The Role Of LncRNA H19 In The Promotion Of Osteogenesis By Mechanical Force

VBackgroundOsteoporosis is a bone metabolic disease characterized by decreased bone density and destruction of bone tissue microstructure.With the accelerated progress of my country’s aging society,the incidence of osteoporosis is increasing year by year,and the medical,care,and economic burdens caused by osteoporotic fractures are gradually increasing.Osteoporosis has gradually become a global clinical problem that needs to be solved urgently.Public health issues.Appropriate intensity exercise training,as a treatment plan that can improve all fracture risk factors(bone strength,fall risk,fall impact)that can be changed,has become a research hotspot in the treatment of osteoporosis.At present,the mechanism of exercise for preventing and treating osteoporosis is still being explored continuously.Long-chain non-coding RNAs,especially lncRNA H19,have been confirmed to play a regulatory role in bone metabolism,bone tumors and other bone physiological and pathological processes,but the mechanism of action in the prevention and treatment of osteoporosis has not been fully elucidated.In this study,an animal model of osteoporosis was prepared by ovariectomized surgery,and progressive treadmill exercise was used to intervene to study the effect of exercise on bone mineral density and bone biomechanics of ovariectomized mice.In the in vitro experiment,mechanical stretch stimulation was used to interfere with bone marrow mesenchymal stem cells and knock down the expression of lncRNA H19 to observe the changes in downstream pathways and osteogenic differentiation.The expression of mechanosensitive protein Antxr1 was further knocked down and overexpressed to study the effect of lncRNA H19 on osteogenic differentiation and downstream pathways after being regulated by Antxr1 under mechanical stretch stimulation.This study integrated the above experiments to explore the mechanism of lncRNA H19 in the process of mechanical stress regulating osteogenic differentiation to prevent and treat osteoporosis.Methods and MaterialsPart 1: The effect of exercise on bone density and strength of ovariectomized mice1.C57BL/6 mice were modeled for ovariectomy at the age of 12 weeks,and were randomly divided into control group,control + exercise group,ovariectomized group,ovariectomized + exercise group,with 8 mice in each group.At 16 weeks of age,a 9-week progressive treadmill exercise(formal treadmill exercise intensity is 8-15m/min,60min/d,slope 25°,5d/week).2.After the treadmill is finished,take materials,use mouse bilateral femur and tibia as specimens,and use micro CT to detect bone density(trabecular bone,cortical bone and whole bone),trabecular bone volume,and trabecular bone thickness;use universal Detect the maximum load,fracture load and deflection;use paraffin sections and HE staining for bone morphology;use RT-q PCR to detect the expression of lncRNA H19,osteogenic markers and related indicators mRNA in bone tissue;use Westen Blot technology to detect bone Expression of osteogenic markers and Wnt/β-Catenin signaling pathway in tissues.Part 2: The effect of mechanical stretch on the osteogenic differentiation of bone marrow mesenchymal stem cells and the regulation of lncRNA H191.Detect the effects of different traction strengths on the osteogenic differentiation of BMSC.The primary bone marrow mesenchymal stem cells of C57BL/6 mice were extracted for culture,and the Flexcell-5000 stretching system was used for mechanical stretching of the cells,using 4% deformation strength,2h,4h,6h,8h and 6%deformation strength,2h,4h,6h,8h mechanical stretch to detect osteogenic indicators and the expression of lncRNA H19.2.After knocking down the expression of lncRNA H19,observe the effect of mechanical stretch on the osteogenic differentiation of bone marrow mesenchymal stem cells.M-H19 Smart Silencer is used to knock down the expression of lncRNA H19,and use the best plan from the previous experiment for mechanical stretch intervention to detect osteogenic markers,Wnt/β-Catenin signaling pathway and micro RNA141,675,188,22 To study the role of lncRNA H19 in the process of mechanical traction to promote osteogenic differentiation and potential downstream micro RNAs.Part 3: The effect of knockdown and overexpression of Antxr1 on the osteogenic differentiation of lncRNA H19 and bone marrow mesenchymal stem cells1.SiRNA is used to knock down the expression of Antxr1 in bone marrow mesenchymal stem cells and perform mechanical stretch to detect the expression of lncRNA H19,osteogenic markers and Wnt/β-Catenin signaling pathway,and study the regulatory effect of Antxr1 on lncRNA H19 and The effect on osteogenic differentiation.2.ADV-Antxr1-OE is used to overexpress Antxr1 and mechanically stretch bone marrow mesenchymal stem cells to detect the expression of lncRNA H19,osteogenic markers and