| Objective:Osteoporosis(OP)is a systemic metabolic bone disease characterized by decreased bone mass,destruction of bone microstructure,increased bone brittleness and easy to cause brittle fracture.Prevention and treatment of OP have always been problems that need to be solved for clinicians.Bone marrow mesenchymal stem cells(BMSCs)are the common precursors of osteoblasts and adipocytesand play an important role in bone metabolism.Mi RNAs can participate in bone formation and angiogenesis by targeting various transcription factors in stem cells,osteoblasts,osteoclasts and chondrocytes.As a new potential therapeutic target or biomarker of OP,miRNAs have received rapid clinical attentionand.More and more studies have shown that miRNAs play an important role in osteogenic differentiation of OP,such as miR-346,miR-20 a,miR-29 b,miR-433 and so on.At present,it has been reported that micro RNA-1286(miR-1286)has been confirmed to be related to the occurrence and progress of cancer and osteoarthritis,but its role in OP has not been studied.We found that the serum of OP patients overexpressed miR-1286,and the expression of miR-1286 gradually decreased after different time stages of osteogenic differentiation of h MSCs,so we speculated that miR-1286 may be related to the progress of OP.Relevant studies suggest that miR-1286 may participate in the process of osteogenic differentiation through some signal pathways(such as NF-k B,Wnt/?-catenin,oxidative stress).However,whether miR-1286 is involved in the occurrence and development of OP has not been confirmed.Frizzled related protein 4(FZD4)can participate in the process of cell proliferation and differentiation through different signal pathways(oxidative stress,methylation,Wnt/?-catenin signal pathway),and affect the direction and process of cell differentiation of BMSCs.There is a binding site between miR-1286 and FZD4 from the Starbase3.0 website,but there is no relevant report on whether there is a correlation between miR-1286 and FZD4 in the regulation mechanism of osteogenic differentiation of BMSCs.Therefore,the purpose of this study is to explore and analyze the relationship between miR-1286 and OP,and to clarify the role of miR-1286 in the regulation of OP bone remodeling by BMSCs osteogenic differentiation and its related mechanism,and to provide a new therapeutic direction for the prevention and treatment of OP.Methods:The first part:(1)Select 50 healthy adults and 50 OP patients,collect 5ml of blood for EDTA anticoagulation,and use real-time quantitative polymerase chain reaction(q RT-PCR)to detect the expression of serum miR-1286 in patients with osteoporosis.(2)Culture the BMSCs cells of OP patients in vitro,culture them with mesenchymal stem cell-osteogenic differentiation medium(MODM).The growth,survival and differentiation of BMSCs were observed.And use alkaline phosphatase(ALP)staining and alizarin red staining to characterize the differentiation of BMSCs in patients with OP.(3)q RT-PCR was used to detect the expression of miR-1286 during the differentiation of BMSCs in patients with OP.The second part:(1)BMSCs from 50 patients with OP were subcultured for 3-4generations and then induced into osteogenic differentiation with MODM medium.They were divided into the overexpression miR-1286 group(miR-1286-mimic group),the control group(miR-NC group),the low-expressing miR-1286 group(miR-1286-Inhibitor group).The BMSCs samples of each OP patient were made 3replicate holes.(2)The miR-1286-mimic group were transfected with miR-1286 to form a BMSCs differentiation environment overexpressing miR-1286;the miR-NC group were transfected with anti-miR-NC for the control study;the differentiation environment of BMSCs with low expression of miR-1286 was formed by transfection of miR-1286 in miR-1286-Inhibitor group.The MODM medium were replaced every2-4 days and cultured for 14 days.(3)q RT-PCR were used to detected the m RNA expression levels of ALP,Runt-related transcription factor 2(RUNX2),Osteocalcin(OCN),and Osterix;Western blotting were used to detected the protein expression levels of RUNX2,OCN,FZD4,and Osterix;q RT-PCR were used to detected the m RNA expression of FZD4 wild-type(FZD4 WT),FZD4 mutant(FZD4 MUT),and FZD4 Level;luciferase reporter gene to detect the effect of miR-1286 expression on FZD4;use ALP kit to detect ALP activity;Alizarin red staining were used to evaluate calcification.The third part:(1)the BMSCs from 50 OP patients were as samples,the BMSCs from passage the 5th generation were induced osteogenic differentiation with MODM medium.And they were divided into groups: overexpression of FZD4 group(pc DNA-FZD4 group),pc DNA-NC control group(pc DNA-NC group),the control group(miR-NC group),overexpression of miR-1286 group(miR-1286-mimic group),overexpression miR-1286 and FZD4(miR-1286-mimic+pc DNA-FZD4 group).The BMSCs samples of each OP patient were made 3 replicate holes.(2)The pc DNA-FZD4 group were transfected with pc DNA-FZD4 to mimic FZD4 overexpression,the pc DNA-NC group were transfected with the pc DNA-NC control group,the miR-NC group were transfected with anti-miR-NC for the control study,and the miR-1286-mimic group were transfected with micro RNA-1286 to mimic overexpression,miR-1286-mimic+pc DNA-FZD4 group were transfected with pc DNA-FZD4 and miR-1286 to mimic overexpression.The MODM medium were replaced every 2-4 days and cultured for 14 days.(3)q RT-PCR were used to detected ALP,RUNX2,OCN,Osterix m RNA expression levels;Western blotting were used to detected RUNX2,OCN,and Osterix protein expression levels;ALP kits were used to detect ALP activity;Alizarin red staining were used to assessed calcification.Result:The first part:(1)The expression of miR-1286 in the serum of patients with OP were significantly higher than that of the normal population,and the difference were statistically significant(p<0.01).