| Background:Osteoporosis(OP)is a complex polygenic genetic disease,and genetic factors are important causes of OP and osteoporotic fractures.In recent years,advances in genomic technology have broadened our understanding of the genetic structure and biological mechanisms of complex diseases.A large number of single nucleotide polymorphisms(SNPs)have been found to be related to osteoporosis.Characterization of the biological functions of osteoporosis-related SNP may provide new ideas for the prevention and treatment of OP.Long intergenic noncoding RNA(Linc RNA)has been identified as a factor closely related to the occurrence and development of OP.Linc RNA SNP can produce or destroy certain mi RNA binding sites,affect target gene expression,and ultimately correlate with disease susceptibility.In our previous study,we screened and found a functional SNP of uc002 yug.2 rs2246640(C>T),from chromosome region21q22 via a case-control study,which has been repeatedly verified to be significantly associated with OP susceptibility.However,the mechanism underlying is unclear.Objective:The aim of this study is to explore the mechanism underlying the role of a functional SNP in uc002 yug.2 rs2246640(C>T)in regulating osteoblast differentiation and OP genetic susceptibility.Methods:1.Linc RNA related to bone mineral density in the 21q22 chromosome region was screened through the UCSC database,NCBI-Pub Med and literature.For the screened functional SNP in Linc RNA-uc002 yug.2,RNAfold was applied to predict the effect of its functional SNP rs2246640(C>T)on the secondary structure and free energy of the uc002 yug.2 transcript.Primary osteoblasts from OP patients were collected and cultured,and the uc002 yug.2 rs2246640 genotype was identified by gene sequencing.Quantitative real-time PCR(Q-PCR)was used to detect the expression level of uc002 yug.2 in osteoblasts from patients with CC and TT genotype OP at the rs2246640.Lnc RNASNP2 and Target Scan were used to predict the effect of uc002 yug.2rs2246640(C>T)on mi RNA binding sites and the interaction between mi RNA and its target gene.For the predicted possible impact of uc002 yug.2 rs2246640(C>T)on mi R-3934-3p and target gene SMAD5,Q-PCR and western blot(WB)methods were further used to detect the expression levels of mi R-3934-3p and SMAD5 in osteoblasts from OP patients with uc002 yug.2 rs2246640 CC and TT genotypes.2.The recombinant vectors uc002 yug.2 rs2246640 [C] and rs2246640 [T] were transfected into the h FOB1.19 osteoblast cell line to observe the effect of uc002 yug.2rs2246640(C>T)on the osteoblastic differentiation of h FOB1.19 cells from the aspects of cell proliferation,differentiation,and mineralization.The cell proliferation was detected by Cell Counting Kit-8(CCK8)method;The activity of alkaline phosphatase(ALP)in the supernatant of cells was detected by using sodium p-nitrophenyl phosphate colorimetry;The expressions of ALP m RNA,runt-related transcription factor2(RUNX2)and osterix(OSX),osteocalcin(OCN)and osteopontin(OPN)m RNA and protein were detected by Q-PCR and western blotting methods;The mineralization level of extracellular matrix was detected by alizarin red S staining.The effect of uc002 yug.2 rs2246640(C>T)on the interaction between uc002 yug.2 and mi R-3934-3p and the interaction between mi R-3934-3p and SMAD5 3’-UTR were determined through a dual luciferase reporter gene experiment.On this basis,h FOB1.19 cells were treated with mi R-3934-3p mimics or mi R-3934-3p inhibitor to analyze the effects of mi R-3934-3p on the expression of SMAD5 protein and the above indicators of osteogenic differentiation in h FOB1.19 cells.Further,the inhibitor NC or mi R-3934-3p inhibitor was co-transfected with the recombinant vector uc002 yug.2rs2246640 [C] or rs2246640 [T] to h FOB1.19 