The Role Of MiR-214 In Mechanical Stress-induced Osteogenic Differentiation

Background:The incidence of osteoporosis is continuing to increase with a growing aging population,and it has become a major global public health problem.A large number of animal studies reported that exercise could decrease bone loss,increase bone mass,bone mineral density,geometric parameters and biomechanical characteristics of bone.Numerous experiment in vitro also showed that mechanical stress induced by exercise could promote osteogenesis,inhibit bone resorption,and improve bone metabolism while the exact mechanism of exercise in prevention and treatment of osteoporosis is not clear.MicroRNAs(miRNA)play a important role in the regulation of bone metabolism.Exercise can induce miRNA express differently in body,but there’s few research on exercise regulates bone metabolism through miRNA.Therefore,the present research synthesizes relative research and hypothesizes that exercise might induce expression of microRNAs in the bone that modulate the expression of osteogenic and bone resorption factors,leading to the positive impact of exercise on bone.MethodsPart 1 Effects of exercise on microRNAs and bone microstructure of tibia in mice1.Six male C57BL/6 mice were randomly assigned into exercise group and control group(3 mice each group).Exercise group was treated with 5 weeks moderate treadmill running.In the first week,the initial speed was 12 m/min,and the speed and running time increased up to 50min and 18 m/min at a 5° slope in the second week.Exercise training was performed 6 days per week with 1 day of rest throughout the program.The mice in the control group were housed under conventional conditions without exercise as control;2.Three right tibias were selected and scaned by microCT for Tb.N,Tb.Sp,Tb.Th,BV/TV,BS/BV and BS/TV;3.Total RNA extracted from three left tibias selected was used for miRNA sequence.And the miRNA differentially expressed was screened,and further verified by qPCR;Part 2 Effect of over-expression of miR-214 on osteogenic differentiation of osteoblast under mechanical strain1.Primary osteoblast from mice was treated with 3%elongation,0.5 Hz,SIN 1/2,4 h for 14 days in strain group,osteoblast in strain group was stimulated by mechanical strain on the 1st,3rd,5th,7th,9th,11th,and 13th day while control group was static culture.Total RNA of strain group and control group was extracted on the 1st,3rd,7th,and 14th day;2.The gene expression of miR-214 and osteogenic factors including ALP,OCN,Osterix,and ATF4 was tested by qPCR;3.Over-expression of miR-214 in osteoblast and observe its effects on the number of ALP positive cells,mRNA expression of osteogenic factors such as ALP,OCN and Osterix,and bone resorption factors like RANKL and RANK.The protein expression of Osterix and Pten was tested by WB.And the osteoblast was divided into control group(C),strain group(S),Agomir-214 control group(CA),Agomir-214 strain group(SA),Agomir-NC control group(CN)and Agomir-NC strain group(SN).Part 3 Effect of over-expression of miR-214 on osteogenic differentiation of BMSC under mechanical strain1.OVX osteoporosis mice were set up.And the primary BMSC from OVX mice was treated with 3%elongation,0.5 Hz,SIN1/2,4 h for 5 days in strain group.BMSC in strain group was stimulated by mechanical strain on the 1st,3rd,and 5th day while control group was static culture.Total RNA of strain group and control group was extracted on the 1st,3 rd,and 5th day;2.The gene expression of miR-214 and osteogenic factors including FGFR1,OCN,Osterix,β-catenin and ATF4 in BMSC from OVX mice was tested by qPCR;3.The gene expression of miR-214 and osteogenic factors including ATF4,OCN,β-catenin,Osterix,FGFR1 and LRP5,and relative bone resorption factors like RANKL,RANK,and adipogenic factors like PPAR-y and C/EBP-a in BMSC from growing mice under 5 days mechanical strain was tested by qPCR;4.Over-expression of miR-214 in BMSC from OVX mice and growing mice.And the gene expression of miR-214 and osteogenic factors including ATF4,FGFR1,OCN,Osterix,β-catenin,LRP5 and Runx2,and the relative bone resorption factors like RANKL,RANK,and adipogenic factors like PPAR-y and C/EBP-awas tested by qPCR.The protein expression of Osterix and Pten was tested by WB.And BMSC was divided into control group(C),strain