| Osteoporosis(OP)is a type of chronic metabolic bone disease that characterized as low bone mineral density(BMD),deterioration of bone microarchitecture and increased bone fragility the loss of bone density.Up to date,the most common recognised diagnosis option for OP was Dual Energy X-ray absorptiometry(DEXA)scan.Given the latent development of OP and the caused irreversible decrease of BMD,early diagnosis of OP is of great potential for clinical and public health in the prevention and treatment of OP.Exosomes(Exos)are novel identified extracellular vesicles,which enable to contain several types of biological small molecules,such as microRNAs(miRNAs),message RNAs(mRNAs),long non-coding RNAs(lncRNAs).Once diseases or pathlogical conditions did happen,the component of Exos could show apprarent changes.Previous literatures have indicated that the altered expression of exosomal miRNAs involved in the mediation of information transmition between different types of cells and regulated the expression of target genes,thus,participating in the pathogenesis and development of a large number of human diseases.Given the previous findings,in the present study,it containd two chapters,the first chaptar was aimed to screen the differential expressions of exosomal miRNAs between OP cases and healthy controls,and the exosomal miR-130a-3p was selected for validation in a large study population,in order to confirm if there were statistical differences of exosomal miR-130a-3p between OP cases and healthy controls.The second chaptar was performed to investigate the effects of exosomal miR-130a-3p on the differentiation of osteoblasts and BMD of ovariectomy(OVX)mice through in vivo and in vitro experiments,further unveiling the cellular and molecular function of exosomal miR-130a-3p on OP.Part â… ObjectiveThe study population was recruited for a community-based epidemiological follow-up study for osteoporosis(OP),the differential expressions of exosomal microRNAs(miRNAs)in plasma were screened in a small sample size between OP cases and healthy controls,and then were validated in an independent study population with a large sample size.In addition,we have also measured the plasma free expression of validated miRNAs.MethodsBiological samples including plasma and urine were collected from all of the elderly population who participated in this follow-up study.The measurements of bone mineral density(BMD)in lumbar(L1 to L4),hip and femoral neck(FN)were implemented by using Dual Energy X-ray absorptiometry(DEXA)scan.Considering the gender differences on the influence of prevalence of OP,the study subjects were elderly women.A total of 200 study subjects(including 100 low-BMD samples and 100 high-BMD samples)were selected.After excluding the influences of other diseases and conditions,there were 9 low-BMD and 9 age-matched high-BMD subjects that were included in further tests.Each 3 plasma samples from high-BMD or low-BMD subjects were randomly mixed into a pool.The Exosomes(Exos)in plasma were extracted,and the RNA of Exos were purified for initial screening of differential expressions of exosomal miRNAs.As for the significantly exosomal miRNAs in plasma,we have also selected 30 low-BMD and 30 age-matched high-BMD subjects and extracted the plasma Exos for first validation.To avoid the instability caused by small sample size,futher second validation for significantly exosomal miRNAs was conducted in a large population that included 100 low-BMD and 100 age-matched high-BMD subjects.In addition,we have also randomly selected 72 low-BMD and 72 high-BMD sujects from the study population of second validation,and expressions of the plasma free miRNAs were also detected.ResultsThe initial screen has identified a number of 15 differential expressions of plasma exosomal miRNAs.After the first and second validations,it found that the expressions of miR-130a-3p were significantly increased in plasma Exos of elderly women with OP(all P<0.05).Furthermore,we have also investigated the expressions of plasma free miR-130a-3p,and the results showed that there were also increased levels of plasma free miR-130a-3p in elderly women with OP.Correlation analysis revealed that the CT values of exosomal miR-130a-3p were positively associated with FN and hip BMD(T values),but the CT value of plasma free only showed the positive