Study On The Effect Of LncRNA MSTRG24062.2 On Implant Osseointegration In Hyperlipidemia Rats

Background:Hyperlipidemia is a kind of metabolic disease characterized by elevated blood lipid levels.In hyperlipidemic rats,peri-implant osteoporosis and new bone formation were reduced,couple with decreased bond strength between bone and implant interface.Therefore,hyperlipidemia has become a potential risk factor affecting peri-implant osseointegration,and it is of great practical significance to study the effect of hyperlipidemia on implant osseointegration.Long non-coding RNAs(lncRNAs),as competitive endogenous RNAs(ceRNAs)or "miRNA sponges",can competitively bind miRNAs to participate in the microregulation of genes.Previous studies of our group have also confirmed that miRNA-125a-5p and miRNA-125b-5p can target FZD93’-Untranslated Regions(UTR)to inhibit the differentiation of bone marrow mesenchymal stem cells(BMSCs)into osteoblasts.Together with the previous RNA sequencing results of our group and bioinformatics software analysis such as Targetscan and miRanda,the uncharacterized lncRNA MSTRG.24062.2 was concluded predictably,which competitively binds to miRNA-125a-5p and miRNA-125b-5p.Therefore,this experiment was aimed to.investigate the specific mechanism by which lncRNA MSTRG.24062.2 competitively binds miRNA-125a-5p and miRNA-125b-5p,which affects osteogenic differentiation of BMSCs under high-fat environment and the implant osseointegration in hyperlipidemic rats.Purpose:(1)To study the differential expression of lncRNA MSTRG.24062.2 in the osteogenic differentiation of BMSCs under high-fat environment and its effect on osteogenic differentiation;(2)To verify the effect target in terms of the bonding of lncRNA MSTRG.24062.2 and miRNA-125a-5p,as well as lncRNA MSTRG.24062.2 and miRNA-125b-5p,and elucidate the regulatory mechanism on the competitively binding in terms of lncRNA MSTRG.24062.2 and miRNA-125a-5p,miRNA-125b-5p;(3)To elucidate the effect of lncRNA MSTRG.24062.2 on peri-implant osseointegration in hyperlipidemic rats.Methods:(1)BMSCs were induced by high-fat and ordinary osteogenic induction,and cells were collected at 3,5,7,and 14 days after induction.RT-qPCR was adopted to detect level of lncRNA MSTRG.24062.2,miRNA-125a-5p,miRNA-125b-5p,ALP,Runx2,PPAR-y and FZD9 mRNA in BMSCs from each group.The osteogenic differentiation of BMSCs was evaluated by alkaline phosphatase(ALP),alizarin red and oil red O staining combined with morphological detection.(2)Overexpressing lncRNA MSTRG.24062.2,the mRNA expression variation of ALP,Runx2,miRNA-125a-5p,miRNA-125b-5p and FZD9 were tested by RT-PCR.The expression variation of ALP,Runx2 and FZD9 protein in BMSCs was detected by western blot.The osteogenic differentiation of BMSCs was evaluated by alkaline phosphatase(ALP)and alizarin red.The target binding regions of lncRNA MSTRG.24062.2 with miRNA-125a-5p and miRNA-125b-5p were predicted by bioinformatics software.Then wild-type and mutant luciferase reporter vectors were constructed based on the predicted results,coupled with the validation of the predicted target binding regions,using the dual fluorescein reporter system.(3)The hyperlipidemia rat model was constructed and tested.After that the lncRNA MSTRG.24062.2 was overexpressed in hyperlipidemia rats and 1 mm periimplant bone tissue was obtained after 2 weeks.Specimens of each group were processed for Micro-CT scan for three-dimensional reconstruction and BV/TV analysis.Hard tissue sections were prepared and stained with HE to observe the bone interface and bone trabecular arrangement peri-implants,following that,PCR was adopted to detect the expression of lncRNA MSTRG.24062.2,miRNA-125a-5p,miRNA-125b-5p,FZD9,ALP and Runx2.Results:(1)Under the hyperlipidemia environment,the osteogenic differentiation of BMSCs was inhibited,and tissue morphology staining results showed that the formation of mineralized nodules decreased and the number of lipid droplets increased.At the same time,the expression of osteogenesis-related factors ALP and Runx2 decreased and adipogenesis-related factor PPAR-γ increased,indicating that the osteogenic differentiation of BMSCs was inhibited by the high-fat environment at the molecular level.And the expression levels of lncRNA MSTRG.24062.2 decreased,suggesting that the expression of lncRNA MSTRG.24062.2 is affected by the hyperlipidemia environment.(2)After lncRNA MSTRG.24062.2 was overexpressed in BMSCs,the expression of lncRNA MSTRG.24062.2,ALP,Runx2 and FZD9 increased and miRNA-125a-5p and miRNA-125b-5p decreased.Furthermore,the formation of mineralized nodules increased upon the overexpression of lncRNA MSTRG.24062.2 as indicated by alizarin red staining.By analyzing the activity results of dual-luciferase assay,lncRNA MSTRG.24062.2 was confirmed bound to the target sites of miRNA-125a-5p and miRNA-125b-5p.Thus LncRNA MSTRG.24062.2 promoted osteogenic differentiation of BMSCs by negatively regulating miRNA-125a-5p and miRNA-125b-5p and upregulating FZD9.(3)The level of serum lipids in the high-fat reared group was higher than which in the ordinary reared group.The BV/TV of the high-fat reared group was decreased with disordered bone arrangement and new bone.Overexpression of lncRNA MSTRG.24062.2 with lentiviral vectors increased BV/TV(P<0.05,dense bone arrangement,and increased new osteogenesis),as well as,lncRNA MSTRG.24062.2,FZD9,ALP,and Runx2 expression in peri-implant bone tissue increased,and miRNA125a-5b and miRNA-125b-5p expression decreased.Conclusion:(1)LncRNA MSTRG.24062.2 promotes the osteogenic differentiation of BMSCs by targeted binding of miRNA-125a-5p and miRNA-125b-5p.The inhibition of osteogenic differentiation of BMSCs in high-fat environment is related to the decreased expression of lncRNA MSTRG.24062.2.(2)Hyperlipidemia inhibits peri-implant osseointegration.The uncharacterized function of lncRNA MSTRG.24062.2 was confirmed for the first time to promoting osseointegration around implants in hyperlipidemia rats and the formation of new bone,revealing the potential value of lncRNA as a bone biomarker,which showed the possibility of targeting lncRNA MSTRG.24062.2 might be an effective method to treat osteoporosis under hyperlipidemia.