MiR-29c-3p Targeted Adjustment Dvl2 On The Differentiation Of Rat BMSCs Into Osteoblasts In Hyperlipidemia Environment

Background:Hyperlipidemia was important risk factors of some diseases occurrence and development,such as atherosclerosis,osteoporosis,hypertension,coronary heart disease,stroke,which now is one of the high incidence of diseases that affect people health.Studies showed that hyperlipidemia has adverse on bone healing,bone mineralization,bone density,and many other bone metabolic process.Hyperlipidemia was one of the risk factors that cause osteoporosis.Wnt signaling pathways played a critical role in the differentiation of the bone marrow mesenchymal stem cells(bone marrow stromal stem cells,BMSCs)into osteoblasts.In recent years,a large number of studies have shown that small RNA(microRNAs,miRNAs)in the process of osteogenic differentiation played an important biological function,which has maintained the balance of bone metabolism.Our previous experiments have established that high-fat feed rats have loose bone around implant,trabecular bone thinning and disordered trabecular bone;the percentage of Ca/P around implant with high-fat diet rats was lower than that in normal;Fluorescence quantitative PCR and Western blot showed Dvl2 gene,Dvl2 protein expression was restrained,suggested that hyperlipidemia interfered early bone implant combined with hyperlipidemia rats in a certain extent.Dvl2 of Wnt/beta-catenin signaling pathway in patients with hyperlipidemia was worthy of being discussed on how to play a role.Wnt signaling pathway and miRNAs existed a lot of intersection signal molecule,its targets and the specific mechanism of action was worth in-depth study.Purpose:(1)The progress that BMSCs differentiated into osteoblasts in hyperlipidemia environment was Observated,RT-PCR was used to evaluate the expression level of Runx2,ALP,SP7,PPAR-?,Dvl2 and microRNAs which targeted to adjust Dvl2 mRNA expression.(2)Using dual luciferase report gene screening identified microRNAs which target to adjust Dvl2.(3)To investigate the microRNAs which targeted to adjustment Dvl2 impacted on the differentiation of BMSCs osteogenesis.Methods:(1)The rat bone marrow mesenchymal stem cells developed the third generation.Flow cytometry were used to detect,At 28 days,Alizarin red staining was applied for observing extra cellular matrix mineralization,oil red O was stained to examine the adipogenic differentiation.Which was used identification of BMSCs,so that used for subsequent experiments.The experiment was divided into two groups:high fat group and control group.Induction line 28 days respectively alizarin red and oil red O was stained to observe osteogenesis differentiation.RT-PCR was used to evaluate the expression level of osteogenesis related gene inside the cell Runx2,ALP,SP7,lipid metabolism related genes PPAR-y.Wnt signaling pathways in Dvl2,the targeted control Dvl2 of miR-21-5p,miR-29c-3p,miR-138-5p,miR-351-5p mRNA expression at 3,5,7,14,21 day,in simulated conditions of high-fat.(2)MicroRNAs associated with target gene prediction software Target Scan,MicroRNA.org,miRDB,Microcosm the Targets and so on.MiRNAs were forecasted by four kind of microRNAs bioinformatics prediction database on-line analysis software,as well as the literature query,which target to adjust Dvl2.Result showed that miRNAs which can combine with Dvl2 gene 3 'UTR region were miR-21-5p,miR-29c-3p,miR-138-5p,miR-351-5p.By in vitro transfection different concentration(10nm,30nm,50nm,100nm)gradient FAM-siRNA,after 6h transfection efficiency was observed by the microscopic,and CCK8 detected cell proliferation to screening suitable transfection concentration.Using the screening concentration transfected miR-21-5p,miR-29c-3p,miR-138-5p,miR-351-5p mimics and inhibitor in vitro,RT-PCR was used to evaluate the expression level of transfection efficiency after 48h,achieved Dvl2 higher expression and lower expression,Western blot was used to detect the expression differences of Dvl2 protein,to identify the most obvious change of Dvl2.Build dual luciferase reporter gene vector,293t cells was transfected to use the simulation of miR-21-5p,miR-29c-3p,miR-138-5p,miR-351-5p and plasmid vector,then the multifunctional enzyme mark was used to detect luciferase activity,which can verify the targeted regulatory role of miRNAs and Dvl2.(3)In hyperlipidemia environment,miR-29c-3p mimics and inhibitir were transfected in vitro,at the same time,BMSCs were used to induce osteoblast,after 3,7 day,Osteogenesis markers expression level of Runx2,ALP protein were detected by Western bolt.Results:(1)The test results shows that:the third generation of BMSCs CD44 positive cells rate was 96.7%,CD45 positive cells rate was 2.8%,CD90 positive cells rate was 95.9%.Mesenchymal stem cell surface antigen expressed positive,which can validate the cells for BMSCs 28 days after,alizarin red staining of fatty group saw less calcified nodules than the control group,oil red O staining of high fat group beaded fat drops than the control group.The RT-PCR results showed that the osteogenesis index Runx2,SP7,ALP mRNA expression of high-fat group was lower than those of control group,fat index PPAR-? mRNA level of high lipid group expressed high than the control group,the difference was statistically significant(P<0.05).MiR-21-5p,miR-29c-3p,miR-138-5p,miR-351-5p mRNA expression level of high-fat group were lower than the control group,at 7,14 days,miR-21-5p,miR-29c-3p,miR-138-5p,miR-351-5p mRNA expression level of high-fat group was relatively lower than control groups.(2)The higher the concentration was,the higher transfection efficiency was,the higher concentration of CCK8,cell proliferation was suppressed.Real-time PCR results showed that cell was transfected by miR-21-5p,miR-29c-3p,miR-138-5p,miR-351-5p mimics,its expression quantity increased respectively 5-6 times,4 times,1000 times and 800 times or so.And after cell was transfected corresponding inhibitor,its expression quantity decreased respectively about 3 times,10 times,10 times and 5 times.Cell was transfected by miR-21-5p,miR-29c-3p,miR-138-5p,miR-351-5p mimics and inhibitor and NC,after 48h,Real-time PCR results showed that cell was transfected by miR-21-5p and miR-29c-3p mimics,Dvl2 mRNA expression decreased,after cell was transfected by miR-138-5p and miR-351-5p mimics,Dvl2 mRNA expression increased,Cell was transfected by miR-21-5p,miR-29c-3p,miR-13 8-5p,miR-351-5p inhibitor,Dvl2 mRNA expression have varying degrees of increase.Western blot results showed that using miR-29c-3p mimics/inhibitor transfected cell,Dvl2 protein expressed differences.(3)Western blot results showed that BMSCS were transfected by miR-29c-3p mimics,protein expression levels of Runx2 and ALP were significantly higher than mimics NC group,while,BMSCs were transfected by miR-29c-3p inhibitor,protein expression levels of Runx2 and ALP were significantly lower than the inhibitor NC group.Conclusion:(1)Under the hyperlipidemia environment,the osteoblast differentiation reduced.During the osteoblast differentiation of BMSCS,miR-21-5p,miR-29c-3p,miR-138-5p,miR-351-5p were involved in bone metabolism and the activity of osteoblast biology under the hyperlipidemia environment.(2)The dual Iuciferase report gene screened and identified microRNAs which targeted to adjust Dvl2,result showed that microRNA were miR-21-5p.While the role of miR-29c-3p,miR-138-5p,miR-351-5p which targeted to adjust Dvl2 was the weak.(3)MiR-29c-3p through targeted adjustment Dvl2 indirectly promoted the differentiation of BMSCs and the mineralization ability.