| Objective : Observational studies examining associations of smoking and alcohol consumption with bone mineral density(BMD)and osteoporosis have generated inconsistent results.We performed two-sample Mendelian randomization(MR)analyses,using smoking or alcohol consumption-associated genetic variants identified in genome-wide association studies,to evaluate whether there are causal associations between smoking/alcohol consumption and BMD.Protein arginine methyltransferases(PRMTs)mediate the transfer of methyl groups to arginine residues in histone and non-histone proteins.PRMT5 is an important PRMT that symmetrically dimethylates arginine 8 in histone H3(H3R8)and arginine 3 in histone H4(H4R3).In previous studies,PRMT5 has been demonstrated to inhibit bone marrow stem cell(BMSC)osteogenic differentiation in W20-17 and ST-2 cells and promote PPARγ2 expression in during adipogenesis in C3H10T1/2 cells.However,it is unknown that whether the PRMT5 level in human adipocytes and rat BMMSCs adipocytes influences the differentiation process.We found that PRMT5 is increased in bone marrow adipocytes in patients with osteoporosis.Next,we extracted rat bone marrow mesenchymal stem cells and used EPZ015666 to inhibit the activity of PRMT5.We found that EPZ015666 inhibited adipocyte differentiation in rat BMMSCs.Furthermore,we found that EPZ015666 also inhibited the expression of PPARγ2 in rat BMMSCs.In conclusion,we have found that EPZ015666 can inhibit the adipogenic differentiation of rat BMMSCs.We conclude that PRMT5 may be an important therapeutic target for the treatment of osteoporosis.Bone metabolism is divided into two aspects: bone formation and bone absorption.In the process of bone formation,osteoblasts differentiation as an important mechanism plays an important role in bone metabolism.Bone morphogenetic proteins(BMPs)are a kind of morphogenetic proteins which are determined by Dlx3,Runx2,OCN regulatory spectrum.Previous studies have confirmed that micro RNA(mi RNAs)and BMP2 synergistically inhibit the cell differentiation pathway including osteoblast differentiation,and mi RNA-133 a regulates the osteoblast differentiation of MC3T3-E1 cells.The role of DLX3 has been shown to promote the synthesis of osteoblasts and osteochondrocytes.Many experiments have proved that DLX3 can promote the osteogenic differentiation and some osteogenic transcription factors through BMP2/Smad pathway in the osteoblasts MC3T3-E1,and has an important influence on the formation of osteoblasts.The increased expression of osteoblast specific markers is an important marker of osteoblast differentiation,including alkaline phosphatase(ALP)activity,osteocalcin(OCN)secretion and Runx2 expression level,as a marker in the early stage of osteogenesis.Regulation of osteogenic differentiation related signaling pathway;BMP/TGF β signaling pathway;phosphorylation of Smad Protein results in up regulation of Runx2 and osterix.FGFs signal path,Wnts signal path,etc.Recent years,some researches have been made on osteoporosis,and the pathways that affect the formation of osteoporosis are also common,including RANKL/RANK/OPG,Wnt/β-Catenin,PTH,TGF-β/BMP signal pathway.Bone formation is mainly affected by BMP / Smads and Wnt / β-Catenin pathway.Bone absorption is mainly affected by RANKL/RANK/OPG pathway.The mi RNAs mentioned above are more advanced at this stage.These mi RNAs may have overlapping or joint regulation on the target proteins.These targets have a positive effect on osteogenic differentiation,including Wnt,BMP,and the conduction factors in FGF pathway.Dlx3 is an important protein for the differentiation of osteoblasts which can enhance the expression through BMP-2 induced pathway and BMP-2/Smad5.DLX3 plays an important role in the process of bone development and the animals knocked out of Dlx3 can show the decrease of bone growth and mineralization,and the decrease of bone density;the expression of Dlx3 can be induced by activating p38/Smad5 in osteoblasts;there is no relevant report that mi R-133 a DLX3 functional axis is involved in the regulation of osteogenesis.On the other hand,protein kinase A can stabilize the protein structure of Dlx3 by phosphorylation of DLX3,which plays an important role in the osteogenic differentiation induced by BMP-2.In addition,DLX3 can be independent of BMP-2/Smad5 system and play an important role in the differentiation of osteoblasts.In recent studies,BMP can jointly regulate the differentiation of osteoblasts through the regulation of some micro RNAs,such as the negative regulation of mi RNA133 a on Runx2 and the negative regulation of mi RNA-135 a on Smad5,which jointly inhibit the differentiation of osteoblasts.Whether mi RNA-133 a has a negative regulatory effect on DLX3,and the possibility that DLX3 is the target protein of mi R-133 a also deserves to further study.TGF-β/BMP signaling pathway plays an important role in the process of osteoblast differentiation and osteogenesis,mi RNAs can inhibit the expression of TGF-β/BMP related genes from the post transcriptional level.Our study of mi R-133 a in osteoblast MC3T3-E1 and C2C12 showed that the expression of mi R-133 a was significantly up-regulated in the process of osteogenic differentiation.Therefore,we