Effects Of WNT1 Gene Mutation On WNT Signaling Pathway And Saos2 Cell Mineralization

Osteogenesis imperfecta（OI）is a rare geneticl disease caused by collagen formation disorder.Patterns of inheritance are autosomal dominant or recessive heredity.The clinical symptoms include fragile bone,blue sclera,dysplasia of dentin,hearing impairment and so on.So far,20 gene were found to be associated with the OI.More than 90%patients were caused by autosomal dominant mutations in one of the COL1A1 and COL1A2,which encoding the type I collagen.The remaining 10%disease are related with type I collagen metabolism,involving the posttranslational modification of collagen,collagen folding,abnormal secretion process,osteoblast differentiation,calcium channel and Wnt signaling pathway in bone development.The WNT1 gene mutations reported resulted in 25 persons.The molecular mechanism of WNT1 gene in osteogenesis is not clear,and needs further study.Objective:To detectthe mutation site of the WNT1 gene in the patients of OI.The WNT1mutant was overexpressed,and TOPflash assay and the dominant negative effect assay of WNT1 protein were carried out.Meanwhile,the circRNA luciferase reporter vectors was constructed and the double luciferase reporter experiment was carried out to detect the sponge function of circRNA to the miRNA of target WNT1.Methods:1.Sanger sequencing was performed to identify candidate mutations of WNT1gene found in high throughput sequencing of previous laboratories..2.The WNT1 mutant were overexpressed transfected in HEK293T cells or Saos2cells by transient transfection,and the assay ofthe TOP-Flash and the dominant negative effect of WNT1 protein andmineralization assaywere carried out.3.The circRNA luciferase reporter vectors were transiently transfected HEK293cells to detect the spongy function of circRNA on the target WNT1 of miRNA.Results:1.Among the OI patients,9 mutation sites were not reported,including c.301C>T,c.382T>G,c.397G>A,c.620G>A,c.677C>T,c.681C>A,c.774C>A,c.937C>T and c.1010G>C.Meanwhile,The overexpression vectors of WNT1 mutants are successfully constructed,including WNT1^R101C,WNT1^F128V,WNT1^A133T,WNT1^R207H,WNT1^S226L,WNT1^R313C,WNT1^R337P,WNT1^C227*,WNT1^Y258*.,the mutants reported in the database are also built.2.In the TOPflash assay,wild WNT1 protein as a positive control,Except for WNT1^S226Land H287Pfs*30 mutant,the rest mutants were significantly different from wild proteins.3.In the dominant negative effect assay,wild WNT1 protein as a positive control,Except for Y258*mutant,the rest mutants was not significantly different from wild type protein,P>0.05.4.In mineralization assay with Saos2 cells,the mineralization ability of mutant WNT1 group was significantly weaker than that of wild type WNT1 group.5.Hsa_circ₀01308 can be combined with miR-148a and miR-152;hsa_circ₀01319 may interact with miR-152;hsa_circ₀01372 is interacting with miR-148a,miR-148b and miR-152;hsa_circ₀01649 is associated with the circRNA1319.Conclution:The new WNT1 mutation sites enrich the mutant profiles of Chinese OI patients and provide new datas for the study of the pathogenesis of WNT1 induced OI.TheTOPflash assay also indirectly reflected that the transcriptional activitywas reduced by beta-catenin in the classical WNT signaling pathway.In vitro,mutantWNT1^Y258*appears to have a mild dominant negative effect.circRNA1308,circRNA1372 and circRNA1649 can combine miRNAs,with testifying miRNA spongy function.