摘要
目的:研究微小RNA-34a(microRNA-34a,miR-34a)对高糖条件下骨髓间充质干细胞(bone marrow-derived mesenchymal stem cells,BMSCs)衰老的调控及潜在机制。方法:从60~80 g的雄性SD大鼠骨髓中分离和培养BMSCs,将细胞分为5组:正常葡萄糖(NG)组、高糖(HG)组、HG+miR-34a mimic组、HG+miR-34a NC组和HG+miR-34a inhibitor组;为了探究miR-34a是否通过沉默信息调节因子1(silent information regulator 1, SIRT1)调控高糖条件下BMSCs的衰老水平,除了上述5组,另增加HG+siRNA-SIRT1组、HG+siRNA-NT组和HG+miR-34a inhibitor+siRNA-SIRT1组。RT-qPCR法检测各组细胞中miR-34a及SIRT1 mRNA的表达,CCK-8法检测细胞的活力,衰老相关β-半乳糖苷酶染色法检测细胞的衰老情况,Western blot检测SIRT1、叉头框蛋白O3a(forkhead boxO3a, FOXO3a)和P21蛋白的表达。结果:与NG组相比,HG组miR-34a表达明显上调(P<0.01),细胞活力减弱(P<0.05),衰老细胞明显增加(P<0.01);与HG+miR-34a NC组相比,HG+miR-34a mimic组的细胞活力明显减弱(P<0.01),衰老细胞明显增加(P<0.01),SIRT1蛋白表达明显降低(P<0.01),FOXO3a蛋白表达明显升高(P<0.01),HG+miR-34a inhibitor组上述各参数则表现相反;与HG组相比,HG+siRNA-SIRT1组P21及FOXO3a蛋白表达均明显升高(P<0.01),当向HG+miR-34a inhibitor组加入siRNA-SIRT1后,miR-34a inhibitor对高糖条件下BMSCs中P21及FOXO3a蛋白表达的抑制作用被部分减弱(P<0.05)。结论:miR-34a通过靶向调节SIRT1的表达实现对高糖条件下BMSCs衰老的调控。
AIM: To detect the effect and potential mechanism of microRNA-34 a(miR-34 a) on the senescence of bone marrow-derived mesenchymal stem cells(BMSCs) under high glucose condition. METHODS: BMSCs were isolated and cultured from 60~80 g male SD rats. The BMSCs were divided into 5 groups: normal glucose(NG) group, high glucose(HG) group, HG+miR-34 a mimic group, HG+miR-34 a NC group and HG+miR-34 a inhibitor group. In order to confirm whether miR-34 a regulated the senescence of BMSCs under high glucose condition by regulating the expression of silent information regulator 1(SIRT1), in addition to the above groups, HG+siRNA-SIRT1 group, HG+siRNA-NT group and HG+miR-34 a inhibitor+siRNA-SIRT1 group were added. The expression of miR-34 a and SIRT1 mRNA was detected by RT-qPCR. CCK-8 assay and senescence-associated β-galactosidase assay were used to detect cell viability and senescence, respectively. The protein expression of SIRT1, forkhead box O3 a(FOXO3 a) and P21 in the BMSCs was analyzed by Western blot. RESULTS: The expression of miR-34 a in HG group was increased significantly compared with NG group(P<0.01), and long-term exposure of the BMSCs to high glucose lead to decreased cell viability and increased senescence(P<0.05). Compared with HG+miR-34 a NC group, the cell viability in HG+miR-34 a mimic group was decreased significantly(P<0.01), the senescence of BMSCs was increased significantly(P<0.01), the protein expression of SIRT1 was decreased significantly(P<0.01) and the protein expression of FOXO3 a was increased significantly(P<0.01). However, inhibition of miR-34 a expression showed the opposite effect to miR-34 a mimic. Similar to the HG+miR-34 a mimic group, the protein expression of P21 and FOXO3 a in HG+siRNA-SIRT1 group were significantly higher than that in HG group(P<0.01). After adding siRNA-SIRT1 into HG+miR-34 a inhibitor group, the inhibitory effect of the miR-34 a inhibitor on the expression of P21 and FOXO3 a in BMSCs were partly weakened(P<0.05). CONCLUSION: miR-34 a regulate the senescence of BMSCs under high glucose condition by regulating the expression of SIRT1.
作者
刘晓虹
张凤云
王坤
王志荣
张卓琦
LIU Xiao-hong;ZHANG Feng-yun;WANG Kun;WANG Zhi-rong;ZHANG Zhuo-qi(Xuzhou Medical University, the Affiliated Hospital, Xuzhou Medical University, Xuzhou 221000, China;Department of Cardiology, the Affiliated Hospital, Xuzhou Medical University, Xuzhou 221000, China)
出处
《中国病理生理杂志》
CAS
CSCD
北大核心
2019年第6期994-1003,共10页
Chinese Journal of Pathophysiology
基金
国家自然科学基金青年基金资助项目(No.81700231)
关键词
微小RNA-34a
骨髓间充质干细胞
糖尿病
衰老
沉默信息调节因子1
MicroRNA-34a
Bone marrow-derived mesenchymal stem cells
Diabetes mellitus
Senescence
Silent information regulator 1