group(S),Agomir-214 control group(CA),Agomir-214 strain group(SA),Agomir-NC control group(CN)and Agomir-NC strain group(SN).5.ALP staining and Alizarin red staining was used for evaluating ALP activity and calcium deposition of BMSC from growing mice after over-expression of miR-214 under mechanical strain.ResultsPart 1 Effects of exercise on microRNAs and bone microstructure of tibia in mice1.Exercise could promote the bone microstructure of tibia like BMD,BS/TV,BS/BV while decrease the level of Tb.Sp;2.Forty miRNA including miR-214,miR-146a and let-7a down-regulated while two miRNA up-regulated significantly in tibia after exercise(p<0.05);3.The miRNA differentially expressed was verified by qPCR and the results showed that miR-214,miR-199a and miR-30d down-regulated while miR-31 up-regulated significantly in exercise group compared to control group(p<0.01).Part 2 Effect of over-expression of miR-214 on osteogenic differentiation of osteoblast under mechanical strain1.3 days and 7 days mechanical strain could inhibit the mRNA expression of miR-214 and up-regulate the mRNA expression of ALP,Osterix and ATF4 in osteoblast(p<0.05);2.The number of ALP positive cells in strain groups(S/SA/SN)was much higher than ALP positive cells in control goups(C/CA/CN).And there’re more ALP positive cells in Agomir-NC groups(CN/SN)than in Agomir-214 groups(CA/SA);3.Compared to C and CN group,the mRNA expression of ATF4,LRP5 andβ-catenin increased significantly in osteoblast in S group and SN group.The protein expression of Osterix up-regulated,and the mRNA expression of M-CSF,RANKL and RANK decreased significantly in S group(p<0.05).Compared to SA group,the mRNA expression of Runx2,LRP5 and β-catenin up-regulated while the mRNA expression of RANK down-regulated significantly in SN group(p<0.05).Part 3 Effect of over-expression of miR-214 on osteogenic differentiation of BMSC under mechanical strain1.5 days mechanical strain could inhibit the mRNA expression of miR-214 and up-regulate the mRNA expression of FGFR1,OCN,Osterix,β-catenin and ATF4 in BMSC from OVX mice(p<0.05);2.5 days mechanical strain could up-regulate the mRNA expression of ATF4,OCN,P-catenin,Osterix,FGFR1 and LRP5,inhibit the mRNA expression of RANK,PPAR-y and C/EBP-a significantly(p<0.05),but could not decrease miR-214 level significantly in BMSC from growing mice(p>0.05);3.Compared to C and CN group,miR-214 level decreased significantly,and the mRNA expression of ATF4,FGFR1,OCN,Osterix,β-catenin,LRP5 and Runx2β-catenin increased significantly in BMSC from OVX mice in S group and SN group while the mRNA expression of RANKL,RANK and C/EBP-a down-regulated significantly(p<0.05).Compared to SA group,the mRNA expression of ATF4,Runx2,OCN,FGFR1,LRP5 and β-catenin up-regulated while the mRNA expression of RANKL,RANK and C/EBP-a down-regulated significantly in SN group in BMSC from OVX mice(p<0.05);4.Compared to control groups(C/CA/CN),miR-214 level in strain groups(S/SA/SN)could not decreased significantly(p>0.05)and the number of positive-ALP cell and calcium deposition increased obviously.The mRNA expression of osteogenic factors including ATF4,Runx2,LRP5 and FGFR1 up-regulated while the mRNA expression of RANK down-regulated significantly in strain groups(S/SA/SN).Compared to SA group,the mRNA expression of ATF4,Runx2,FGFR1,LRP5 and P-catenin up-regulated while the mRNA expression of RANK,M-CSF and PPAR-y down-regulated significantly in SN group in BMSC from growing mice(p<0.05).Conclusion1.Exercise could promote the BMD and bone microstructure,inhibiting the expression of miR-214;2.Mechanical strain could inhibit the expression of miR-214 in osteoblast and BMSC from OVX mice,promote the gene expression of osteogenic factors,and inhibit the gene expression;3.Over-expression of miR-214 inhibit the gene expression of osteogenic factors,promote the gene expression of bone resorption factors and adipogenic factors,and weaken the positive effect of mechanical strain on osteogenic differentiation of BMSC from OVX-mice and osteoblast but not from growing mice;4.The inhibition of miR-214 may be one of the ways in which exercise promote osteogenic differentiation of BMSC from OVX-mice and osteoblast.And exercise may not be the main way in which exercise promote osteogenic differentiation of BMSC from growing mice.