Regulation Of MiRNAs On Wnt/β-catenin Signaling Pathway In Plastrum Testudinis Preventing And Treating GIOP

Objective:1.The differential expression of miRNAs in vertebrae of GIOP patients was screened and the target genes related to Wnt/β-catenin signaling were predicted by bioinformatics analysis forpreliminarily revealing the pathogenesis of GIOP.2.To investigate the role of plastrum testudinis(PT)in the prevention and treatment of GIOP,and to explore regulation of miRNA(let-7f)on Wnt/β-catenin signaling pathway in plastrum testudinisagainst GIOP.3.To investigate the effect of extracts of of plastrum testudinis on the proliferation and osteogenic differentiation of BMSCs and the targeting effect of let-7f on TNFR2.Methods1.Clinical research(1)High-throughput sequencing for miRNA and target gene predictionSix patients undergoing spinal surgery from Spine Orthopedic of the first Aff iliatedHospital of Guangzhou University of Chinese Medicine,three of which were suffer from GIOP and other patients were in the normal control group,wereincluded in this study from 20140ct.-2015 Oct.The total RNAs were extracted from the vertebrae for miRNA screened by high-throughput sequencing.Bioinformatics for clustering analysis of differential miRNAs and predictingits target genes was for GO analysis,Pathway analysis and prediction of new miRNAs.(2)RT-qPCR validated differential miRNAs by high-throughput sequencing screening,while Wnt/β-catenin pathway-related target genes were predicted20 patients undergoing spinal surgery fromthe first Affiliated Hospital ofGuangzhou University of Chinese Medicine,10 of which were suffer from GIOP and other patients were in the normal control group,wereincluded in this study from 20140ct.-2016 Oct.RT-qPCR validated differential miRNAs by high-throughput sequencing screening,while Wnt/β-catenin pathway-related target genes were predicted by bioinformatics.2.Animal Experimental Studies200 female SD rats aged 3 months were randomly assigned to two experiments:preventive and therapeutic experiments,in which rats respectively received oral PT at the same time of glucocorticoid injection(preventinve experiment)or after glucocorticoid injections inducing osteoporosis(therapeutic experiment).In the two experiments,rats were euthanized for experimental analysis at the end of M1,M2,M3 of oral PTE,respectively.(1)The role of PTin the prevention and treatment of GIOP① The body weight,uterus,bilateral adrenal glands were weighed after sacrifice.②The Bone mineral densityof L1-3 separated from rats were analyzed using dual-energy X-ray absorptiometry.③ L2separated was scanned by a cone-beam-type desktop micro-CT.After scan,cancellous bone of the vertebrae was chosen as the volume of interest(VOI)and the three-dimensional images were obtained through multi-planar reformation.④Subsequent to micro-CT analysis,L2 vertebrae were subjected to compression test todetermine themechanical parameters with a materials testing machine.Both end-plates of the vertebrae body and its appendix were removed to obtain acentral cylinder with planoparallel ends and height of approximately5 mm.⑤L4 vertebraes were fixed in 4%phosphate-buffered paraformaldehyde for more than 24 hours and then were decalcified with 10%EDTA for 4-6 weeks.The samples were dehydrated by standard graded alcohol solutions and embedded in LEICA High melt Paraffin.Bone tissues in paraffin were sectioned longitudinally to 5um thin sections.Then HE stains were carried outto use for histomorphometry.⑥ TRAP stains were carry out for elvaluating the osteoclastic activity on bone surface(2)Effect of PT on AKP activity and bone turnover markers of serum①Blood was collected fromabdominal aorta and serum wereobtained by centrifuge.Serum were use to assessment of AKP activity.② ELISA tests were carried out for expressive level of PINP and β-CTX.(3)Regulation of PT on expression of let-7f and genes and protein related to Wnt/β-catenin signaling pathway in veterbrae of rats① Both end-plates of the vertebrae body,its appendix were removed and soft tissue were removed in liquid nitrogen preservation.