The Role Of MiR-203-3p In The Regulation Of Osteogenesis In Diabetic Jawbone And Its Molecular Mechanism

BackgroundDiabetes mellitus(DM)is a serious threat to human health.It is usually caused by an absolute deficiency of insulin(type 1 diabetes),or insulin resistance and a relative deficiency(type 2 diabetes),leading to hyperglycemia.Diabetes is often accompanied by bone metabolism disorders,often leading to osteopenia,osteoporosis,or poor structure and other bone diseases.Diabetes,on the other hand,also interferes with bone formation,increases the risk of fractures in the patient and hinders fracture healing.The bones of diabetic patients can be characterized by osteoporosis,alveolar bone resorption,and loose teeth.When teeth loose or even fall off,dental implants are an important treatment to repair missing teeth.However,the abnormal bone physiology and pathological changes caused by the diabetic environment are a major challenge for dental implants.Therefore,to study the mechanism of jaw abnormalities caused by diabetes and put forward possible interventions has great significance for oral clinical practice.In terms of its source,the jaws differ from the bones of the extremities: the jaws develop from the neural crest of the embryo,and the limb bones originate from the mesoderm.Studies on jaw bone BMSCs and tibial BMSCs have shown that different sources of BMSCs have different biological characteristics.BMSCs from the above two sources differed in their osteogenic and proliferative capacities,with neural crest-derived BMSCs being more osteogenic.Furthermore,the jaw and skeletal limbs have different patterns of bone formation,which proceed mainly via intramembrane osteogenesis in the jaw and mainly via endochondral osteogenesis in skeletal limbs.At present,the study of diabetic bone disease mainly focuses on the cells or tissues of the bones of the extremities,while the research on the mechanism of the disease of the jaw bone in diabetes is less.Therefore,we hypothesized that during bone formation,the expression and regulation mechanism of related osteogenic genes may be different in these two originated bone tissues.MicroRNAs(miRNAs/miRs)are noncoding single-stranded RNA molecules consisting of ~22 nucleotides.miRNAs are widely distributed in all types of eukaryotic organisms.By binding to the 3’-untranslated region(3’-UTR)of the target gene mRNA,mi RNAs may affect their stability or translational repression and post-transcriptionally regulate the process of gene expression,which is associated with individual growth,development and diseases.Various miRNAs have important roles in numerous human diseases,including diabetes,and are aberrantly expressed in affected tissues of patients.Studies have indicated that certain miRNAs are important in the maintenance of bone development and metabolism.They regulate osteogenesis by modulating osteogenic transcription factors and their associated factors or regulates osteogenesis-associated signaling pathways.Although numerous factors and pathways associated with osteogenesis have been identified to be regulated by miRNAs,few studies have assessed miRNA expression in the jaw bone.Therefore,whether miRNA is involved in the loss of diabetic alveolar bone and osteoporosis of the jaw remains unclear.ObjectivesTo screen differentially expressed miRNAs in type 2 diabetes and normal jaw bones,clarify the role of mi RNAs in regulating type 2 diabetes jaw bone formation and explore the mechanisms involved.The results will help reveal the mechanism by which diabetes affects osteogenesis and provide an effective target for the clinical application and improvement of diabetic osteoporosis.MethodsPart Ⅰ: Real-time fluorescence quantitative RT-PCR was used to detect the mRNA expression of osteogenesis-related genes,including ALP,OC,Runx2,Osterix,Smad1,and Col1a1,in jaw bones of diabetic rats and normal rats.ALP staining was used to determine ALP activity of BMSCs cultured in high-glucose and low-glucose environment in vitro after 7 days of osteogenic induction.RT-q PCR was used to detect the expression of mRNA of the osteogenesis-related genes,including ALP,OC,Runx2 and Osterix,after 0,3,6,9,12,15,18,and 21 days of osteogenic induction of mandibular BMSCs cultured in high-glucose and low-glucose environment in vitro.Western blot was used to detect the protein expression level of Runx2,a key osteogenic transcription factor,after 1,4,7,10,14 and 21 days of osteogenic induction.Part Ⅱ: small RNA high-throughput sequencing was used to find differentially expressed miRNAs in jaws of experimental type 2 diabetic rats and normal rats.Differentially expressed miRNAs were verified by RT-q PCR.On this basis,Targetscan,mi Randa,and PITA were used to predict mi RNA target genes and crossovers were taken.The function of predicted target genes was analyzed,and osteogenesis-related mi RNAs were selected for further study.Part Ⅲ: Agomir-203-3p,antagomir-203-3p,agomir NC or antagomir NC were transfected into C3H10T1/2 cells or rat jaw derived BMSCs.On day 7 of osteogenic induction,ALP staining was performed to detect the activity of ALP.Western blot was used to detect the protein expression level of Runx2 on day 4,7,and 14 of osteogenic induction.RT-qPCR was used to detect the mRNA expression of osteogenesis-related genes,including ALP,OC,Runx2,and Osterix,on day 1,4,7,and 14 of osteogenic induction.Part Ⅳ: A highly conserved putative binding site for miR-203-3p in vertebrate was found in the 3′-UTR of the candidate target gene Smad1 mRNA by using Targetscan software.AgomiR-203-3p,antagomir-203-3p,agomir NC or antagomir NC was