| Objectice:The purpose of this study is to screen out the lncrna which is closely related to the pathogenesis of postmenopausal osteoporosis,and to verify and explore the possible mechanism and molecular mechanism of different lncrna in the pathogenesis of osteoporosis.Furthermore,the intervention effect of icariin was further studied to explore the effect of Icariin on the above differential lncrna.It provides a new molecular marker for genetic diagnosis of osteoporosis and a new potential target for the treatment of osteoporosis.Methods:(1)According to the inclusion criteria,6 postmenopausal patients with traumatic hip fracture were selected to construct the culture system of human BMSCs,which were identified by flow cytometry and osteogenic and adipogenic differentiation induction.BMSCs were extracted from the two groups of patients.High throughput sequencing technology was used for sequencing and bioinformatics analysis to find the differential lncrna,and qPCR technology was used to verify the differential lncrna.(2)To establish ovariectomized osteoporosis rat model and verify the correlation between the changes of differential lncrna and osteogenic ability;The intervention effect of Icariin on ovariectomized osteoporosis rats was observed by intragastric administration.He staining and micro CT were used to observe the bone microstructure;The effects of Icariin on Runx2,OCN,colla and lncrna were observed by PCR and WB.(3)BMSCs were transfected with overexpression and knockdown plasmids to observe the effect of differential lncrna on osteogenic differentiation of BMSCs,and the effect of differential lncrna on osteogenic markers was observed by PCR and WB;The differential lncrna was located by fish;Clinical samples and animal samples were used to predict and verify the downstream miRNA,and further analyze the correlation;The targeted binding of lncrna and miRNA was verified by luciferase and pull down experiments;After CO transfection of miRNA and lncrna mimics,osteogenesis was induced.Furthermore,the target genes were found by transcriptome high-throughput sequencing and related bioinformatics analysis,and the relationship between the target regulatory axis was comprehensively analyzed and verified by bioinformatics site prediction,luciferase binding site prediction and mRNA complementation experiments.Result:(1)Bone marrow mesenchymal stem cells were successfully extracted from postmenopausal osteoporosis patients,and their biological level was verified.The results of lncrna sequencing of BMSCs were analyzed and different lncrnas were screened.RT qPCR was used to further verify the results.Compared with normal samples,lncrna or 1111,lncrna slc9a4,lncrna SFRP4,lncrna kcna3 and lncrna hcg27 were significantly increased in the sequencing results,lncrna samd12,lncrna pp1r9b,lncrna neatl,lncrna tim3 and lncrna malatl were significantly decreased in the sequencing results,and lncrna tim3 was selected as the research target of downstream experiment.(2)In OVX rat bone tissue and BMSCs,the endogenous expression levels of lcnrna tim3 and related osteogenic markers decreased,but increased during the osteogenic differentiation of BMSCs;Through the intervention of icariin,it was observed that icariin could improve bone microstructure by promoting osteogenic differentiation and the expression of related factors.(3)The results showed that lncrna tim3 could promote the osteogenic differentiation and the expression of osteogenic markers;The downstream miRNA of lncrna tim3 was predicted and verified to be mirnal22-5p by bioinformatics software and luciferase;Fish was used to verify the localization of tim3 in the cytoplasm,and the mechanism of Cerna was speculated;It was found that lncrna tim3 was negatively correlated with mirna-214-5p expression,and its targeted binding was verified.Lncrna tim3 can promote osteogenic differentiation by adsorbing mir-214-5p expression;Smad4 was identified by mRNA sequencing;Through the prediction of Shengxin site and luciferase,the targeted binding ability of Smad4 was successfully verified,and the specific regulatory relationship between Smad4 expression and lncrna tim3 adsorption of mir-214-5p expression to promote osteogenic differentiation was confirmed through the complementation experiment.Conclusion:LncRNA TIM3/mir-214-5p/samd4 axis plays an important role in PMOP.Icariin treatment can alleviate PMOP by regulatingLncRNA TIM3/mir-214-5p/samd4 axis. |
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