Wnt/β-Catenin signaling pathway,and further study the regulation of lncRNA H19 by Antxr1 Role and influence on osteogenic differentiation.ResultsPart 11.Micro CT showed that compared with the control group,the femoral trabecular bone mineral density and trabecular bone volume percentage of the ovariectomized mice were significantly reduced,and the cortical bone mineral density and total bone mineral density were significantly reduced.The above indicators of the ovariectomized exercise group were significantly increased compared with the ovariectomized group.It shows that exercise can increase bone density,and exercise can improve the reduction of bone density caused by ovariectomy.2.The three-point bending experiment was used to test the bone biomechanics.The maximum load,fracture load and deflection of the femur of the ovariectomized mice were significantly lower than those of the control group.The above indicators in the ovariectomized + exercise group increased significantly,indicating that exercise can improve the ovariectomized Biomechanical properties of ovarian mouse bone.3.The results of HE staining showed that the number of bone trabecula in the bone marrow cavity of the ovariectomized mice decreased,and the fat cells increased.Compared with the control group,the control + exercise group and the ovariectomized+ exercise group had more bone trabeculae and significantly reduced fat cells.4.Perform mRNA and protein detection on bone tissue samples.The expressions of lncRNA H19,Wnt1,β-catenin and osteogenic markers OCN,Runx2,and OSX in the bone samples of the ovariectomized mice were reduced.The above indicators in the exercise group were compared with the corresponding controls Compared with the group,the expression of Antxr1 increased;the expression of Antxr1 increased in the ovariectomized mice and decreased in the exercise group.Part 21.mRNA and protein detection showed that the expression of osteogenic markers OCN and Runx2 increased with the increase of mechanical stretch strength,but when the stretch strength was greater than 4% deformation strength 6h/d and 6% deformation strength 4h/d,Bone effect will no longer increase.In this experiment,the best stretch plan to promote osteogenic differentiation of bone marrow mesenchymal stem cells is6% deformation strength,a single stretch time of 4h,a frequency of 0.5Hz,and continuous stretch for 7 days.2.After knocking down the expression of lncRNA H19,mRNA and protein detection showed that the expression of Wnt1,β-catenin,osteogenic markers OCN and Runx2 in the inhibitor group decreased,and the expression of the above indicators in the inhibitor + stretch group increased.ALP staining showed that the ALP activity of the inhibitor group decreased,and the ALP osteogenic activity of the inhibitor+stretch group increased.3.After knocking down the expression of lncRNA H19,the expression of micro RNA 141,675,188 in the inhibitor group increased,and the expression of the above three micro RNAs in the inhibitor + stretch group decreased.Compared with the control group,the expression of the above three micro RNAs in the control + stretch group was reduced,but there was no statistical difference.Part 31.After knocking down the expression of Antxr1,the detection of mRNA and protein showed that the expression of lncRNA H19,Wnt1,β-catenin,osteogenic markers OSX and Runx2 in the inhibitor group increased,and the expression of the above indicators in the inhibitor + stretch group increased.ALP staining showed that ALP activity increased after Antxr1 was knocked down.2.After the overexpression of Antxr1,the mRNA and protein detection of the overexpression group showed that the expression of Wnt1,β-catenin,osteogenic marker OSX,and Runx2 decreased,and the expression of the above indicators in the overexpression + stretch group increased.ALP staining showed that ALP activity decreased after overexpression of Antxr1.Conclusion1.Moderate-intensity progressive treadmill exercise can increase the bone density and bone biomechanical properties of C57BL/6 normal mice and ovariectomized mice by promoting bone formation,effectively preventing postmenopausal osteoporosis and possibly reducing osteoporosis Occurrence of sexual fractures.2.Mechanical stretch stimulation can promote the osteogenic differentiation of bone marrow mesenchymal stem cells.The best stretch plan is 6% deformation strength,4h/d,0.5Hz,and continuous stretch for 7 days.lncRNA H19 promotes the osteogenic differentiation of bone marrow mesenchymal stem cells by positively regulating the Wnt/β-Catenin signaling pathway,and negatively regulates micro RNA141,675,and188.3.Antxr1 can positively respond to mechanical stretch stimuli,and negatively regulate the expression of lncRNA H19 and the osteogenic differentiation of bone marrow mesenchymal stem cells.The mechanism may be related to the negative regulation of Wnt/β-Catenin signaling pathway.