transfected into C3H10T1/2 cells or rat jaw derived BMSCs respectively,and the protein expression of Smad1 was detected by Western blot.AgomiR-203-3p or agomir NC was transfected into C3H10T1/2 cells or rat jaw derived BMSCs.The BMP/Smad signaling pathway related proteins Smad1 and pSmad1/5/8 were detected by Western blot,and the expression of Smad1 mRNA was detected by RT-q PCR.Then cells were osteogenic induced after transfection of agomi R-203-3p,antagomir-203-3p,agomir NC or antagomir NC.Western blot was used to detect the protein expression level of Smad1 under osteogenic induction.Luciferase reporter plasmids pGL3-Smad1-WT/pGL3-Smad1-mut containing the mi R-203-3p putative binding site or mutation site in the Smad1 3’-UTR,phRL-TK and agomir-203-3p/agomir NC was co-transfected into C3H10T1/2.A dual luciferase reporter assay was used to analyze the putative binding site of miR-203-3p in the 3’-UTR of Smad1 mRNA.The Smad1 overexpression plasmid or negative control was transfected into C3H10T1/2 cells with agomi R-203-3p or agomi R NC.After transfection,osteogenic induction was performed for 7 days.The mRNA expression of Runx2,Alp,Osterix and OC was analyzed by RT-q PCR.ALP activity was detected by ALP staining.ResultsPart Ⅰ: The results of RT-q PCR showed that the osteogenic gene in the mandible of diabetic rats was significantly down-regulated,which is consistent with the results of existing studies of limb bones.The results of ALP staining showed that the ALP activity of osteoblast-induced BMSCs was down-regulated in high glucose medium.Western blot analysis showed that the protein expression of Runx2 was down-regulated at each time point in cells cultured in high glucose medium.RT-qPCR results showed that the mRNA expression of osteogenic genes,including ALP,OC,Runx2,and Osterix,was down-regulated,except that there was no significant difference in the expression of Runx2 and Alp at the end of osteogenic induction.These results indicate that diabetes and high glucose levels inhibit osteogenisis.Part Ⅱ: Small RNA high-throughput sequencing cluster analysis of heat maps showed that 23 miRNAs were expressed differently between the two groups of rats.Eight miRNAs were up-regulated and 15 miRNAs were down-regulated in the jaws of diabetic rats.Rno-mi R-203a-3p,rno-mi R-23a-3p,rno-miR-320-3p,rno-miR-1-3p,rno-miR-1b,rno-mi R-872-5p,rno-miR-185-5p and rno-miR-345-5p were upregulated and rno-miR-30c-5p,rno-miR-96-5p,rno-mi R-17-5p,rno-mi R-148a-3p,rno-let-7b-5p,rno-miR-181a-5p,rno-miR-128-3p,rno-mi R-199a-5p,rno-mi R-181b-5p,rno-miR-200b-3p,rno-miR-142-3p,rno-mi R-674-3p,rno-mi R-149-5p,rno-miR-139-5p and rno-miR-375-3p were downregulated.RT-q PCR validation results revealed consistent with the results of high-throughput sequencing.We use Targetscan,miRanda,and PITA to predict mi RNA targets,and it was found that miR-203a-3p may target multiple osteogenic related genes.Whether miR-203a-3p modulates osteogenesis has not been reported in previous studies and was initially determined to be a further study subject.Part Ⅲ: ALP staining showed that agomi R-203-3p reduced ALP activity,while antagomir-203-3p enhanced ALP activity.The protein expression of Runx2 was inhibited by agomir-203-3p during osteogenesis and promoted by antagomir-203-3p.RT-q PCR results showed that agomi R-203-3p inhibited the expression of Runx2 and ALP,while antagomir-203-3p promoted the expression of Runx2 and ALP.Similarly,the expression of Osterix and OC was delayed by agomir-203-3p and promoted by antagomir-203-3p.These results indicate that mi R-203-3p inhibits the formation of bone in C3H10T1/2 cells and BMSCs.Part Ⅳ: By using Targetscan software,putative binding site for miR-203-3p,CAUUUCA,was found in the 3′-UTR of the predicted target gene Smad1 mRNA,which is widely conserved in vertebrates.Western blot showed that Smad1 protein expression was inhibited with agomir-203-3p transfection and was promoted with antagomir-203-3p transfection and that the protein levels of Smad1 and p-Smad1/5/8 were significantly inhibited after being transferred into agomiR-203-3p.RT-qPCR results showed that agomir-203-3p transfection suppressed Smad1 mRNA expression.It was suggested that mi R-203-3p inhibits the protein expression of Smad1 by affecting the stability of Smad1 mRNA.In addition,western blot results showed that agomi R-203-3p inhibited or delayed the expression of Smad1 in the osteogenic process of C3H10T1/2 cells and rBMSCs,while inhibition of miR-203-3p promoted the expression of Smad1.Compared with the agomi R-NC group,the luciferase activity of the pGL3-Smad1 vector was decreased in the agomiR-203-3p group,while agomir-203-3p had no effect on the luciferase activity of the pGL3-Smad1 mut vector.These results are consistent with bioinformatics predictions and the putative binding site in the 3’-UTR of Smad1 mRNA is a direct target of mi R-203-3p.The RT-q PCR and ALP staining results showed that ectopic expression of Smad1 attenuated miR-203-3p-mediated inhibition of osteogenic differentiation.These results indicate that miR 203-3p inhibits osteogenic differentiation,in part through down-regulation of Smad1 expression.ConclusionMiR-203-3p plays an inhibitory role in the of osteogenesis of diabetic rat jaw bones,and is highly expressed in mandible of diabetic rats or high-glucose cultured cells.Smad1 is an important mediator of the BMP/Smad pathway.MiR-203-3p inhibits its post-transcriptional expression through targeted binding to Smad1,which in turn affects BMP/Smad signaling pathways,which may be the mechanism by which miR-203-3p inhibits osteogenesis in diabetic conditions.