| Background:Aging is often accompanied by a gradual decrease in bone mass and bone strength,which usually leads to related diseases such as senile osteoporosis.Bone marrow mesenchymal stem cells(BMSCs)are multifunctional stem cells that can differentiate into chondrocytes,osteoblasts,and fat cells.Their differentiation potential is very important for maintaining the normal balance of bone metabolism in the human body.As the number of aging BMSCs cells decreaseing,their potential to differentiation into osteoblasts in the bone marrow is reduced,and age-related secretion phenotype molecules are secreted into the bone microenvironment,inhibiting their osteogenic differentiation,which leads to age-related loss of bone mass,eventually leading to osteoporosis.Therefore,to determine the regulation mechanism of age-related BMSC aging and osteogenic differentiation direction is of great significance for the treatment of senile osteoporosis.Mature bone tissue is maintained by the balance between bone formation of osteoblasts,derived from BMSCs,and bone resorption by osteoclasts,produced by hematopoietic stem cells(HSCs).In addition to these cellular-level mechanisms,bone function is also subjected to epigenetic control mechanisms,including the regulation of genes through DNA methylation,MiRNA transcriptional regulation,and chromatin architecture modification.MiRNA,considered to be an important molecule for gene regulation in epigenetics,controls the dimension of skeletal cell remodeling by inhibiting mRNA translation after transcription.However,at present,there is limited research on how MiRNA mediates the interaction between different cells and the same kind of cells in regulating bone remodeling,and the role of MiRNA in regulating bone cell differentiation and functional maturation of BMSCs or HSC remains to be elucidated.MicroRNAs(MiRNA,miRs)are a class of small endogenous(19-25 nt)non-coding RNAs that bind to the 3’untranslated region(UTR)of mRNA and negatively regulate their targets by inhibiting translation or disrupting mRNA stability Gene expression.Studies have shown that MiRNA controls a wide range of physiological and pathological processes,and several MiRNA have been found to participate in and affect the bone formation process.Davis et al.reported that the expression of MiR-183-5p in bone-derived extracellular vesicles increasesd with age,reduced the proliferation,osteogenic differentiation of BMSCs,and promoted their aging.Li et al et al.had shown that MiR-188 regulateed the transition between osteogenic differentiation and adipocyte differentiation during osteoblast senescence.The overexpression of BMSCs-specific MiR-188 in mice reduced bone formation and increased bone marrow fat accumulation.Through targeted regulation of FoxO1,MiR-182 is a negative regulator of osteoblast proliferation,differentiation and bone formation,while MiR-216a promoted osteoblast differentiation and enhanced bone formation.Despite these findings,the mechanism of MiRNA-induced senescence of bone marrow mesenchymal stem cells and reduced osteogenic differentiation capacity still needs further study.In the early experiments,we used the target prediction software TargetScan to screen out the abnormal expression of MiR-384-5p in aging rat BMSCs,but its effect on age-related osteoporosis and its mechanism of action have not been reported.Therefore,further research on the specific mechanism of action of MiR-384-5p in the direction of osteogenic differentiation and cell senescence of BMSCs,and to clarify the effect of MiR-384-5p on the occurrence and progression of osteoporosis,will not only help to deeply understand the pathogenesis of osteoporosis,and at the same time provide a potential therapeutic target for age-related osteoporosis.Method and result:Part Ⅰ Biological effects of MiR-384-5p and the effects of aging and osteoporosis in BMSCs of rats.1.MiR-384-5p expression in BMSCs of aged and ovariectomized rats.In order to evaluate the relationship between MiR-384-5p and the aging and osteoporosis in rats,we measured the expression of MiR-384-5p in BMSCs of young(3 month old)and old(21 month old)rats by qRT-PCR.The results showed that MiR-384-5p was significantly upregulated in older rats compared to young rats.In addition,we constructed a rat model of the sham-operation group and the ovarian ovariectomy group,and verified the successful modeling by TRAP and Masson staining.At the same time,qRT-PCR detection results showed that MiR-384-5p expression was significantly higher in the bone marrow mesenchymal stem cells of the ovariectomized group,compared with the sham-operated group.2.In vivo injection of KD-MiR-384-5p can delay the progress of osteoporosis in elderly rats.In order to further evaluate the effect of MiR-384-5p on the progress of osteoporosis,an in-vivo experiment with KD-MiR-384-5p or KD-NC injected into the femoral bone marrow cavity of an 18-month-old rat was used.Three months after administration,qRT-PCR analysis confirmed that the expression of MiR-384-5p in BMSCs of