes associated with osteogenic differentiation of BMSCs were significantly higher than NC group(RUNX2,OSX,OCN,BSP).Relevant data indicate that overexpression of let-7i-5p regulates osteogenic differentiation of BMSCs cells.To further confirm the regulation of let-7i-5p,we examined changes in the expression of CKIP-1 after the expression of let-7i-5p in mesenchymal stem cells.Promote or reduce the expression of let-7i-5p,respectively,using the corresponding miRNA mimics or inhibitor to treat BMSCs cells,and detect the transfection efficiency by fluorescence.The results showed that after treatment with let-7i-5p mimics or let-7i-5p inhibitor,RT-PCR and Western blot analysis demonstrated that let-7i-5p overexpression reduced the expression of CKIP-1 in bone marrow mesenchymal stem cells,while after inhibition of let-7i-5p,CKIP-1 mRNA and protein expression was increased.These results indicate that the expression of Let-7i-5p can significantly regulate CKIP-1 gene expression and alter the osteogenic activity of BMSCs.Combined with the conclusion that the let-7i-5p is highly targeted to the CKIP-1 gene.Therefore,we conclude that let-7i-5p can target the CKIP-1 gene to regulate osteogenic differentiation of BMSCs.3.Targeting promotes the expression of miR-98-5p in bone tissues of fractured mice and tail-suspended mice,which can effectively promote bone formation.RT-PCR and WB assays confirmed that the miR-98-5p expression was promoted in experimental group.moreover,the osteogenic differentiation-related genes(ALP,RUNX2,OCN)of the mouse femur were more active;X-ray scan results showed that the experimental group revealed the more rapidly speed in bone Healing(fracture line and epiphyseal morphology);HE staining showed that the experimental group had more strengths in bone structure;Three-point mechanical test the mice that 1 week after tail suspension,the mechanical properties of the bones showed that the mechanical properties of the femur in the experimental group were significantly different in bending load,bending strain and fracture energy.The Micro-CT test confirmed that the bone density and bone structure of the experimental group in the tail suspension which was better than the control group and the sham experimental group;the above-mentioned bone tissue test results can confirm that the miR-98-5p regulator can target and regulate the CKIP-1 gene,which can significantly improve the bone formation process in mice.Conclusion:We screened several miRNAs which most closely related to CKIP-1 by bioinformatics methods and RT-PCR.Moreover,it was found that let-7i-5p and miR-98-5p changed significantly during the osteogenic differentiation of BMSCs.Cell-experiments confirmed that the target gene of the small molecule regulator let-7i-5p is CKIP-1;and let-7i-5p targeting CKIP-1 can promote bone marrow mesenchymal stem cell osteoblast differentiation.In animal-experiments,we demonstrated in the mouse folding model and mouse tail suspension model that targeting the promotion of miR-98-5p expression can significantly promote the bone formation process in mice and effectively slow the bone loss caused by weightlessness.The development of the experiment can explore new ideas for molecular treatment for astronomer's osteoporosis and fracture repair of wounded,and provide corresponding theoretical data for the treatment and repair of clinical bone diseases. |
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