布鲁氏菌侵染过程中has-miR-5196-3p对炎症小体的调控作用

Regulatory Effect of has-miR-5196-3p on Inflammasome During Brucella Infection

试验旨在研究miRNA分子has-miR-5196-3p在布鲁氏菌侵染THP1细胞过程中对NOD样受体蛋白3炎症小体(NLRP3)的调控作用。运用TargetScan、MiRDB等生物信息学预测网站预测has-miR-5196-3p和NLRP3-3′UTR的结合位点,构建其野生型及突变型双荧光素酶报告载体,分别与has-miR-5196-3p mimics及阴性对照共转染293T细胞,通过双荧光素酶报告系统检测各组相对荧光素酶活性的变化,验证has-miR-5196-3p和NLRP3-3′UTR的靶向关系;布鲁氏菌侵染THP1细胞,实时荧光定量PCR检测NLRP3、半胱氨酰天冬氨酸特异性蛋白酶(caspase1)及has-miR-5196-3p mRNA的表达情况,Western blotting检测NLRP3和caspase1蛋白表达水平;将has-miR-5196-3p mimics、has-miR-5196-3p inhibitor及阴性对照转染至THP1细胞,实时荧光定量PCR及Western blotting检测NLRP3和半胱氨酰天冬氨酸特异性蛋白酶(caspase1)的表达水平。结果显示,试验成功构建含有NLRP3-3′UTR的野生型及突变型双荧光素酶报告载体;has-miR-5196-3p mimics极显著降低pmirGLO-NLRP3-3′UTR-wt的相对荧光素酶活性(P<0.01);布鲁氏菌侵染THP1细胞后,NLRP3炎症小体被激活,而has-miR-5196-3p无明显变化(P>0.05);has-miR-5196-3p mimics能显著抑制NLRP3的表达(P<0.05),而has-miR-5196-3p inhibitor则相反。结果表明,miRNA分子has-miR-5196-3p可与NLRP3-3′UTR结合并抑制其表达,布鲁氏菌侵染THP1细胞过程中通过has-miR-5196-3p靶向负调节NLRP3炎症小体的表达及炎症的发生。

This study was aimed to explore the regulation of the miRNA molecule has-miR-5196-3p on the NOD-like receptor protein 3 inflammasome(NLRP3)during the process of Brucella infecting THP1 cells.Use TargetScan,MiRDB and other bioinformatics prediction sites to predict the binding sites of has-miR-5196-3p and NLRP3-3′UTR.Its wild-type and mutant dual luciferase reporter vectors were construced,respectively,has-miR-5196-3p mimics and negative control were transfected into 293T cells,and the relative luciferase activity changes of each group were detected by the dual luciferase reporter system to verify the targeting relationship between has-miR-5196-3p and NLRP3-3′UTR.THP1 cells were infected with Brucella,the mRNA expressions of NLRP3,caspase1 and has-miR-5196-3p were detected by Real-time PCR,and the protein expressions of NLRP3 and caspase1 were detected by Western blotting..has-miR-5196-3p mimics,has-miR-5196-3p inhibitor and negative control were transfected into THP1 cells,Real-time PCR and Western blotting were used to detect the expression level of NLRP3 and cysteinyl aspartate specific protease(caspase1).The results showed that the wild-type and mutant dual luciferase reporter vector containing NLRP3-3′UTR was successfully constructed.has-miR-5196-3p mimics extremely significantly reduced the relative luciferase activity of pmirGLO-NLRP3-3′UTR-wt(P<0.01).After Brucella infecting THP1 cells,NLRP3 inflammasome was activated,while has-miR-5196-3p did not change significantly(P>0.05).has-mi-R-5196-3p mimics could significantly inhibit the expression of NLRP3(P<0.05),while has-miR-5196-3p inhibitor was the opposite.It showed that the miRNA molecule has-miR-5196-3p could bind to NLRP3-3′UTR and inhibit its expression.During the process of Brucella infecting THP1 cells,has-miR-5196-3p targets and negatively regulated NLRP3 inflammasome expression and inflammation happened.