②RT-qPCR was detected the gene expression of let-7f、TNFR2、GSK3β、β-catenin、AP-1、CTSK、MMP9、OPG、Runx2、SP7.③IHC was used for observing the protein expression of Runx2、CTSK.④Western-blot was detected the protein expression of TNFR2、GSK3β、β-catenin、Runx2、CTSK.3.BMSCs experimental study(1)Role of PTE on proliferation and osteogenic differentiation of BMSCsBMSCs were obtained from the femur or tibial of 4-week-old SD rats,cultured and passaged.CCK8 was used to detect the proliferation of BMSCs at 1,3,5,7,14 and 21 days of PTE intervention.The optimal concentration of PTE was selectedfor osteogenic induction and the effect of PTE on osteogenic differentiation of BMSCs was detected by ALP staining and alizarin red staining.(2)The targeting relationship between let-7f and TNFR2TNFR2(TNFRSF1B)mutant vector and TNFR2(TNFRSF1B)3’UTR vector constructed were co-transfected with NC mimics,let-7f-5pmimics using Lipofectamine TM 2000 reagent,and the dual luciferase reporter assay system was used to verify the targeting effect of let-7f-5p on TNFRSF1B.Results1.Clinical research(1)High-throughput sequencing for miRNA and target gene prediction Eighteen miRNAs of differential expression were screened out by high-throughput sequencing,of which 2 miRNAs were up-regulated and 16 miRNAs were down-regulated.By predicting target genes of differentialmiRNA in miranda,mirbase and targetscan databases,488 common target genes ofup-regulated miRNAs and 475 common target genes of down-regulated miRNAs was found.In addition,three new miRNAs were foundon chromosome 1,11 and 12,respectively.(2)RT-qPCR validated differential miRNAs by high-throughput sequencing screening,while Wnt/β-catenin pathway-related target genes were predictedAccording to the sequencing results and the original data standardized tag reads,selected 25 miRNAs were validated.hsa-miR-186-5p,hsa-miR-21-5p,hsa-miR-214—5p,hsa-miR-10b-5p and hsa-miR-451a were significantly up-regulated while hsa-let-7f-5p,hsa-let-7a-5p,hsa-miR-27a-3p were down-regulated significantly(P<0.05),of which the significant difference in let-7f expression was most of all,and TNFR2 targeted by let-7f was closely associated with Wnt/β catenin signaling pathway.2.Animal Experimental Studies(1)preventive and therapeutic effect of plastrum testudinis on GIOP in rats①Comparisonof body weight,adrenal weight,and uterine weight among all groupsIn preventive experiment,weights of body,adrenal and uterine in GIOP rats were significantly lower at M1,M2 and M3 than that in SHAM group(P<0.05);whereas weights of body,adrenal and uterine in PT or PT+ALN group were higher significantly than that in GIOP group at the same time points(P<0.05).In therapeutic experiment,in GIOP group body weight at Ml and M2 and adrenal weight at M1 are remarkably lowered than in SHAM group,while adrenal weight in all therapeutic groups were higher significantly than in GIOP group at M1.② bone mineral density of lumbar vertebra in ratsIn preventive experiment,BMD in GIOP group was significantly lower at Ml,M2 and M3 while AREA and BMC were significantly lower at M1 and M2 than that in SHAM group(P<0.05).BMD in PT or PT+ALN group was higher significantly than that in GIOP group(P<0.05),and there was no significance among the therapeutic groups.In therapeutic experiment,both BMC and BMD were significantly reduced in GIOP group compared with SHAM group(P<0.05).At M1 both AREA and BMC are remarkably elevated in the therapeutic groups while both BMC and BMD are remarkably elevated at M3 compared to GIOP group(P<0.05),and AREA in both PT and ALN groups are remarkably elevated compared to GIOP group(P<0.05).