relations with hip BMD(T values).The increase of both exosomal miR-130a-3p and plasma free miR-130a-3p levels were related with the decreased BMD.Conclusions1 There were increased levels of miR-130a-3p in both plasma and plasma Exos of elderly women with OP as compared to healthy controls.2 The levels of plasma exosomal miR-130a-3p were correlated with the FN and hip BMD,but the plasma free miR-130a-3p only showed an association with hip BMD.The increases of plasma exosomal miR-130a-3p and plasma free miR-130a-3p levels were associated with the decreased BMD in elderly women with OP.Part â…¡ObjectiveBased on the previous fingdings in Part 1,we perfomed cells and animal experiments to investigate the biological effects of miR-130a-3p on osteoblasts(MC3T3-E1),and to evaluate the changes of bone mineral density(BMD)in ovariectomy(OVX)mice when treated with AgomiR-130a-3p or AntagomiR-130a-3p by intravenous injection.In addition,we have also predicted and validated miR-130a-3p regulated targe genes,which may partily explain the molecular regulate mechinsm of miR-130a-3p in osteoporosis(OP).MethodsThe high and low expressions of miR-130a-3p in MC3T3-E1 cells were achieved by liposome transfection of AgomiR-130a-3p and AntagomiR-130a-3p.The effect of miR-130a-3p on osteoblastic differentiations were determined by qRT-PCR and alkaline phosphatase(ALP)activity staining.Cell counting kit(CCK)8 was used to evaluate the effect of miR-130a-3p on the proliferation of MC3T3-E1 cells,Annexin V-PE/7AAD double-staining kit and cell cycle detection kit were used for cell apoptosis and cell cycle alteration detection,respectivey.Animal experiments were performed in the use of C57BL/6 female mice,bilateral ovariectomy(OVX)was conducted to constructe OVX mouse model.Plasma Exos were extracted from 16-week-old OVX and wild type(WT)mice,and the expression of plasma exosomal miR-130a-3p was detected by qRT-PCR.In addition,to investigate the potential sources of miR-130a-3p,the expressions of miR-130a-3p were detected in different tissues/organs of both OVX and WT mice(including heart,liver,spleen,lung,kidney,aortic vascular tissue,femoral skeletal muscle tissue and bone marrow).Considered the Exo-miRNAs database and our findings,we confirmed that vascular endothelial cells may be regarded as one of the sources of cells.The cell culture supernatant of the vascular endothelial cell line(b.END)was collected,the Exos in the supernatant were extracted and labeled with fluorescent membrane dye and added into the culture medium of MC3T3-E1 cells to observe whether the Exos secreted by b.END cells could be endocytosed by MC3T3-E1 cells.Lentivirus overexpressing miR-130a-3p was constructed and used to transfect b.END cells(LV-miR-130a-3p b.END)to achieve high expression of Exo-miR-130a-3p in b.END cells.LV-miR-130a-3p b.END cells were co-cultured with MC3T3-E1 cells,after adding exosomes secretion inhibitor(GW4869),the mRNA levels of osteoblastic differentiation markers were examined.Nanomaterial aptamers complex conjugated with AgomiR-130a-3p or AntagomiR-130a-3p were used to mimic the function of Exos,and then were injected into the mice via the tail vein.After injection,mouse femurs were isolated and examined for femoral BMD by microCT.Target gene prediction and validation of miR-130a-3p were performed by using miRNA database,qRT-PCR and Western Blot(WB)were used to examine the mRNA level and protein expression of target gene,respectively.Dual fluorescent reporter system was applied to detect whether miR-130a-3p could bind to the target gene.ResultsTransfection with AgomiR-130a-3p or AntagomiR-130a-3p showed significantly higher or lower levels of miR-130a-3p in MC3T3-E1 cells,respectively.The elevated levels of miR-130a-3p decreased mRNA expression of ALP,collagen type I(Coll),osteocalcin(OCN)and Runt-related transcription factor 2(Runx2),but increased the mRNA expression of osteopontin(OPN)(all P<0.05).The decrease of miR-130a-3p showed a significant increase in the mRNA expression of ALP,Coll,OCN and Runx2(all P<0.05).After 7 days of transfection with AgomiR-130a-3p or AntagomiR-130a-3p,ALP staining results showed a lighter stained in AgomiR 130a-3p group than AgomiR NC group,while AntagomiR-130a-3p group showed a slightly darker stained than AntagomiR NC group.Cell proliferation assay revealed that