miR-199a-5p靶向MagT1在慢性HBV感染中引起T细胞功能耗竭的作用机制

miR-199a-5p Targets Mechanism Transporter 1 to Regulate the Function of Activated Human T Cells in Chronic Hepatitis B Virus Infection

目的研究miR-199a-5p靶向镁离子转运蛋白1(Magnesium transporter 1,MagT1)在慢性乙型肝炎病毒(hepatitis B virus,HBV)感染中引起T细胞功能耗竭的分子作用机制。方法①选取经临床确诊的慢性乙型肝炎患者10例,经检测其HBV-DNA检测值持续6月达1.0×10~5 IU/ml,且排除其它类型的病毒感染、肝硬化、肝癌等,并选取体检者10例作为正常对照组。②采集患者外周血,分离单个核细胞,用γ-干扰素(interferon gamma,IFN-γ)、白细胞介素2(interleukin 2,IL-2)、CD3抗体、白细胞介素1α(interleukin 1αlpha,IL-1α)活化T细胞;流式细胞仪进行T细胞亚群分析。③荧光定量逆转录聚合酶链反应(real-time reverse transcription quantitative polymerase chain reaction,RT-qPCR)检测慢性HBV感染组与正常对照组T细胞中miR-199a-5p、程序性细胞死亡因子1(programmed cell death 1,PD-1)和MagT1基因表达情况。④双荧光素酶报告基因系统鉴定miR-199a-5p与MagT1的靶向关系。⑤构建包装miR-199a-5p/miR-199a-5p sponge慢病毒载体,感染T细胞后RT-qPCR分别检测慢病毒感染后T细胞miR-199a-5p、PD-1和MagT1基因表达;⑥Western blot检测慢病毒感染T细胞前后MagT1、PD-1蛋白表达水平。结果流式细胞仪检测表明慢性HBV感染组及正常对照组T细胞均活化成功。RT-qPCR检测表明慢性HBV感染组miR-199a-5p表达显著高于正常对照组(P<0.01),PD-1表达也显著高于正常对照组(P<0.01),但MagT1表达显著低于正常对照组(P<0.01)。双荧光素酶报告基因系统证实miR-199a-5p靶向MagT13’UTR并抑制其表达(P<0.05)。RT-qPCR检测表明慢病毒构建成功,miR-199a-5p/miR-199a-5p sponge分别显著上调或下调miR-199a-5p表达(P<0.01)。上调miR-199a-5p表达后,RT-qPCR表明MagT1基因表达显著低于对照组(P<0.01),PD-1基因表达显著高于对照组(P<0.01),且正常对照组均高于HBV感染组(P<0.01);Western blot表明MagT1蛋白表达水平显著下降(P<0.01),PD-1蛋白水平显著升高(P<0.01),且正常对照组高于HBV感染组(P<0.01)。下调miR-199a-5p表达后,MagT1表达量显著高于对照组(P<0.05),PD-1表达量显著低于对照组(P<0.01),且HBV感染组低于正常对照组(P<0.01);Western blot表明MagT1蛋白表达水平显著升高(P<0.01),PD-1蛋白水平显著降低(P<0.01),且HBV感染组低于正常对照组(P<0.01)。结论在慢性HBV感染中,miR-199a-5p可以负向调节靶基因MagT1的表达,并对活化后T细胞功能耗竭具有重要的调节作用。

Objective To research the molecular mechanism of miR-199 a-5 p targets Mechanism transporter 1 to regulate the function of activated human T cells in chronic hepatitis B virus(HBV)infection.Methods①Ten chronic HBV infection patients with HBV-DNA continuously positive for more than 6 months,the detection value more than 1.0×10~5 IU/ml were selected,and which had excluded other virus infection,hepatitis and cirrhosis.On the other hand,ten normol people were randomly selected as the normal group.②Monocytes were isolated from peripheral blood of patients with chronic HBV and normal peoples,T cells were activated with interferon gamma(IFN-γ),interleukin 2(IL-2),CD3 antibody and interleukin 1 alpha(IL-1α).The activated T cells were analyzed by flow cytometry to compare the activation capacity of T cells between HBV-infected group and the normal group.③The expressions of miR-199 a-5 p,programmed cell death 1(PD-1)and MagT1 were detected by real-time reverse transcription quantitative polymerase chain reaction(RT-qPCR)with T cells in each group.④The targeted sites of miR-199 a-5 p and MagT1 were identified by the dual-luciferase reporter gene system.⑤miR-199 a-5 p/miR-199 a-5 p sponge lentivirus vector were constructed.After lentivirus infected activated T cells,the expression of miR-199 a-5 p and MagT1 gene were detected by RT-qPCR.⑥The expression of MagT1 protein and PD-1 protein were detected by Western blot.Results Flow cytometry showed that T cells were activated successfully.RT-qPCR showed that the expression of miR-199 a-5 p(P<0.01)and PD-1(P<0.01)in the HBV-infected group was significantly higher than that in the normal group,the expression of MagT1 was significantly lower than that in the normal group(P<0.01).The dual luciferase reporter system confirmed that miR-199 a-5 p was targeted at MagT13’UTR and inhibited its expression(P<0.05).RT-qPCR indicated that the lentivirus was constructed successfully,and miR-199 a-5 p/miR-199 a-5 p sponge respectively up-regulated and down-regulated the expression of miR-199 a-5 p(P<0.01).After up-regulated the expression of miR-199 a-5 p,the expression of MagT1 was significantly lower than that in normal group(P<0.01),the expression of PD-1 was significantly higher than that in normal group(P<0.01),and normal group was higher than HBV-infected group(P<0.01).Western blot showed that the MagT1 protein level in the HBV-infected group and normal group were significantly decreased(P<0.01),the PD-1 protein level was significantly increased(P<0.01)and HBV-infected group lower than that in normal group(P<0.01).Conclusion miR-199 a-5 p may negatively regulate the expression of target gene MagT1 in chronic HBV infection and play an important regulatory role in the functional exhaustion of activated T cells.