Role Of Different Point Mutations Of FAM20C On Osteoblasts And Odontoblasts Behavioral Research

Backround:The family with sequence similarity 20?FAM20?represents a new family of secreted proteins,including FAM20A,FAM20B,FAM20C.Mutations in each family member can lead to the corresponding diseases,and the disease caused by Fam20c mutation is called Raine Syndrome.There are two completely opposite clinical manifestations of Raine Syndrome,one of which is lethal osteosclerosis,and the other is non-lethal hypophosphatemic rickets.It is still unclear why the same etiology causes opposite manifestations.The Fam20c knock-out osteoblast and odontoblast cell lines already possessed by our group,as well as the Fam20c lethal mutation plasmid Fam20c-L383R?FAM20C 383 leucine mutation to arginine?,Fam20c-G374R?FAM20C 374 glycine mutation to arginine?and Fam20c nonfatal mutation plasmid Fam20c-D446N?FAM20C 446 aspartate mutation to asparagine?,Fam20c-P323S?FAM20C 323 proline mutation to serine?.So this study aims to explore why the same gene mutation will cause different clinical manifestations,and if the mutations at different sites have different effects on cell function.Objective:To explore the effects of different point mutations of Fam20c on the biological behaviors of Fam20c knock-out osteoblast and odontoblast cells,such as cell proliferation,migration,mineralization and regulatory gene expression.Methods:1.The Fam20c knock-out osteoblast and odontoblast cells were transfected with the above four plasmids,and the expression level of total Fam20c gene was detected by real-time PCR to verify the successful transfection of the plasmids into cells.2.BrdU staining was used to observe the proportion of BrdU-positive cells after transfecting Fam20c-L383R,Fam20c-G374R,Fam20c-D446N and Fam20c-P323S into the Fam20c knock-out osteoblast and odontoblast cells,and then,calculate the proliferation of cells.3.The Fam20c knock-out osteoblast and odontoblast cells transfected with plasmids were subjected to a scratch test,and the width of the scratch was measured after 36hours of culture to observe the effect of the above four mutant plasmids on cell migration.4.Real-Time PCR detects Dmp1,Dspp,Mepe,Opn,Alp,Fgf23,Col1a1 and Ocn Changes in expression to observe different types of mutations have different effects on gene expression.5.The control group and the Fam20c knock-out osteoblasts and odontoblasts transfected with plasmids were subjected to osteogenic induction for 21 days,and subjected to alizarin red staining.The effects of mutations at different sites on cell mineralization were compared by the number of calcium nodules.Results:1.Real-Time PCR results showed that the total Fam20c gene expression level was5-10 times higher than that of the control group after transfection of the above four mutant plasmids,which proved that the condition was the optimal transfection condition.2.The results of BrdU staining showed that Fam20c deletion and mutation had no significant effect on osteoblast proliferation,and lethal osteosclerosis and hypophosphatemia mutations had no effect on the proliferation of osteoblasts?OB?.There was no difference between them.However,the loss of Fam20c reduced the proliferation of odontoblasts?OD?.There was no significant difference in proliferation between the OD Fam20c^f/f/f transfected with the four mutant plasmids,though the proliferation was enhanced compared with OD Fam20c^KO.Thus,there was no difference in OD proliferation between lethal and non-lethal mutations.3.Cell scratch assay showed that both Fam20c deletion and mutation reduced the migration ability of osteoblasts and odontoblasts,but there was no difference between lethal and non-lethal mutations in the two cells.4.Real-Time PCR results show that different types of mutations cause different expression levels of different genes in osteoblasts and odontoblasts,which leads to different behaviors of cells.5.The results of Alizarin red staining showed that Fam20c deletion reduced the mineralization ability of osteoblasts,and there was no difference in mineralization between lethal and non-lethal osteoblasts.Because of the weak mineralization ability of odontoblasts,Fam20c deletion has less effect on dentin mineralization.So the lethal and non-lethal mutations of Fam20c do not differ in mineralization of odontoblasts.Conclusion:FAM20C mutations at different sites cause two opposite clinical manifestations of Raine syndrome.In vitro experiments have shown no significant effect on osteoblast proliferation,but can cause cytological behavior and functional changes of osteoblast migration and mineralization;There were no significant differences between the lethal mutations and non-lethal mutations in the proliferation and migration of dentin cells and the cytological behavior and function of mineralization.