| 刘朱美卉,陈晓宇,杨元芳.干扰微小 RNA-320表达通过蛋白激酶 B/哺乳动物雷帕霉素通路保护高糖诱导的胰岛 β细胞损伤[J].安徽医药,2021,25(10):2039-2043. |
| 干扰微小 RNA-320表达通过蛋白激酶 B/哺乳动物雷帕霉素通路保护高糖诱导的胰岛 β细胞损伤 |
| Interference with miR-320 expression protects high glucose-induced pancreatic β cell injury through Akt/mTOR pathway |
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| DOI：10.3969/j.issn.1009-6469.2021.10.030 |
| 中文关键词: 胰腺疾病 微小 RNA-320 高糖刺激 胰岛 β细胞损伤 蛋白激酶 B/哺乳动物雷帕霉素 小鼠 |
| 英文关键词: Pancreatic diseases MiR-320 High glucose stimulation Pancreatic β cell damage Akt/mTOR Mice |
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| 中文摘要: |
| 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、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/哺乳动物雷帕霉素高(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，其机制与激活 Akt/mTOR信号通路有关。 |
| 英文摘要: |
| Objective To explore the role and molecular mechanism of microRNA-320 (miR-320) in high glucose-induced pancreatic β-cell injury.Methods This study started from January 2019 and ended in January 2020. Mouse pancreatic β cell line MIN6 cellswere purchased from Type Culture Collection Chinese Academy of Sciences, and assigned into control group, high glucose group, highglucose + 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 wasdetermined 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 miR320 [(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-mTORC1 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-mTORC1 protein [(0.32±0.02) vs. (0.18±0.01)] expressions were increased in the high glucose + anti-miR-320 group. Compared with |
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