The Role Of P16 Gene Methylation In The Abnormal Proliferation Of Osteoblast Induced By Fluoride

Objective:The study made full use of the resources of coal-burning fluorosis area in Guizhou Province,taken the key molecular p16 gene in cell cycle regulation as the point,detected the transcription expression and methylation level of p16 gene in fluorosis population,and constructed the fluorosis model of human osteoblasts to detect the effect of sodium fluoride on the activity and cell cycle distribution of human osteoblasts.To observe the transcription expression of p16 gene and the change of methylation level,and further use DNA methyl transferase inhibitor 5-AZA-dC to intervene to explore the role of p16 gene methylation level in the development of fluorosis,and to provide new ideas and directions for the prevention and treatment of fluorosisMethods:1 Population based research on fluorosis caused by coal-burning1.1 Objects According to the standard of endemic fluorosis area(GB 17018-201 1),we choose the HH village of zhijin county as the burning coal pollution-type fluorosis survey place,and ZG village of Anshun city as control place.We screened 380 subjects(fluorosis patients 295 cases,control 85 cases)according to their time of fluoride exposure,dental health,clinical symptoms,X-rayexamination1.2 The questionnaire survey,environmental and biological samples Under the principle of informed consent,they will be asked to fill in a questionnaire,and biological samples(blood and urine)1.3 The fluorine content of environmental and urine samples The fluoride content of urine samples was examined using a fluoride ion-selective electrode(PXJ-1B digital ion meter).According to the urine content of fluoride,the responders were divided into 4 groups:UF<1.96 mg/g Cr(140 cases),1.96 mg/g Cr?UF<3.92 mg/g Cr(93 cases),3.92 mg/g Cr?UF<7.84 mg/g Cr(82 cases),UF?7.84 mg/g Cr(65 cases)1.4 Diagnosis of skeletal fluorosis Forearm and shank X-ray examinations of 295 participants from Hehua Village were conducted.Degrees of skeletal fluorosis was assessed by experts at Guizhou Orthopedics Hospital according to diagnostic criteria for endemic skeletal fluorosis.The participants were divided into four groups based on their X-ray manifestations:normal,mild,moderate,severe1.5 Detecting p16 expression and DNA methylation Peripheral blood p16 mRNA transcription expression was examined with real-time quantitative PCR(qRT-PCR)Peripheral blood P16 protein expression was examined with enzyme linked immunosorbent assay(ELISA).Peripheral blood p16 methylation level was examined with methylation-sensitive high-resolution melting(MS-HRM)2 The relative research on the methylation of p16 gene in human osteoblasts with fluoride exposureThe human osteoblasts were treated with 0,125,250,500,or 1000 ?mol·L-1 NaF for 72h,the model of fluorosis has been constructed successfully.Among the different groups were treated with NaF,MTT assay and flow cytometry were used to detected effects of NaF on the cell vitability and the cell cycle,real-time quantitative PCR(qRT-PCR)were performed to detect the mRNA levels of p16,western blot were performed to detect the protein levels of P16,bisulfite sequencing PCR(BSP)was performed to detect the levels of methylation levels of p163 The research on methylation inhibitors 5-AZA-dC intervention in vitroThe human osteoblasts were treated with 1000 ?mol/L NaF for 72 h,the fluoride model was established.According to the results of MTT and the references,these cells continue to been processed for 72h with 5,10,20?mol/L 5-AZA-dC.Among the different groups were treated with the intervention,BSP were performed to detect the methylation levels of p16,qRT-PCR were performed to detect the mRNA levels of p16,Western blot were performed to detect the protein levels of P16,MTT assay and flow cytometry were used to detected effects of NaF on the cell vitality and the cell cycle after osteoblasts were treated with 5-AZA-dC at different concentrations of groups.Results:1 Population based research on fluorosis caused by coal-burningWith higher UF concentrations,the mRNA and protein expression of p16 gene was lower(?2 mRNA?protein =22.812?45.785,P all<0.05),methylation of the p16 promoter was more extensive(?2=40.942,P<0.05),and the severity of skeletal fluorosis was greater(?2= 16.791,P<0.05).On this basis,the differences of expression and methylation levels of the p16 gene were further analyzed among the groups with skeletal fluorosis.With greater seveity of skeletal fluorosis,the expression of the pl6 gene was loweKi(?2 mRNA?protein =8.535?15.963 P all<0.05)and methylation of the p16 promoter was higher(?2=9.242,P<0.05).Thus,abnormal methylation of the promoter of the p16 gene is correlated with fluorosis,and involved in the development of skeletal fluorosis.The methylation levels of all samples were below 75%,and sub-classified into three groups:0-1%methylation,1-12.5%methylation,and 12.5-75%methylation.The results showed that,with higher UF concentrations,methylation of the pl6 promoter was more extensive(?2mRNA?protein =7 176?7.017,P all<0.05).It was shown that p16 gene hypermethylation correlated with lower gene expression.2 Fluorosis experiment in vitroWith increasing NaF concentrations,the proliferation index(PI for short)increased in a concentration-dependent manner(F=66.801,P<0.05),the number of cells in the G0/G1 phase decreases while the S phase cells gradually increase,these results indicate that NaF promotes osteoblast cell cycle progression from the G1 to the S phase,leading to a proliferative effect.With increasing concentrations of NaF,the nRNA and protein ofpp6 was gradually decreased(FmRNA?protein =41.663s 49.844,P all<0.05),the methylation levels ofpl6 was gradually increased(?2=73.034,P<0.05).3 Fluorosis intervention experiment in vitroWith increases of 5-AZA-dC concentration,the methylation ofpl6 was decreased(?2=97.695,P<0.05)and the mRNA and protein expression of p16 was increased(FmRNA?protein=42.850,30.560,P all<0.05).The cell vitality was gradually decreased(F=40.455,P all<0.05),the number of cells in the G0/G1 phase increases while the S phase cells gradually decrease,the proliferation index of osteoblasts gradually decreases(F=56.277,P all<0.05).Conclusion:1.Fluoride exposure could induce aberrant methylation and decrease the expression of p16 gene,which accounts for fluoride increasing cell viability and promoting proliferation in human osteoblasts.That might be one of the important mechanisms of cell proliferation change in skeletal fluorosis.2.DNA methyltransferase inhibitor,5-AZA-dC,could effectively reverse the hypermethyaltion levels ofpl6 promoter region with fluoride exposure,which accounts for reverseing p16 expression,finally reverse proliferation of human osteoblasts caused by fluoride.