摘要
目的探讨微小RNA-320(miR-320)在高糖诱导的胰岛β细胞损伤中的作用和分子机制。方法本研究起止时间为2019年1月至2020年1月。小鼠胰岛β细胞株MIN6细胞购于中国科学院典型培养物保藏中心,将其分为对照组、高糖组、高糖+miRNA抑制剂阴性对照(anti-miR-NC)组、高糖+miR-320抑制剂(anti-miR-320)组、高糖+蛋白激酶B/哺乳动物雷帕霉素(Akt/mTOR)通路抑制剂(LY294002)组、高糖+anti-miR-320+LY294002组。实时荧光定量PCR(RT-qPCR)检测miR-320的表达水平。噻唑蓝(MTT)法、流式细胞术分别检测细胞存活和凋亡;酶联免疫吸附测定(ELISA)试剂盒检测乳酸脱氢酶(LDH)的活力;蛋白质印迹法(Western blotting)检测磷酸化Akt(p-Akt)、磷酸化的mTOR复合物1(p-mTORC1)的表达水平。结果与对照组比较,高糖组胰岛β细胞miR-320表达升高[(2.69±0.14)比(0.98±0.06)],存活率降低[(54.03±4.34)%比(99.74±9.16)%],凋亡率[(20.80±1.83)%比(7.75±0.86)%]、LDH活力[(257.76±18.94)U/L比(104.31±10.98)U/L]升高,p-Akt[(0.22±0.01)比(0.65±0.04)]和p-mTORC1蛋白[(0.18±0.02)比(0.51±0.03)]表达降低;与高糖+anti-miR-NC组比较,高糖+anti-miR-320组胰岛β细胞miR-320表达[(1.43±0.06)比(2.76±0.12)]降低,存活率[(89.43±7.11)%比(54.01±4.21)%]升高,凋亡率[(10.95±1.10)%比(20.72±1.76)%]、LDH活力[(139.77±13.24)U/L比(258.21±20.73)U/L]降低,p-Akt[(0.43±0.02)比(0.23±0.01)]和p-mTORC1蛋白[(0.32±0.02)比(0.18±0.01)]表达升高;与高糖组比较,高糖+LY294002组胰岛β细胞存活率[(33.60±3.67)%比(53.97±4.96)%]降低,凋亡率[(32.13±2.02)%比(20.89±2.23)%]、LDH活力[(337.05±24.15)U/L比(257.95±19.10)U/L]升高。与高糖+anti-miR-320组比较,高糖+anti-miR-320+LY294002组胰岛β细胞存活率[(64.00±5.77)%比(89.54±7.10)%]降低,凋亡率[(18.77±1.89)%比(10.93±1.12)%]、LDH活力[(223.38±23.35)U/L比(139.75±13.44)U/L]升高(P<0.05)。结论干扰miR-320表达能够减轻高糖诱导的胰岛β细胞损伤,其机制与激�
Objective To explore the role and molecular mechanism of microRNA-320(miR-320)in high glucose-induced pancreaticβ-cell injury.Methods This study started from January 2019 and ended in January 2020.Mouse pancreaticβcell line MIN6 cells were purchased from Type Culture Collection Chinese Academy of Sciences,and assigned into control group,high glucose group,high glucose+miRNA inhibitor negative control(anti-miR-NC)group,high glucose+miR-320 inhibitor(anti-miR-320)group,high glucose+protein kinase B/mammalian rapamycin(Akt/mTOR)pathway inhibitor(LY294002)group,high glucose+anti-miR-320+LY294002 group.Real-time fluorescent quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression level of miR-320.Cell survival and apoptosis were tested by MTT(methyl thiazolyl tetrazolium)and flow cytometry.Lactate dehydrogenase(LDH)activity was determined by enzyme-linked immunosorbent assay(ELISA)kit.The expression levels of phosphorylated Akt(p-Akt)and phosphomTOR complex 1(p-mTORC1)were measured by Western blotting.Results Compared with the control group,the expression of miR-320[(2.69±0.14)vs.(0.98±0.06)]in MIN6 cells was increased,the survival rate[(54.03±4.34)%vs.(99.74±9.16)%]was reduced,the apoptosis rate[(20.80±1.83)%vs.(7.75±0.86)%]and LDH activity[(257.76±18.94)U/L vs.(104.31±10.98)U/L]were increased,and the expressions of p-Akt[(0.22±0.01)vs.(0.65±0.04)]and p-mTORCl proteins[(0.18±0.02)vs.(0.51±0.03)]were reduced in the high glucose group.Compared with the high glucose+anti-miR-NC group,the expression of miR-320[(1.43±0.06)vs.(2.76±0.12)]in MIN6 cells was reduced,the survival rate[(89.43±7.11)%vs.(54.01±4.21)%]was increased,and the apoptosis rate[(10.95±1.10)%vs.(20.72±1.76)%]and LDH activity[(139.77±13.24)U/L vs.(258.21±20.73)U/L]were reduced,p-Akt[(0.43±0.02)vs.(0.23±0.01)]and p-mTORCl protein[(0.32±0.02)vs.(0.18±0.01)]expressions were increased in the high glucose+anti-miR-320 group.Compared with the high glucose group,the survival rate[(33.60±3.67)%vs.(53.97±4.96)%]of
作者
刘朱美卉
陈晓宇
杨元芳
LIU ZHU Meihui;CHEN Xiaoyu;YANG Yuanfang(Department of Endocrinology,The First Affiliated Hospital of Zhengzhou University,Zhengzhou,Henan 450000,China)
出处
《安徽医药》
CAS
2021年第10期2039-2043,共5页
Anhui Medical and Pharmaceutical Journal
