microRNA-96-5p对染氟成骨肉瘤细胞c-Jun氨基末端激酶通路的影响  

Effects of miR-96-5p on JNK pathway in UMR-106 cells treated with sodium fluoride

作　　者：陈宣好 杨璐珲 代佑罡 吴彦秋 王楠兰[2,3] 韦艳 CHEN Xuan-hao;YANG Lu-hui;DAI You-gang;WU Yan-qiu;WANG Nan-lan;WEI Yan(Department of Occupational Health and Environmental Hygiene, School of Public Health, Guizhou Medical University,Guiyang, Guizhou 550025, China)

机构地区：[1]贵州医科大学公共卫生学院职业卫生与环境卫生学系,贵州贵阳550025 [2]贵州医科大学环境污染与疾病监控教育部重点实验室 [3]贵州医科大学公共卫生学院营养与食品安全学教研室

出　　处：《环境与健康杂志》2018年第10期857-862,共6页Journal of Environment and Health

基　　金：国家自然科学基金(81560515)

摘　　要：目的探讨miR-96-5p对染氟大鼠成骨肉瘤(UMR-106)细胞c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)通路的影响。方法将处于对数生长期的UMR-106细胞分别转染mi R-96-5p反义寡核苷酸(anti-miRNA oligonucleotide,AMO)、过表达质粒以达到抑制和过表达作用,再以0(对照)、2 000μmol/L NaF对细胞进行染毒,培养24 h后通过实时荧光定量PCR检测miR-96-5p及JNK通路上各因子基因的变化;培养48 h后,采用蛋白免疫印迹法检测JNK通路上各因子蛋白的变化。结果与无氟对照组相比,氟染毒组UMR-106细胞mi R-96-5p、天冬氨酸蛋白水解酶3(cysteinyl aspartate specific proteinase 3,caspase-3)mRNA表达水平升高(P<0.05),cAMP直接活化交换蛋白(exchange protein directly activated by cAMP,Epac)mRNA表达水平下降,差异均有统计学意义(P<0.05);转染mi R-96-5p质粒后,与质粒对照组相比,质粒+NaF组UMR-106细胞miR-96-5p mRNA表达水平升高,差异有统计学意义(P<0.05);转染AMO后,与AMO对照组相比,AMO+NaF组UMR-106细胞mi R-96-5p mRNA表达水平升高,Epac和caspase-3 mRNA表达水平下降,差异均有统计学意义(P<0.05)。与无氟对照组相比,氟染毒组UMR-106细胞腺苷酸环化酶6(Adenylate Cyclase 6,AC6)、Epac、p-JNK蛋白表达水平均下降,差异均有统计学意义(P<0.05);与质粒对照组相比,质粒+NaF组UMR-106细胞AC6、Epac、天冬氨酸蛋白水解酶9(cysteinyl aspartate specific proteinase 9,caspase-9)蛋白表达水平均下降,而p-JNK、磷酸化Jun癌基因(Jun oncogene,c-Jun)、细胞色素C(cytochrome C,cyt C)和非活化caspase-3蛋白表达水平升高,差异均有统计学意义(P<0.05);转染AMO后,与AMO对照组相比,AMO+NaF组UMR-106细胞AC6、Epac、caspase-9、p-JNK蛋白表达均下降,p-c-Jun、cyt C和非活化caspase-3蛋白表达升高,差异均有统计学意义(P<0.05)。结论 mi R-96-5p可能促进染氟UMR-106细胞JNK通路磷酸化,从而介导细胞凋亡。Objective To explore the effects of miR-96-5 p on JNK pathway in UMR-106 cells treated with NaF. Methods UMR-106 cells,transfected with miR-96-5 p vector and anti-miRNA oligonucleotide(AMO),exposed to 0 and 2 000 μmol/L NaF respectively,were determined for the gene expression of miR-96-5 p and JNK pathway by real-time quantitative PCR based on 24-hour culture,as well as the expression of proteins in JNK pathway by Western blot based on 48-hour culture.Results Compared with the control group,mi R-96-5 p and caspase-3 gene expression increased and exchange protein directly activated by cAMP(Epac) decreased significantly(P<0.05) in Naf exposure group. Compared with miR-96-5 p vector control group,miR-96-5 p was up-regulated(P <0.05),other factors’ m RNA did not change in mi R-96-5 p vector +NaF group.Compared with AMO control group,mi R-96-5 p gene was up-regulated(P<0.05),Epac and caspase-3 were down-regulated(P<0.05) in AMO+NaF group. Compared with the control group,the expression of AC6,Epac and p-JNK decreased(P<0.05) in Naf exposure group. Significantly lower expression of AC6,Epac and caspase-9,higher expression of p-JNK,p-c-Jun,cyt C,non-activated caspase-3 were found in miR-96-5 p vector+NaF group compared with those in mi R-96-5 p vector control group(P<0.05). Significantly lower expression of AC6,Epac,caspase-9 and p-JNK,higher expression of p-Jun,cyt C,non-activated caspase-3,were found in AMO+NaF group compared with those in AMO control group(P<0.05). Conclusion mi R-96-5 p may promote apoptosis via phosphorylating JNK pathway in UMR-106 cells exposed to NaF.

关 键 词：氟化钠 成骨肉瘤细胞 microRNA-96-5p c-Jun氨基末端激酶通路 

分 类 号：R994.6[医药卫生—毒理学]