miR-30a-5p启动子区DNA甲基化在同型半胱氨酸致肝损伤中的作用

目的:探讨微小RNA-30a-5p(miR-30a-5p)启动子区DNA甲基化在肝损伤中的作用。方法:随机选取4周龄胱硫醚β-合成酶(CBS)基因正常(CBS+/+)小鼠(n=12)及单基因敲除(CBS+/-)小鼠(n=12),均给予高蛋氨酸饮食8周。HL-7702细胞体外常规培养,分为对照(control)组、同型半胱氨酸(Hcy)组和Hcy+5-氮杂胞苷(AZC)组。全自动生化分析仪检测小鼠血清Hcy、丙氨酸氨基转移酶(ALT)和天门冬氨酸氨基转移酶(AST)水平;微板法测定肝细胞ALT和AST水平;小鼠肝脏石蜡切片行HE染色观察肝脏损伤情况;细胞活力染色检测肝细胞活力;RT-qPCR法检测小鼠肝脏组织和肝细胞中miR-30a-5p的表达;Pearson相关性分析肝脏miR-30a-5p表达与血清ALT和AST水平的相关性;巢式降落式甲基化特异性PCR(nMS-PCR)检测小鼠肝脏组织以及肝细胞中miR-30a-5p启动子区DNA甲基化水平的变化。结果:与CBS+/+对照组相比,CBS+/-组小鼠血清的Hcy、ALT和AST水平显著升高(P<0.05);HE染色显示肝细胞肿胀,可见核碎裂、溶解;肝脏miR-30a-5p表达明显降低(P<0.01);小鼠肝脏组织中miR-30a-5p的表达与血清ALT和AST的水平呈负相关(r2=0.4557,P=0.0003;r2=0.4626,P=0.0003);miR-30a-5p启动子区DNA的甲基化水平升高(P<0.01)。在细胞水平,与control组相比,Hcy组的ALT和AST水平升高(P<0.05,P<0.01),细胞活力显著降低,肝细胞miR-30a-5p启动子区DNA的甲基化水平升高(P<0.01),而使用AZC后miR-30a-5p启动子区DNA的甲基化水平降低(P<0.05),miR-30a-5p的表达上调(P<0.05)。结论:miR-30a-5p启动子区DNA高甲基化可能在肝损伤中发挥重要作用。

Effect of DNA methylation of miR-30a-5p promoter region on hepatic injury induced by homocysteine

AIM:To explore the role of DNA methylation of microRNA-30 a-5 p(miR-30 a-5 p)promoter region in hepatic injury.METHODS:Four-week-old normal mice and cystathionineβ-synthase(CBS)single gene knockout mice were used and divided into normal(CBS+/+,n=12)group and single gene knockout(CBS+/-,n=12)group,and the mice were fed with high methionine diet for 8 weeks.HL-7702 hepatic cells were routinely cultured in vitro and divided into control group,homocysteine(Hcy)group and Hcy+5-azacytidne(AZC)group.Serum Hcy,alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels were measured by automatic biochemical analyzer.The levels of ALT and AST in the cells culture medium were determined by the microplate method.Hepatic injury in the mice were observed with HE staining.Cell viability staining was used to measure the viability of hepatocytes.RT-qPCR was used to detect the expression of miR-30 a-5 p in the liver tissues and hepatocytes.The correlation between the expression of miR-30 a-5 p and serum ALT and AST levels was analyzed by Pearson correlation analysis.DNA methylation level of miR-30 a-5 p promoter region in the liver tissues and hepatocytes was detected by nested landing methylation-specific PCR(nMS-PCR).RESULTS:Compared with the CBS+/+mice,the serum levels of Hcy,ALT and AST in the CBS+/-mice were significantly increased(P<0.05).HE staining showed the hepatocyte swelling and nuclear fragmentation and dissolution.The expression level of miR-30 a-5 p in the liver tissues was decreased(P<0.01).Besides,the expression level of miR-30 a-5 p in the mice was negatively correlated with serum ALT and AST levels(r2=0.4557,P=0.0003,r2=0.4626,P=0.0003),and the DNA methylation of miR-30 a-5 p promoter region was increased(P<0.01).In the HL-7702 cells,compared with control group,the ALT and AST levels were increased in Hcy group(P<0.05,P<0.01),and the cell viability was remarkablely decreased.DNA methylation of miR-30 a-5 p promoter region was increased(P<0.01),which decreased after treated the cells with AZC(P<0.05),while the expression level of miR-30 a-5 p in the cells was increased(P<0.05).CONCLUSION:Hypermethylation of miR-30 a-5 p promoter region may play an important role in hepatic injury.