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《Chinese Journal of Animal and Veterinary Sciences》 2019-01
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Effects of GSTO1 Gene Silencing on Autophagy and Apoptosis of Fluoride-induced Osteoblasts

WANG Jinming;GAO Yufeng;CHENG Xiaofang;YANG Jiarong;XU Huimiao;ZHAO Yangfei;LI Yanyan;College of Animal Science and Veterinary Medicine,Shanxi Agricultural University;College of Arts and Sciences,Shanxi Agricultural University;  
To investigate the role of GSTO1 in the fluoride-induced autophagy and apoptosis in the osteoblasts,RNA interference (RNAi)technology was used to inhibit the expression of GSTO1 gene in mouse osteoblasts,and the expressions of autophagy and apoptosis related genes were detected.Three pairs of 21 nucleotide sequence siRNA (siRNA1,siRNA2,siRNA3)were designed and synthesized according to the mouse GSTO1 mRNA sequence,and the best effective fragment was screened.Then,the mRNA expression levels of GSTO1 under different concentrations of sodium fluoride (NaF) (10~4,10~3,10~2,10,and 1mg·L~(-1))were evaluated to determinethe optimal fluoride concentration.This experiment included control group,negative control group (NC-siRNA),NaF group (1mg·L~(-1)),silencing target gene group (siRNA-GSTO1)and NaF+silencing target gene group (NaF/siRNA-GSTO1).RT-PCR was used to detect the transcription of autophagy-related genes (LC3,p62,Beclin1,Atg3,Atg5)and apoptosis-related genes (BCL2,Caspase-3)in osteoblasts.The protein expression of LC3,p62,Beclin1,Atg3 and Atg5were detected by Western blot.Results were as follows:Osteoblasts were identified by HE,Giemsa,alkaline phosphatase,and alizarin red staining.In the experimental model,50nmol·L~(-1) was found to be the optimal transfection concentration of siRNA,and siRNA2 was the best silent transfection fragment,at 1mg·L~(-1) NaF optimal concentration.The NaF treatment group and siRNA-GSTO1 group inhibited the transcription of LC3,Beclin1,Atg5 and BCL2,and decreased the transcription of p62 and Caspase-3.However,compared with the NaF group,the transcription of LC3,Beclin1 and Atg5mRNA were significantly decreased (P0.05),while the transcription of p62 and Caspase-3 were significantly increased in the NaF/siRNA-GSTO1 group (P0.05).The expression of autophagy-related proteins LC3,Beclin1,Atg5 and p62 in osteoblasts were completely consistent with the gene transcription.In summary,both low-dose fluoride and silencing of GSTO1 expression increased autophagy and decreased apoptosis in the osteoblasts.GSTO1 had synergistic effect on low-dose fluoride-induced autophagy enhancement and apoptosis inhibition in the osteoblasts.
【Fund】: 国家自然科学基金青年项目(31001089);; 山西省自然科学基金面上项目(2014011027-3);; 山西省优秀博士来晋工作奖励资金(SXYBKY201731)
【CateGory Index】: S856.9
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