AgomiR-13 0a-3p could inhibit the proliferation of MC3T3-E1 cells.In addition,flow cytometry revealed that AgomiR-130a-3p enabled to incude the apoptosis of MC3T3-E1 cells.Cell cycle assay results did not find any differences of AgomiR-130a-3p and AntagomiR-130a-3p on the influence of G1,G2 and S stages of MC3T3-E1 cells.In addition,during the osteoblastic differentiation of MC3T3-E1 cells,we found significantly decreased levels of miR-130a-3p.Theses evidence suggested that high levels of miR-130a-3p could inhibit the differentiation of MC3T3-E1 cells by suppressing cell proliferation and inducing apoptosis.Plasma exosomal miR-130a-3p was significantly elevated in OVX mice compared to WT mice,which is consistent with the results of our previous findings of the olderly women OP population in Part I.In addition,we found that there were significantly elevated miR-130a-3p levels in spleen,lung,bone marrow,aortic vascular endothelial tissue and liver of OVX mice compared to WT mice.Considering the results from Exosomes database,we confirmed that the sources of exosomal miR-130a-3p might derive from vascular endothelial cells.When extracted and labelled the Exos from the supernatant of b.END cells and added labbled Exos into the cell culture of MC3T3-E1 cells,we observed that the Exos secreted by b.END cells could be endocytosed by MC3T3-E1.By transfecting b.END cells with lentiviral overexpression of miR-130a-3p(LV-miR-130a-3p),we found that exosomal miR-130a-3p was significantly elevated in the supernatant of LV-miR-130a-3p b.END cells culture as compared to LV-NC b.END cells.Cells co-culture(LV-miR-130a-3p b.END or LV-NC b.END in the upper cell layer,MC3T3-E1 cells in the lower cell layer)experiments,adding GW4869(an inhibitor that can inhibit Exo secretion)or DMSO(a solvent control),found that LV-miR-130a-3p b.END/MC3T3-El+DMSO group showed a significant decrease in the mRNA levels of ALP,OCN and Runx2 in MC3T3-E1 cells,this results confirmed that exosomal miR-130a-3p could also exert an inhibitory effect on the differentiation of osteblasts.All of the OVX mice were randomly assigned into four groups,and then injected with Aptamer-conjuncted AgomiR-130a-3p/AgomiR NC or AntagomiR-130a-3p/AntagomiR NC,respectively.The measurements for femoral BMD revealed that,AgomiR-130a-3p group had significantly decreased levels of femoral BMD in compared to the AgomiR NC group,however,as compared to AntagomiR NC group,AntagomiR-13 0a-3p group showed a significant increase in femoral BMD.These findings of in vivo study indicated that the increased miR-130a-3p levels can aggravate bone loss in OVX mice.Nevertheless,the inhibitors of miR-130a-3p can partially reverse and increase BMD in OVX mice.The target genes of miR-130a-3p were predicted by RNA database,and the target genes of estrogen receptor 1(ESR1),transforming growth factor beta receptor 1(TGFBR1)and TGFBR2,which involved in the bone metabolic process,were selected for further qRT-PCR and WB validation.The results revealed that AgomiR-130a-3p could inhibit both the mRNA and protein expressions of ESR1,TGFBR1 and TGFBR2 genes,while AntagomiR 130a-3p could promote the expressions of ESR1,TGFBR1 and TGFBR2 genes.Dual fluorescein reporter systems demonstrated that miR-130a-3p could bind to ESR1,TGFBR1 and TGFBR2 genes.Conclusions1.miR-130a-3p could reduce osteogenesis process by inhibiting proliferation and differentiation of osteoblasts and inducing osteoblasts apoptosis,contributing to the onset and development of OP.2.Plasma exosomal miR-130a-3p was significantly higher in OVX mice than that of control mice.3.Exosomal miR-130a-3p may accumulated and secreted from vascular endothelial cells,and vascular endothelial cells derived Exos could also be endocytosed by osteoblasts.4.Increased exosomal miR-130a-3p from vascular endothelial cells also showed an inhibitory effect on osteoblastic differentiation.5.AgomiR-130a-3p could aggrevate the bone loss and reduce BMD in OVX mice,wheras,AntagomiR-130a-3p showed the ability to inhibit the bone loss and increase the BMD in OVX mice.Inhibition of miR-130a-3p may serve as a promising target for the treatment of OP.6.The function of miR-130a-3p in the development of OP might be realized by suppressing the expression of ESR1,TGFBR1 and TGFBR2 genes... |
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