LXR异常激活上调p27蛋白致胰岛β细胞周期阻滞

Activation of liver X receptors induced pancreatic β cell cycle arrest by up-regulating the expression of p27 protein

目的探讨肝X受体(liver X receptors,LXR)激动剂对小鼠胰岛β细胞系MIN6的作用。方法 LXR激动剂T0901317作用于MIN6细胞后,CCK-8法检测细胞活力;流式细胞仪检测细胞周期变化;实时定量RT-PCR法检测S期激酶相关蛋白2(Skp2)和p27 mRNA的表达水平;western blot检测Skp2和p27蛋白的表达情况。结果与control组相比,1、5和10μmol/L药物处理后的细胞存活率分别为(98.54±0.94)%、(87.03±0.93)%和(75.57±1.85)%,各组间差异有统计学意义(F=301.90,P<0.01)。与control组G1期(35.93±2.25)%和S期(52.87±1.19)%相比,1、5和10μmol/L药物处理组G1期细胞比例分别为(38.45±0.91)%、(45.46±1.34)%和(53.28±1.14)%,差异有统计学意义(F=80.83,P<0.01)。S期细胞比例分别为(48.65±0.85)%、(36.31±1.37)%和(31.45±1.22)%,差异有统计学意义(F=221.30,P<0.01)。10μmol/L T0901317处理组p27蛋白表达水平(2.84±0.14)明显高于control组(2.28±0.10),差异有统计学意义(t=4.54,P<0.05)。但两组间p27 mRNA水平差异无统计学意义(t=0.28,P>0.05)。10μmol/L T0901317下调Skp2 mRNA(0.52±0.02,t=29.22,P<0.01)和蛋白质水平(相对灰度值为0.98±0.12,control组为1.89±0.01,t=10.98,P<0.01)。以对照组(100%)为参照,转染组、药物处理组和干扰组的细胞存活率分别为(100.97±1.08)%、(75.03±1.83)%和(86.67±2.45)%,与对照组比较,转染组间差异无统计学意义(t=1.542,P>0.05),药物处理组细胞活力下降(t=23.58,P<0.01);药物处理组和干扰组间差异亦有统计学意义(t=7.77,P<0.01)。结论 LXR异常激活可导致胰岛β细胞增殖抑制,p27蛋白表达量增加,其机制可能为降解调控p27蛋白的Skp2 mRNA和蛋白质表达水平下降。

Objective To investigate the effects of liver X receptor （LXR） agonist on the proliferation of mouse pancreatic 13 cell line MIN6 cells. Methods The viability, changes of cell cycle, mRNA levels of S phase kinase associated protein 2 （Skp2） and p27, and protein levels of Skp2 and p27 in MIN6 cells treated with LXR agonist T0901317 were determined by the CCK-8 method, flow cy- tometry, real-time RT-PCR and western blot, respectively. Results The viability of MIN6 cells treated with 1 μmol/L, 5 μmol/L and 10 μmol/L of T0901317 were （98.54_±0.94）%, （87.03 ±0.93）% and （75.57 ±1.85）% of the controls, respectively, and there was significant difference among them （ F = 301.90, P 〈 0.01 ）. The percentages of G1 phase ceils in the MIN6 ceils treated with 0 μmol/L, 1 μmol/L, 5 μmol/L and 10 μmol/L of T0901317 were （35.93 ±2.25）%, （38.45 ±0.91）%, （45.46 ±1.34）% and （53.28 ± 1.14） %, respectively, and there was significant difference among them （ F = 80.83, P 〈 0.01 ）. Similarly, the percentages of S phase ceils in the MIN6 cells treated with 0 μmol/L, 1 μmol/L, 5 μmol/L and 10 p, mol/L of ＂I＂0901317 were （52.87 ±1.19） %, （48.65 ±0.85 ）%, （36.31 ±1.37 ）% and （31.45 ± 1.22 ）%, respectively, and there was also significant difference a- mong them （ F = 221.30, P 〈 0.01 ）. The protein levels of p27 in the MIN6 cells treated with 10 μ mol/L of T0901317 （2.84 ±0.14） were significantly higher than that in the controls （2.28 ± 0.10） （ t = 4.54, P 〈 0.05 ）, while there was no significant difference in the mRNA levels of p27 between them （t =0.28, P 〉0.05）. However, 10 mol/L of T0901317 significantly decreased mRNA （0.52 ± 0.02, t =29.22, P 〈0.01 ） and protein levels （0.98 μ0.12 vs 1.89 ±0.01, t = 10.98, P 〈0.01 ） of Skp2 in MIN6 cells. Based on the control siRNA transfection group as a reference （ 100% ）, the cell survival rates of the p27 siRNA transfection group, 10 p.mol/L of T0901317 treatment group and the intervention group （ 1）27 siRNA transfection ± 3＂0901317 treatment） were （ 100.97 ± 1.08 ） %,（75.03 ±1.83 ） % and （86.67 ± 2.45 ） %, respectively. There was no significant difference between the control siRNA and p27 siR- NA transfection groups （t = 1. 542, P 〉 0.05 ）. Compared with the control siRNA transfection group, the cell survival rates of the T0901317 treatment group decreased （ t = 23.58, P 〈 0.01 ）. There was also significant difference in the cell survival rates between the T0901317 treatment group and the intervention group （ t = 7.77, P 〈 0.01 ）. Conclusion The activation of LXR may induce pancre- atic 13 cell cycle arrest by up-regulating the expression of p27 and down-regulating the expression of Skp2.