concluded that mi R-133 a played an important role in the process of osteogenic differentiation and osteogenesis.Then we confirmed that the key factor DLX3 of TGF-β/BMP signaling pathway is one of the target genes of mi R-133 a by bioinformatics and make double luciferase detection.Methods : 1.Genetic variants associated with smoking amount(n=3),alcohol consumption(n=2),and levels of excessive alcohol intake biomarker carbohydrate-deficient transferrin(CTD)(n=3 for CTD percent;1 for CTD concentration;and 2 for total transferrin)identified in published GWAS were used as instruments in MR analyses.Summary statistics data obtained from 32735,28498,and 8143 individuals of European ancestry included in The Genetic Factors for Osteoporosis Consortium were used to generate associations of the genetic instruments with femoral neck(FN-BMD),lumbar spine(LS-BMD),and forearm(FA-BMD),respectively.2.(1)Bone tissue specimens from patients undergoing hip replacement in the Department of Spine and Joint Orthopedics of Shengjing Hospital were obtained and divided into osteoporosis group and non-osteoporosis group.Adipocyte tissue from femoral head was taken.After washing with saline,coronal section was cut and bone marrow tissue samples from cartilage cap to lower femur were collected longitudinally for tissue fixation and decalcification.Immunohistochemical staining was used.The sections were observed by optical microscopy at magnification of 10,20 and 40.Each section collects 4 to 5 views.The brown staining was positive around the nucleus and in the nucleus of adipocytes.(2)Bone marrow adherence method was used to isolate and culture the P3 generation of rat BMMSCs.Flow cytometry was used to detect the low expression of cell surface markers CD29 and the high expression of CD45 and CD90.(3)BMMSCs in good growth state were divided into normal control group and AM group(adipogenic induction).Different concentrations of EPZ015666,P1 group(0.1 μmol/L EPZ015666)and P2 group(1μmol/L EPZ015666)were added to induce adipogenic differentiation,and the number of positive cells was changed by oil red O staining.(4)Real-time fluorescence quantitative PCR was used to detect the expression of PPARγ2 pathway,including FABP4,in BMMSCs of rats with different concentrations of EPZ015666.The expression of PPARγ2 and FABP4 was detected by Western Blot.3.In the process of osteogenic differentiation,the level of mi R-133 a decreased significantly.q RT-PCR was used to detect the expression of mi R-133 a in the process of osteogenic differentiation.C2C12 and MC3T3-E1 cells were used as cell models.In order to induce the differentiation of C2C12 cells,DMEM without serum and with BMP-2 was used instead of culture medium.In order to induce the differentiation of MC3T3-E1 cells,α-MEM supplemented with 10% FBS,50 μ m ascorbic acid and 5 mm β-glycerophosphate was used instead of culture medium.Then we detected the changes of several phenotypic markers related to osteogenic differentiation,including Runx2,ALP and OCN.mi R-133 a inhibited osteogenic differentiation.We also examined the effect of mi R-133 a on osteogenic differentiation using corresponding mimics and inhibitors.The efficiency of mi RNAs mimics/inhibitors and the expression of Runx2,ALP and OCN were detected by q RT-PCR.Western blot was used to detect the expression of OCN protein.ALP staining and alizarin red staining were used to detect the osteogenic differentiation of C2C12 cells.mi R133 a target DLX3.We searched for potential targets of mi R-133 a according to Mirdb.org.DLX3 was found to be a potential target of mi R-133 a.Therefore,we carried out the luciferase activity Assa to verify this point.Results: 1.Genetic prediction of daily smoking(CPD)was associated with lower FN-BMD(-0.014 SD in BMD increased by each cpd(95% CI,-0.027 to-0.0002;P = 0.047)).LS-BMD and FA-BMD had similar effects.But they did not reach statistical significance when the sample size was small(P = 0.17 and 0.27).By using alcohol consumption related variables or CTD level related variables as tools,we did not find any significant associated BMD results for alcohol consumption.2.(1)the expression of PRMT5 in bone marrow adipose tissue of osteoporosis patients was higher than that of non-osteoporosis patients.The expression of PRMT5 in osteoporotic patients was significantly higher than that in the control group.(2)The level of PRMT5 was relatively constant during the differentiation of rat bone marrow mesenchymal stem cells(BMMSCs)into adipocytes.BMMSCs with good growth in vitro were selected for subsequent experiments.The expression of CD29 and CD90 in MSCs was positive,the positive rates were 99.92% and 99.95%,respectively.In contrast,the positive rate of CD45 was 2.51%.Therefore,we believe that the isolated cells are high-purity rat bone marrow mesenchymal stem cells.Bone marrow mesenchymal stem cells of P3 generation were cultured in adipocyte induction medium(AM)and then differentiated into adipocytes by adding differentiation mixture inducer consisting of dexamethasone,insulin and 3-isobutyl-1-methylxanthine(IBMX).Compared with the control group,the number of red lipid droplets increased.Western blot analysis showed that PRMT5 protein level increased significantly after lipogenesis induction.We detected the protein level of PRMT5 adipocyte differentiation model.The level of PRMT5 protein increased after adding differentiation cocktail inducer for 2 weeks.Glyceraldehyde-3-phosphate dehydrogenase(GAPDH)was used as the control.Our conclusion is that PRMT5 protein level increases during the adipogenesis of BMMSCs.