③ Effects on bone microarchitecture of lumbar vertebra in all groupsIn preventive experiment,BS/TV,Tb.N,Tb.Th and vBMD of GIOP rats were significantly decreased while SMI and Tb.Sp were significantly increased compared with SHAM group(P<0.05)SMI in both PT and PT+ALN groups are significantly reduced whereas Tb.Th in PT group and Tb.N in PT+ALN group are notably increased compared to GIOP group(P<0.05).In therapeutic experiment,BV/TV,Tb.Th and vBMD are significantly lowered while SMI and Tb.Sp were significantly higher in GIOP group that in SHAM group(P<0.05).BS/TV,Tb.N,Tb.Th and vBMD were significantly elevated whereas SMI and Tb.Sp were reduced significantly in therapeutic groups compared with GIOP group.In both preventive and therapeutic experiments,compared to SHAM group,bone microstructure in GIOP group rats were severely deteriorative,while microstructure in therapeutic groups are improved as presented by both micro-CT 2D and 3D images.④Effect on bone biomechanics of lumbar vertebra in all groupsIn preventive experiment,compressive strength were significantly reduced in GIOP group compared with SHAM group(P<0.05).Both PT and PT+ALN groups showed higher compressive strength and PT groupshowed remarkably elevated compressive stiffness in comparison with GIOP group(P<0.05).In therapeutic experiment,compressive strength were significantly lower in GIOP group than that in SHAM group(P<0.05).Compressive strengths and energy absorption values of all therapeutic group rats are notably elevated compared to GIOP group(P<0.05).⑤ Improvement on histological structure of lumbar vertebra in all groupsIn both preventive and therapeutic experiments,compared to SHAM group at several time points,trabeculas in GIOP group were destroyed in different degrees,which manifested as sparse,inconsecutive,fractured and wide space,and accompanied by sparse osteoblast and osteoclast in trabecula surface,reduced stem cells and full adipocytes in marrow cavity.Trabecula deterioration could be improve to some extent in both PT and PT+ALN groups at several time points.⑥osteoclastic activity in lumbar vertebra in all groupsAt different time points in both preventive and therapeutic experiments,lumbar vertebra of GIOP group showed lower osteoclastic activity at different time points particularly at M3 than SHAM does as presented by positive TRAP staining.TRAP stainings in all therapeutic groups showed enhanced osteoclastic activity with aging compared with GIOP group,and in both PT and PT+ALN groups TRAP staining showed stronger osteoclastic activity than in ALN group.(2)Effect of PT on serum AKP activity and bone turnover markers in GIOP rats①Results of serum AKP activity of each groupAt M1 and M2 in preventive experiment,compared with SHAM group,GIOP group showed significant higher AKP activity(P<0.05).Compared with GIOP group,PT group and PT+ALN group showed significantly low AKP activity(P<0.05).In therapeutic experiment,compared with SHAM group,GIOP group showed higher AKP activity at M1(P<0.05).And compared with GIOP group and PT group at M2,PT+ALN group showed significantly high AKP activity(P<0.O5).②Comparison of PINP and β-CTX levels among each groupIn preventive and therapeutic experiment,compared with SHAM group,GIOP group showed rising trend in PINP and β-CTX levels,though no significant difference was observed.Compared with GIOP group,each therapeutic group showed the downtrend in PINP and β-CTX levels,though no significant difference was observed.(3)Regulation of PT on let-7f and Wnt/β-catenin related factors in rats vertebrae①mRNA expression of let-7f,TNFR2,GSK3β,β-catenin,AP-1,CTSK,MMP9,OPG,Runx2 and SP7 in preventive and therapeutic experimentevaluated by RT-PCRAt M3 time point in preventive and therapeutic experiment,compared with SHAM group,GIOP group showed significantly low let-7f and β-catenin expression levels(P<0.01),significantly high TNFR2 and GSK3 β expression levels(P<0.05),and showed the down trending in AP-1,CTSK,MMP9,OPG,Runx2 and SP7 expression levels,though no significant difference was observed.Compared with GIOP group,PT group and PT+ALN group showed significantly high let-7f and β-catenin expression levels(P<0.05),significantly low TNFR2 and GSK3 β expression levels(P<0.05),and showed the rising trend in AP-1,CTSK,MMP9,OPG,Runx2 and SP7 expression levels,though no significant difference was observed.