A20突变体对前列腺癌细胞炎症反应因子及凋亡抑制基因表达的影响

Effect of A20 mutant on expression of inflammatory factors and anti-apoptosis genes in PC-3M cells

目的通过构建A20突变体真核载体,获取A20突变体,研究其对前列腺癌细胞PC-3M细胞炎症反应因子及凋亡抑制基因表达的影响。方法构建A20突变体pEGFP-C1表达载体,提取质粒,限制性内切酶酶切鉴定并测序。质粒DNA转染COS7细胞,用LPS诱导PC-3M细胞产生炎症反应,利用Western blot法检测PC-3M细胞A20,IκB-α、pERK1/ERK2、pJNK、Bcl-2;利用ELISA检测NF-κB、TNF-α、IL-6等的水平。结果限制性内切酶酶切鉴定与DNA测序证实合成A20突变体真核载体寡核苷酸链插入正确。Western blot检测结果表明转染后PC-3M细胞A20蛋白表达显著增。A20突变体抑制IκB-α的降解,ERK1/ERK2、JNK磷酸化及凋亡抑制基因Bcl-2的表达。ELISA法检测结果表明,A20突变体能显著抑制NF-κB、TNF-α、IL-6的表达(P<0.05,P<0.01)。结论成功构建人A20突变体真核表达载体,A20突变能抑制炎症反应因子及凋亡抑制基因。

Objective To construct an eukaryotic expression vector of A20 mutant,which expresses A20 stably,and evaluate its effects on the expression of inflammatory factors and anti-apoptosis genes in prostate cancer PC-3M cells. Methods The domain of A20 in inhibiting NF-κB activity was designed based on the gene sequence of natural A20. Then the sequence of A20 mutant was subcloned into the eukaryotic expression vector pEGFP-C1,which was identified and confirmed by restriction enzyme digestion and DNA sequencing. Subsequently,the constructed recombinant plasmid pEGFP-C1-hA20 mutant was transfected into COS 7 cells to replicate the plasmid,and then the expression of protein A20,IκB-α,pERK1 / ERK2、pJNK and Bcl-2 were detected in PC-3M cells after transfection by Western blotting. The concentrations of NF-κB,TNF-α and IL-6 in the supernatant were determined by ELISA. Results Restriction enzyme digestion and DNA sequencing confirmed that the obtained sequence of mutant was correct. Western blotting indicated that the expression of protein A20 was enhanced after the pEGFP-C1 plasmid was transfected into the PC-3M cells. After stimulation with LPS,the degradation of IκB-α and phosphorylation of ERK1 / ERK2 and JNK were inhibited after A20 mutant pretreatment. The expression of Bcl-2 was obviously increased,but decreased significantly after A20 mutant pretreatment. After stimulation with LPS for 24 h,the concentrations of NF-κB,TNF-α and IL-6 in the supernatants of PC-3M cells were obviously increased in the blank and control groups,but those levels were significantly reduced in mutant group（ P〈0. 05,P〈0. 01）. Conclusion The eukaryotic expression vector of A20 mutant is successfully constructed,which expresses A20 stably and suppresses the expression of inflammatory factors and anti-apoptosis genes in PC-3M cells.