(2)The BMSCs of OP patients grew adherently after4-6 hours of passage,gradually protruding from pseudopodia,and the adherent cells had a clear outline and showed a spindle-shaped arrangement;after 7 days of subculture,the number of cells gradually increased and cells appeared after further fusion and growth,the morphology was like tadpoles and grass bundles,closely arranged,and vortex-like patterns can be seen in some areas.Seven days after osteogenic induction,ALP-positive cells stained dark blue;Alizarin red stained orange-red,with obvious calcified nodules.(3)The expression level of miR-1286 on the 3rd,7th,and 14 th day after inducing osteogenic differentiation of BMSCs were lower than that on the 1st day;the expression level of miR-1286 on the 7th and 14 th day after inducing the osteogenic differentiation of BMSCs were lower than that on the 3rd day;The expression level of miR-1286 on the 14 th after inducing osteogenic differentiation of BMSCs were lower than that on the 7th day,and the difference were statistically significant(p<0.01).The second part:(1)The m RNA expression levels of ALP,RUNX2,OCN,and Osterix in the miR-1286-mimic group were lower than those in the miR-NC group,and the m RNA expression levels of ALP,RUNX2,OCN,and Osterix in the mi-1286-Inhibitor group were higher than the miR-NC group,the difference were statistically significant(p<0.01).(2)The ALP activity of the miR-1286-mimic group was lower than that of the miR-NC group;the ALP activity of the miR-1286-Inhibitor group were higher than that of the miR-NC group,the difference were statistically significant(p<0.01).(3)The protein levels of RUNX2,OCN and Osterix in the miR-1286-mimic group were lower than those in the miR-NC group,and the protein levels of RUNX2,OCN and Osterix in the miR-1286-Inhibitor group were higher than those in the miR-NC group,the difference were statistically significant(p<0.01).The luciferase reporter gene experiment showed that the overexpression of miR-1286 significantly quenched the fluorescence of FZD4 WT;the expression level of FZD4 MUT in the miR-1286-mimic group were not significantly different from that in miR-NC group(P >0.05);the expression level of FZD4 WT in the miR-1286-mimic group were lower than that in the miR-NC group.The expression level of FZD4 WT in the miR-1286-Inhibitor group were higher than that in the miR-NC group,the difference were statistically significant(p<0.01).The ALP staining of the miR-1286-mimic group showed that the ALP content were significantly reduced,and the Alizarin Red staining showed that the formation of mineralized nodules was significantly reduced.The third part:(1)The m RNA expression levels of ALP,RUNX2,OCN and Osterix in the pc DNA-FZD4 group were higher than those in the pc DNA-NC group,and the difference were statistically significant(p<0.01).The ALP activity of the pc DNA-FZD4 group werer higher than that of the pc DNA-NC group,and the difference werer statistically significant(p<0.01).In addition,Western blotting analysis also showed that the protein levels of RUNX2,OCN,and Osterix in the pc DNA-FZD4 group were higher than those in the pc DNA-NC group,and the difference were statistically significant(p<0.01).ALP staining showed a significant increase in ALP content in the pc DNA-NC group,and Alizarin red staining showed a significant increase in the formation of mineralized nodules.(2)The expression levels of ALP,RUNX2,OCN and Osterix m RNA in the miR-1286-mimic+pc DNA-FZD4 and miR-1286-mimic groups were lower than those in the miR-NC group;and the levels of ALP,RUNX2,OCN and Osterix m RNA in the miR-1286-mimic group were significantly lower than those in miR-1286-mimic+pc DNA-FZD4 group.ALP activity,RUNX2 protein,OCN protein and Osterix protein also showed the same changes.Conclusions:(1)The expression of miR-1286 in OP patients were significantly higher than that of healthy controls.And the expression of miR-1286 gradually decreased after 1d,3d,7d and 14 d of inducing osteogenic differentiation of BMSCs.These results suggest that miR-1286 is negatively correlated with osteogenic differentiation of OP,which may be related to the occurrence and development of OP.(2)The BMSCs extracted from OP patients in this study were cultured and found that the passage effect were good,the cell growth ability were vigorous,and the functional status were good,which can meet the observation and study of cell morphology,and can be used as the experimental target for subsequent in vitro experiments on osteogenic differentiation.(3)The overexpression of miR-1286 inhibited the osteogenic differentiation ability of BMSCs in OP patients,resulting in the down-regulation of the key enzymes and protein expression downstream of osteogenic differentiation;while the low expression of miR-1286 promoted the osteogenic differentiation of BMSCs in OP patients,resulting in the up-regulation of key enzymes genes and proteins downstream of osteogenic differentiation.These suggest that miR-1286 may be involved in osteogenic differentiation of OP.(4)Mi R-1286 can directly bind to FZD4 m RNA,and then affect the expression of FZD4.The overexpression of FZD4 promotes the osteogenic differentiation of BMSCs in OP patients and accelerates the osteogenesis of OP.Mi R-1286 can regulate and inhibit the osteogenic differentiation of h BMSCs by interacting with FZD4,which indicates that miR-1286 and FZD4 have the characteristic of targeted binding,and can be targeted combined with FZD4,to participate in the process of BMSCs osteogenic differentiation of OP through Wnt/FZD4 pathway,thus regulating and affecting the occurrence and development of OP. |
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