雄激素受体负向调节雄激素非依赖性前列腺癌细胞FN1和ZNF438基因表达的研究

Study of FN1 and ZNF438 gene expression negatively regulated by androgen receptor in androgen-independent LNCaP cells

目的 验证FN1和ZNF438是否为雄激素非依赖性前列腺癌细胞雄激素受体靶基因,并探索雄激素受体对FN1和ZNF438基因的表达调节作用.方法 采用染色质免疫共沉淀-实时荧光定量PCR及琼脂糖凝胶电泳技术验证雄激素受体（AR）作用于FN1和ZNF438基因.用双氢睾酮（DHT）刺激LNCaP-AI细胞及慢病毒干扰LNCaP-AI细胞AR的表达,通过逆转录荧光定量PCR（RT-qPCR）和蛋白质免疫印迹（Western blot）分析FN1和ZNF438基因的表达情况.结果 AR-ChIP实验组所富集到的FN1和ZNF438基因分别为7.274±0.290和6.843±0.078,显著高于IgG-ChIP对照组的1.004±0.113和1.000±0.014,差异有统计学意义（t=34.91、128.377,均P＜0.05）.在双氢睾酮（DHT）刺激LNCaP-AI细胞后,FN1和ZNF438基因的mRNA及蛋白表达水平分别为0.434±0.050和0.069±0.042,显著低于对照组的1.000±0.016和1.025±0.277;差异有统计学意义（t=18.532、5.905,均P＜0.05）.而在慢病毒转染干扰LNCaP-AI细胞AR的表达后,FN1和ZNF438基因的mRNA及蛋白表达水平分别为17.579±4.415和1.895±0.424,显著高于对照组的1.028±0.445和1.041 ±0.190;差异有统计学意义（=6.461、3.184,均P＜0.05）.结论 FN1和ZNF438为雄激素非依赖性前列腺癌LNCaP-AI细胞雄激素受体负向调节的靶基因,可为进一步研究雄激素非依赖性前列腺癌细胞的生长发展机制提供新的研究思路.

Objective To verify whether FN1 and ZNF438 are the androgen receptor（AR） target genes in LNCaP-AI cells and investigate the effects of AR exerts on FN1 and ZNF438 gene expression.Methods ChIP-PCR and agarose gel electrophoresis were performed to verify the fact that AR acted on FN1 and ZNF438 gene.Subsequently,LNCaP-AI cells were treated with dihydrotestosterone（DHT） and lentivirus transfection to down-regulate AR expression.The expression of FN1 and ZNF438 gene in mRNA and protein levels were analyzed by RT-qPCR and Western blot.Results FN1 and ZNF438 gene enrichment of AR-ChIP group were 7.274 ± 0.290 and 6.843 ± 0.078,significantly higher than the IgG-ChIP group of 1.004 ±0.113 and 1.000 ± 0.014 （t（FN1） =34.91,t（ZNF438） =128.377,P 〈 0.05）.Differences were statistical significance.After DHT stimulation,the expression of FN1 and ZNF438 gene in mRNA and protein levels were 0.434 ± 0.050 and 0.069 ± 0.042,significantly lower than the control group of 1.000 ± 0.016 and 1.025 ± 0.277 （t（FNI） =18.532,t（ZNF,438） =5.905,P 〈 0.05）.Differences were statistical significance.Moreover,after AR down-regulation,the expression of FN1 and ZNF438 gene in mRNA and protein levels were 17.579 ± 4.415 and 1.895 ± 0.424,significantly higher than the control group of 1.028 ±0.445 and 1.041 ±0.190（t（FN1） =6.461,t（ZNF438） =3.184,P〈0.05）.Differences were statistical significance.Conclusions FN1 and ZNF438 gene are AR negatively regulated target genes in LNCaP-AI cells.The study might provide a new sight to further explore the mechanism of LNCaP-AI cells grow in androgen-depleted conditions.