(3)PRMT5 inhibitor EPZ015666 can inhibit the adipogenic differentiation of rat bone marrow mesenchymal stem cells.The primary mesenchymal stem cells of rats were extracted and cultured in fat forming medium.In vitro,P3 rat bone marrow mesenchymal stem cells were cultured in adipogenic medium for 2 weeks,and lipid droplets appeared in the cytoplasm,and gradually increased.Oil red O staining was used to observe the lipid droplets in the cells after 2 weeks of lipogenic induction.It was found that with the increase of EPZ015666 concentration(0.1 μM,1 μM),the lipid droplets in the cells with 1 μM EPZ015666 concentration decreased significantly.We found that PRMT5 can promote the adipogenic differentiation of rat bone marrow mesenchymal stem cells,while EPZ015666,the inhibitor of PRMT5,can inhibit the adipogenic differentiation of rat bone marrow mesenchymal stem cells.(4)PRMT5 promotes the expression of PPARγ2 downstream genes and proteins in BMMSCs adipocytes.QRT-PCR was used to detect the expression of PPARγ2 in bone marrow mesenchymal stem cells of rats incubated with 0.1 μM and 1 μM EPZ015666 for 2 weeks.Compared with the control group,the expression level of PPARγ2 in BMSCs treated with PRMT5 inhibitor EPZ015666 was significantly decreased.High concentration of EPZ015666 had stronger inhibition on PPARγ2.Under the same conditions,the expression level of FABP4 was detected.The cells treated with high concentration of EPZ015666 showed low expression level of FABP4.Therefore,PRMT5 can regulate the expression of PPARγ2 and FABP4 in BMMSCs.3.(1)In the process of osteogenic differentiation,the expression of mi R-133 a decreased significantly.After the osteogenic differentiation of C2C12 cells and MC3T3-E1 cells induced by BMP-2,we found that the m RNA levels of Runx2,ALP and OCN were significantly up-regulated.These results indicate that we have successfully induced osteogenic differentiation.Compared with the control group,the expression of mi R-133 a in BMP-2-induced group decreased significantly.Our study shows that mi R-133 a may play an important role in the regulation of osteogenic differentiation.(2)mi R-133 a can inhibit osteogenic differentiation.48 hours after mi R-133 a mimics/inhibitor and negative control were transfected into C2C12 cells,we verified the interference efficiency of mi RNA mimics/inhibitor.The expression of Runx2,ALP and OCN were detected by q RT-PCR after 24 hours serum-free culture in C2C12 cells transfected with mir-133 a inhibitor.On the contrary,transfection of mi R-133 a mimics in C2C12 cells showed that the m RNA expression level of osteogenic differentiation gene was significantly down regulated.In C2C12 cells,the expression of OCN protein was significantly increased after mir-133 a inhibitor was transfected,and the expression of OCN protein was significantly decreased after mi R-133 a inhibitor was transfected.After 7 days of induction and differentiation,ALP staining showed that the blue of mi R-133 a overexpression group was significantly lighter than that of the control group,while the blue of mi R-133 a inhibitor group was significantly darker.After 14 days of induced differentiation,alizarin red staining showed that the mineralization level of matrix was similar.Our data show that mir-133 a can inhibit the differentiation of osteoblasts.(3)Mir-133 a targets DLX3.When mir-133 a and DLX3 plasmids were co transfected into MC3T3-E1 cell line,DLX3 wild type group(WT)showed significant inhibition of luciferase activity,but mut showed no change of luciferase activity.It was found that overexpression of mi R-133 a in MC3T3-E1 cells significantly inhibited the level of DLX3 protein,while in mi R-133 a inhibition group,the expression of DLX3 protein increased.These data suggest that mi R-133 a may inhibit the expression of DLX3 gene by target.Conclusion:1.Although a potential causal association between alcohol consumption and BMD were not observed in our study,smoking amount may be causally associated with BMD.2.(1)The content of PRMT5 in adipocytes of femoral head in osteoporosis group was significantly higher than that in non-osteoporosis group.(2)The level of PRMT5 is relatively constant during the differentiation of rat bone marrow mesenchymal stem cells into adipocytes.(3)PRMT5 inhibitor EPZ015666 can inhibit adipogenic differentiation of rat bone marrow mesenchymal stem cells.(4)PRMT5 promotes the expression of PPARγ2 downstream genes and proteins in rat BMMSCs adipocytes.3.(1)During the process of osteogenic differentiation,the levels of mi R-133 a is obviously downregulated.(2)mi R-133 a suppresses osteogenic differentiation.(3)mi R-133 a target DLX3.(4)mi R-133 a suppresses osteogenic differentiation by targeting DLX3.Our result demonstrated that mi R-133 a could inhibit the process of osteogenic differentiation by targeting DLX3 in vitro. |
PDF Full Text Request