②Protein expression of Runx2 and CTSK evaluated by IHCAt M3 time point in preventive and therapeutic experiment,compared with SHAM group,GIOP group showed significantly lower Runx2 expression and higher CTSK expression.Compared with GIOP group,each therapeutic group showed higher Runx2 expression and lower CTSK expression,and PT+ALN group showed the most significantdifference.③Protein expression of TNFR2,p-GSK3β,p-β-catenin,Runx2 and CTSK of each group in preventive and therapeutic experiment evaluated by WBIn preventive and therapeutic experiment,compared with SHAM group,GIOP group showed significant higher TNFR2 and p-β-catenin expression(P<0.01),significant lower p-GSK3β expression(P<0.01).Compared with GIOP group,PT group and PT+ALN group showed significant lower TNFR2 and p-β-catenin expression(P<0.05)and significant higher p-GSK3β expression(P<0.05).3.BMSCs experimental study(1)The effect of extracts from plastrum testudinis(PTE)on the proliferations and osteogenic differentiations of BMSCsThe proliferations of BMSCs increased with the increasing of the concentration of the PTE intervention in the first week,whereafter the proliferations in each groups showed dose-dependent reduction.Theoptimal concentration of PTE intervention is 30ug/ml.After osteogenic differentiated in two weeks,the positive staining of ALP in PTE group was stronger and the number of positive cells increased obviously.Alizarin red staining showed the number of positive staining cells in PTE group was obviously more than in the control group.(2)The targeting relationship between let-7f and TNFR2In transfected TNFRSF1B 3’ UTR vector cells,the expression of TNFRSF1B that co-transfereed let-7f-5p mimics was significantly down-regulated than which co-transfereed NC mimics(P<0.001);However,there is no statistical difference between the expression of TNFRSF1B that co-transfered let-7f-5p mimics and NC mimics in the vector cells transfered mutant TNFRSF1B-3;UTR(P>0.05).Conclusion1.Clinical researchTwo up-reglulated and 16 down-regulated miRNAs were screened out by high-throughput sequencing.After valided by RT-qPCR,miR-186-5p、miR-21-5p、miR-214-5p、miR-10b-5p、miR-451a were up-reglulated while let-7f-5p、let-7a-5p、miR-27a-3p were down-reglulated,which might play an important regulatory role in the pathogenesis of GIOP.Based on the results of sequencing data,verification results and bioinformatics analysis,and according to KEGG Pathway data,We predicted that Let-7f-5p targeting TNFR2 to regulate Wnt/β-catenin pathway may be,to a certain extent,a pathogenesis of GIOP.2.Animal Experimental Studies(1)PT,which could control GIOP obviously,combined with ALN have shown a good joint effect and advantages in bone mass,bone microstructure,bone biomechanics and bone histomorphology and other aspects;(2)In.this study,AKP activity showed a tendency to increase first and then decreased gradually regardless of the application or withdrawal of GC in rats,while PT or PT+ALN can reverse the effect of the beginning ofGC-induced AKP activityand later GC-induced AKP activity decreased.(3)This study has verified that inthe GIOP rat lumbar spine,gene expression of Let-7f,β-catenin were down-regulated while TNFR2 and GSK3 β were up-reglulated,and at the protein level,expression of TNFR2 and p-β-cateninwere upregulated while p-GSK3 3 was down,which resulted in the decrease of free β-catenin in the cytoplasm,the degradation of β-catenin phosphate and the inhibition of Wnt/β catenin signling pathway.And finally we detected the decrease of the expression of Runx2 protein in the nuclear transcription factor.PT or PT+ALN can reverse the GC-induced let-7f down-regulation and Wnt-β-catenin pathway inhibition,which thus verifying our hypothesis.3.BMSCs Experimental Studies(1)This study verified the proliferation and osteogenic differentiation of BMSCs by CCK8 test,ALP and alizarin red staining(2)It was found that let-7f-5p targets the binding site of TNFRSF1B(TNFR2)gene by dual luciferase reporter assay,and it clarified the targeting effect of let-7f-5p on TNFR2.This result may lay the foundation for investigating the effect of let-7f-5p on Wnt/β-catenin pathway.