rats,treated with KD-MiR-384-5p,was significantly reduced,compared with the KD-NC group.In addition,Micro-CT analyzed the surface of rat tibia skull volume.Compared with the control group,MiR-384-5p knockdown significantly increased the bone volume and number of trabecular bones in elderly rats,and reduced the separation of trabecular bones.PartⅡ MiR-384-5p regulated the internal mechanism of BMSCs aging and osteogenic differentiation.1.MiR-384-5p overexpression can promote the expression of aging factors and inhibit osteogenic differentiation of rat BMSCs.The study used MiR-384-5p overexpressed lentiviral particles(OE-MiR-384-5p)and negative control lentiviral particles(OE-NC)to transfect rat BMSCs cells.Verification of the establishment of MiR-384-5p overexpression cell model in BMSCs was executed by qRT-PCR;analysis of osteogenic differentiation ability of BMSCs was based on quantitative calcium deposition of Westin red staining(ARS);Aging status was assessed by β-galactosidase staining(SA-β-gal);Detection of osteogenesis and aging factor expression was executed by qRT-PCR and WB.The results showed that compared with the OE-NC group,MiR-384-5p expression of BMSCs in the OE-MiR-384-5p group was significantly increased,calcium salt deposition was significantly less,and showed more cell senescence,while the mRNA and protein expression levels of specific genes Ocn,Alp and Osx were significantly reduced,while the expression of senescence-related factors p21 and p16 was higher.2.MiR-384-5p knockdown can inhibit the expression of BMSCs aging factor and promote its osteogenic differentiation.In this study,MiR-384-5p knockdown lentiviral particles(KD-MiR-384-5p)and negative control lentiviral particles(OE-NC)were used to transfect rat BMSCs cells.The results showed that compared with OE-NC group,MiR-384-5p expression of BMSCs in KD-MiR-384-5p group was significantly reduced,calcium salt deposition was significantly increased,the number of cell senescence was reduced,and bone-specific genes Ocn,Alp and Osx MRNA and protein expression levels was increased significantly,while the expression of aging-related factors p21 and p16 was decreased.Part Ⅲ.MiR-384-5p Regulated BMSCs Aging and Osteoblastic Differentiation through GLI2.1.MiR-384-5p binded to Gli2’s 3’UTR and inhibits its expression.To determine whether mili-384-5p down-egulated Gli2,we constructed a luciferase reporter vector,containing the Wt or Mut MiR-384-5p target sequence of Gli2 3’UTR.MiR-384-5p overexpression significantly inhibited the luciferase activity of the Wt Gli2 3’UTR reporter gene,but not the Mut reporter gene.In addition,qRT-PCR and WB detection results in cell experiments showed that BMSCs in the OE-MiR-384-5p group significantly reduced the expression of Gli2 at both mRNA and protein levels,while BMSCs in the KD-MiR-384-5p group increased.Gli2 mRNA and protein expression.2.Inhibition of MiR-384-5p can up-regulate Gli2 and promote the osteogenic differentiation of BMSCs.In order to determine the role of GLI2 in MiR-384-5p-mediated differentiation and senescence of BMSCs,BMSCs were used to co-transfect cells with shGli2 and shGli2+KD-MiR-384-5.Analysis by qRT-PCR and Western blot analysis showed that the mRNA and protein expression levels of Gli2 in BMSCs of shGli2 group were significantly reduced;the results of ARS and SA-β-gal staining showed that BMSCs of shGli2 group showed less calcium salt deposition and more Cell senescence;simultaneous qRT-PCR and WB detection results showed that the mRNA and protein expression levels of the specific genes Ocn,Alp and Osx in BMSCs of the shGli2 group decreased significantly,while the expression of senescence-related factors p21 and p16 increased significantly.However,the BMSCs in the shGli2+KD-MiR-384-5 group reversed the above biological effects of cells.In conclusion:In this study,we observed that MiR-384-5p was significantly upregulated in BMSCs of elderly and ovariectomized rats compared to young rats.The vivo tests have shown that by inhibiting the action of MiR-384-5p,bone loss can be prevented and the osteogenic ability of elderly rats can be improved.The vitro functional assays showed that after overexpression of MiR-384-5p in BMSCs of young mice,they can inhibit the differentiation of BMSCs into osteoblasts and accelerate their aging,while knocking down MiR-384-5p in aging BMSCs had the opposite effect.In addition,we demonstrated that MiR-384-5p directly inhibited the expression of Gli2 at the mRNA and protein levels by directly binding to the 3’untranslated region(3’UTR)of Gli2 mRNA.By inhibiting the expression of MiR-384-5p,Gli2 can restore the osteogenic ability of BMSCs.Overall,our research showed that MiR-384-5p can inhibit the differentiation of BMSCs osteoblasts and accelerate their aging by targeting Gli2,and as a negative regulator of bone formation,eventually promoting the progress of osteoporosis.Therefore,inhibition of MiR-384-5p function is a treatment strategy for age-related osteoporosis. |
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