cells,and the expressions of SMAD5 protein and the above indicators of osteogenic differentiation in h FOB1.19 cells were measured to determine whether uc002 yug.2 rs2246640(C>T)affected osteogenic differentiation of h FOB1.19 cells through the mi R-3934-3p/SMAD5 axis.To clarify the mediating role of mi R-3934-3p/SMAD5 axis in influencing osteogenic differentiation of rs2246640 [T] h FOB1.19 cells,uc002 yug.2 rs2246640 [T]recombinant vector was transfected into h FOB1.19 cells with mi R-3934-3p inhibitor or(and)SMAD5 sh RNA,respectively.The effects of mi R-3934-3p/SMAD5 axis on the above osteogenic differentiation indexes of rs2246640 [T] h FOB1.19 cells were analyzed.Results:1.The expression of uc002 yug.2 in OP patients with rs2246640 TT genotype was significantly lower than that in patients with CC genotype(p < 0.01).uc002 yug.2rs2246640(C>T)changes the secondary structure of uc002 yug.2 transcripts(rs2246640 [C] transcript secondary structure MFE=-613.39 kcal/mol vs.rs2246640[T] transcript secondary structure MFE=-609.39 kcal/mol).The predicted result of Target Scan database showed that rs2246640(C>T)affected the combination of uc002 yug.2 and mi R-3934-3p.The expression of mi R-3934-3p in OP patients with rs2246640 TT genotype was significantly higher than that in CC genotype patients(p< 0.01).The predicted result of Target Scan database showed that the SMAD5 3’-UTR region contains sites that bind to mi R-3934-3p.The expression of SMAD5 protein in OP patients with rs2246640 TT genotype was significantly lower than that in CC genotype(p < 0.01).2.uc002 yug.2 rs2246640(C>T)inhibited the proliferation of h FOB1.19 cells(p< 0.05),decreased ALP m RNA expression(p < 0.01)and supernatant ALP activity(p< 0.05),decreased the m RNA and protein expression of osteogenic differentiation related factors RUNX2,OSX,OCN and OPN(all p < 0.01),and inhibited the formation of mineralized nodules in h FOB1.19 cells(p < 0.01);mi R-3934-3p mimics significantly decreased the relative luciferase activity of h FOB1.19 cells transferred with rs2246640 [C](p < 0.01),but had no significant effect on the relative luciferase activity of h FOB1.19 cells transferred with rs2246640 [T] vector(p > 0.05);mi R-3934-3p mimics inhibited the relative luciferase activity of h FOB1.19 cells transferred with SMAD5 3’-UTR WT vector(p < 0.01),but had no significant effect on the relative luciferase activity of h FOB1.19 cells transferred with SMAD5 3’-UTR MUT vector(p >0.05);mi R-3934-3p mimics inhibited the expression of SMAD5 protein and the abovementioned index of osteogenic differentiation in h FOB1.19 cells(all p < 0.05),while mi R-3934-3p inhibitor promoted the expression of SMAD5 protein and the abovementioned index of osteogenic differentiation in h FOB1.19 cells(all p < 0.05);mi R-3934-3p inhibitor inhibited the expression of SMAD5 protein in h FOB1.19 cells transferred with uc002 yug.2 rs2246640 [C] vector and the above-mentioned index of osteoblastic differentiation(all p < 0.05),but had no significant inhibitory effect on the expression of SMAD5 protein and the above-mentioned index of osteoblastic differentiation in h FOB1.19 cells transferred with rs2246640 [T] h FOB1.19 cells vector(all p > 0.05);mi R-3934-3p inhibitor significantly promoted the abovementioned index of osteoblastic differentiation in h FOB1.19 cells transferred with rs2246640 [T](all p < 0.05),which could be reversed by SMAD5 sh RNA(all p < 0.01).sh RNA NC had no significant effect on mi R-3934-3p inhibitor-induced osteoblastic differentiation in h FOB1.19 cells transferred with rs2246640 [T](all p > 0.05).Conclusion:Linc RNA uc002 yug.2 functional SNP rs2246640(C>T)regulates osteoblast differentiation through the mi R-3934-3p/SMAD5 axis. |
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