微RNA-155在db/db小鼠体内的表达及其作用

Expression and role of microRNA-155 in db/db mice

目的探讨微RNA-155(miR-155)在C57BLKS/db(db/db)小鼠血清和肾脏中的表达及其在糖尿病肾病(DKD)发病机制中的作用。方法选取24只db/db小鼠,按随机数字表法分为6、8和10周龄组,每组8只,并设同期同周龄C57BL/6小鼠作为对照组。使用实时荧光定量PCR测定小鼠血清和肾组织miR-155的表达。通过免疫组化、实时荧光定量PCR和Western印迹测定小鼠肾组织中Ets-1、内皮型一氧化碳合酶(eNOS)和血管紧张素Ⅱ的Ⅰ型受体(AGTR1)mRNA和蛋白的表达。结果与对照组相比,6、8、10周龄db/db小鼠血清中miR-155的表达水平均明显增加(均P<0.01),10周龄时miR-155表达最明显(P<0.01);6、8和10周龄db/db小鼠肾组织中的miR-155的表达均明显上调(均P<0.01),10周龄时上调最明显(P<0.01)。免疫组化结果显示,Ets-1、eNOS和AGTR1均定位于肾小球内皮细胞;实时荧光定量PCR结果显示,与对照组相比,6、8和10周龄db/db小鼠肾组织中Ets-1、eNOS、AGTR1 mRNA的表达水平均下调(均P<0.05),10周龄时下调均最为明显。Western结果显示,与对照组相比,6周龄db/db小鼠肾组织Ets-1、eNOS和AGTR1表达均无明显变化,8周龄时eNOS蛋白表达水平下调(P<0.05),10周龄时AGTR1蛋白表达水平开始下调(P<0.05),Ets-1和eNOS蛋白表达水平均明显下调(均P<0.01)。结论db/db小鼠血清、肾组织中miR-155的表达水平随着DKD的进展逐渐升高,而miR-155靶基因Ets-1、eNOS和AGTR1随着DKD的进展表达逐渐降低。miR-155可能通过抑制其靶基因Ets-1、eNOS和AGTR1,影响内皮细胞功能而参与DKD的发生和发展。

Objective To investigate the expression of microRNA-155(miR-155)in serum and kidney of C57BLKS/db(db/db)mice and its role in the pathogenesis of diabetic kidney disease(DKD).Methods The db/db mice(n=24)were divided into 6,8,and 10 weeks old groups(n=8)with age increasing according to the random number table,and C57BL/6 mice of the same age were used as control group.The expression of miR-155 in mouse serum and kidney tissue was determined using real-time quantitative PCR.The mRNA and protein expression of Ets-1,eNOS,AGTR1 in renal tissues was verified by real-time quantitative PCR,Western blotting and immunohistochemistry.Results Compared with the control group,the expression of miR-155 in serum of db/db mice at 6,8 and 10 weeks of age were significantly increased(all P<0.01),and the increase of miR-155 was most obvious at 10 weeks of age(P<0.01).Meanwhile the expression of miR-155 in kidney tissues of 6,8 and 10 weeks old db/db mice was significantly up-regulated(all P<0.01),and the highest expression of miR-155 was at 10 weeks of age(P<0.01).Immunohistochemistry showed that Ets-1,eNOS and AGTR1 were localized in glomerular endothelial cells.The results of real-time quantitative PCR showed that the mRNA expression of Ets-1,eNOS and AGTR1 were down-regulated in the kidney tissues of db/db mice at 6,8 and 10 weeks of age compared to the control(all P<0.05),and the level of down-regulation was the most obvious at 10 week.Western blotting results showed that there was no significant change in Ets-1,eNOS and AGTR1 in 6-week-old db/db mice compared to the control group;the eNOS protein expression was down-regulated at 8 weeks of age(P<0.05);the expression of AGTR1 protein was down-regulated(P<0.05),and the protein expression of Ets-1 and eNOS were significantly down-regulated at 10-week age(both P<0.01).Conclusions The expression of miR-155 in serum and kidney tissues of db/db mice increases during the progression of DKD,while the expression of miR-155 target genes Ets-1,eNOS and AGTR1 decreases with the progression of DKD.MiR-155 may participate in the development and progression of DKD by inhibiting its target genes Ets-1,eNOS and AGTR1,affecting endothelial cell function.