miR-148a-5p参与了高蛋氨酸饮食诱导ApoE^-/-小鼠的肝细胞凋亡

Involvement of miR-148a-5p in ApoE^-/-mouse hepatocyte apoptosis induced by high methionine diet

背景:高蛋氨酸饮食可以导致ApoE^-/-小鼠发生肝损伤,微小RNA(miRNA)参与细胞存活、分化和细胞凋亡等各种细胞过程,具有重要意义。目的:探讨miR-148a-5p在高蛋氨酸饮食诱导ApoE^-/-小鼠肝细胞凋亡中的作用。方法:12只ApoE^-/-小鼠随机分为2组,每组6只,ApoE^-/-对照组为普通饮食,ApoE^-/-高蛋氨酸组为高蛋氨酸饮食。苏木精-伊红染色观察2组肝组织形态学变化;TUNEL染色观察肝细胞的凋亡情况;Western blot测定Bax、Bcl-2的表达改变;荧光定量PCR检测miR-148a-5p的表达;运用Target Scan靶基因预测软件预测miR-148a-5p的靶基因;双荧光素酶活性实验明确其靶向关系。结果与结论:①与对照组相比,ApoE^-/-高蛋氨酸组肝脏组织肝小叶结构发生明显紊乱,部分细胞呈胞浆疏松化变性,肝细胞凋亡增加,Bax的表达明显上升且Bcl-2的表达显著下降(P<0.01);②荧光定量PCR结果显示,ApoE^-/-高蛋氨酸组miR-148a-5p的表达增加(P<0.05);③靶基因预测软件提示,Bcl-2为miR-148a-5p的潜在靶基因;④双荧光素酶活性实验确定了miR-148a-5p与Bcl-2的靶向关系(P<0.05);⑤肝细胞中过表达miR-148a-5p后Bax的表达显著上升且Bcl-2的表达明显下降(P<0.01);⑥提示在高蛋氨酸饮食诱导的ApoE^-/-小鼠肝细胞中,miR-148a-5p通过负调控Bcl-2促进肝细胞凋亡,这可能是造成肝损伤的机制之一。

BACKGROUND:High methionine diet can cause liver damage in ApoE^-/-mice,and microRNAs(miRNAs)are involved in various cell processes such as cell survival,differentiation,and apoptosis,which is of great significance.OBJECTIVE:To investigate the effect of miR-148a-5p on ApoE^-/-mouse hepatocyte apoptosis induced by high methionine diet.METHODS:Twelve ApoE^-/-mice aged 5 weeks were randomly divided into two groups with six ApoE^-/-mice in each group:ApoE^-/-control group was fed with a normal diet,and ApoE^-/-high methionine diet group(ApoE^-/-+HMD)was fed with a high methionine diet.Hematoxylin-eosin staining was used to observe the morphological changes of liver tissues in the two groups.TUNEL staining was used to observe the apoptosis of hepatocytes.The expressions of Bax and Bcl-2 were detected by western blot.Fluorescence quantitative PCR was used to detect the expression of miR-148a-5p.Target Scan prediction software was used to predict the target gene of miR-148a-5p,and double luciferase activity assay was used to verify the targeted relationship.RESULTS AND CONCLUSION:Compared with the control group,the hepatic lobular structure of the ApoE^-/-mice with high methionine diet was obviously disturbed,and some cells showed cytoplasmic osteoporosis;hepatocyte apoptosis increased;the expression of Bax significantly increased and the expression of Bcl-2 significantly decreased(P<0.01).Fluorescence quantitative PCR results showed that the expression of miR-148a-5p increased in the ApoE^-/-mice with high methionine diet(P<0.05).The target gene prediction software indicated that Bcl-2 was a potential target gene of miR-148a-5p,and the targeted relationship between miR-148a-5p and Bcl-2 was confirmed by the double luciferase activity assay(P<0.05).After miR-148a-5p was overexpressed in hepatocytes,Bax expression increased and Bcl-2 expression decreased significantly(P<0.01).In conclusion,miR-148a-5p promoted hepatocyte apoptosis through negative regulation of Bcl-2 in ApoE^-/-mouse hepatocytes induced by high methionine diet,which may be one of the mechanisms causing liver injury.