中国血液净化

中国血液净化 ›› 2018, Vol. 17 ›› Issue (10): 694-698.doi: 10.3969/j.issn.1671-4091.2018.10.010

• 基础研究 • 上一篇    下一篇

miR-29 对腹膜间皮细胞EMT 的作用

杨丽娜1,樊怡2,马健飞2   

  1. 1. 中国医科大学附属第一医院老年医学科
    2. 中国医科大学附属第一医院肾内科
  • 收稿日期:2018-07-13 修回日期:2018-07-27 出版日期:2018-10-12 发布日期:2018-10-12
  • 通讯作者: 马健飞 majiangfei@medmail.com.cn E-mail:majiangfei@medmail.com.cn
  • 基金资助:

    沈阳市科学计划项目(17-230-9-35)，国家自然科学基金(81300636)

Effects of miR-29a on epithelial to mesenchymal transition in human peritoneal mesothelial cells

  • Received:2018-07-13 Revised:2018-07-27 Online:2018-10-12 Published:2018-10-12

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摘要： 【摘要】目的观察miR- 29a 对高糖刺激人腹膜细胞(human peritoneal mesothelial cells，HPMCs)导致上皮细胞-间充质细胞转分化(epithelial to mesenchymal transition，EMT)的影响。方法应用高糖刺激HPMCs，建立HPMCs EMT 模型，运用Real time PCR 检测高糖刺激HPMCs 不同浓度及不同时间后上皮细胞标志物E-cadherin 及间充质细胞标志物α-SMA 和FN 信号分子的表达水平及miR-29a表达水平变化。然后用miR-29a inhibitor 转染HPMCs 下调miR-29a 表达，运用Real time PCR 检测miR-29a 表达水平及EMT 相关标志物E-cadherin、α-SMA、FN 等信号分子的变化水平。结果Real time PCR 结果显示，不同浓度及不同时间葡萄糖刺激均可使HPMCs 的间充质细胞标志物α-SMA(不同浓度:1.5% 1.970 ± 0.153 比1,F=7.692，P=0.008；2.5% 3.291 ± 0.265 比1,F=4.479，P＜0.001；4.25% 4.301±0.346 比1, F=5.496, P＜0.001；不同时间:6h 2.161±0.202 比1, F=6.563, P=0.001;12h 2.820±0.285 比1, F=5.111, P＜0.001; 24h 3.291±0.265 比1, F=4.479, P＜0.001；48h 3.980±0.407 比1, F=9.377, P=0.006)、FN(不同浓度:1.5% 1.630±0.157 比1, F=7.092, P=0.002；2.5% 2.910±0.199 比1, F=4.000，P＜0.001；4.25% 3.601±0.301 比1,F=5.575，P＜0.001；不同时间:6h 1.761±0.172 比1，F=7.144，P=0.002；12h 2.390±0.170 比1,F=4.499，P＜0.001；24h 2.910±0.199 比1,F=4.000，P＜0.001；48h 3.601±0.300 比1,F=4.570，P＜0.001)表达逐渐增加,上皮细胞标志物E- cadherin(不同浓度:1.5% 0.693±0.065 比1，F=4.665, P=0.001；2.5% 0.452±0.045 比1, F=4.994, P＜0.001；4.25% 0.302±0.030 比1, F=4.000, P＜0.001；不同时间:6h 0.802±0.084 比1，F=4.000，P=0.012；12h 0.630±0.070比1，F=7.030，P=0.001；24h 0.452±0.045 比1,F=4.994，P＜0.001；48h 0.290±0.030 比1,F=4.000，P＜0.001)表达逐渐减少，提示HPMCs 发生EMT 改变。并且随着葡萄糖浓度升高及作用时间延长，miR-29a(不同浓度:1.5%1.452±0.147 比1，F=13.411，P =0.001；2.5% 3.120±0.320 比1, F=10.372, P＜0.001；4.25% 4.130±0.368 比1, F=12.497, P＜0.001；不同时间:6h 1.330±0.114 比1, F=5.788, P=0.001；12h 2.310±0.173 比1, F=5.546, P＜0.001；24h 3.100±0.236 比1, F=10.372, P＜0.001；48h 3.310±0.211比1, F=4.929, P＜0.001)表达逐渐升高。Real time PCR 结果表明，与高糖组相比，应用miR-29a inhibitor转染HPMCs 后，α- SMA(1.871±0.206 比3.291±0.265，F=0.104，P=0.002)、FN(1.782±0.156 比2.910±0.199，F=0.091，P=0.002)表达减少，E-cadherin(0.873±0.085 比0.452±0.045, F=0.760, P=0.002)表达升高。结论高糖以浓度依赖及时间依赖的方式诱导EMT，并且EMT 与miR-29a 表达正相关。应用miR-29a inhibitor下调miR-29a 表达能够部分逆转HPMCs EMT 的发生。

关键词: 人腹膜间皮细胞, EMT, miR-29a

Abstract: 【Abstract】Objective To investigate the effect of miR-29a on high glucose induced epithelial to mesenchymal transition (EMT) in human peritoneal mesothelial cells (HPMCs). Methods HPMCs were treated with high glucose to create a EMT model of HPMCs. Real-time PCR was used to quantify the expression of miR-29a and EMT makers including E-cadherin, α-SMA and fibronectin (FN) in HPMCs stimulated with various concentration of glucose for various period as well as in control cells. miR- 29a inhibitor was used to downregulate miR-29a expression in HPMCs, and the expression of miR-29a and EMT markers in these cells were then measured by real-time PCR. Results High glucose stimulated HPMCs for various concentration and period promoted the expression of α-SMA (1.5% glucose: 1.970±0.153 vs. 1, F=7.692, P=0.008; 2.5% glucose: 3.291±0.265 vs. 1, F=4.479, P＜0.001; 4.25% glucose: 4.301±0.346 vs. 1, F=5.496, P＜0.001; high glucose for 6h: 2.161±0.202 vs. 1, F=6.563, P=0.001; high glucose for 12h: 2.820±0.285 vs. 1, F=5.111, P＜0.001; high glucose for 24h: 3.291±0.265 vs. 1, F=4.479, P＜0.001; high glucose for 48h: 3.980±0.407 vs. 1, F=9.3769, P=0.006) and FN (1.5% glucose: 1.630 ± 0.157 vs. 1, F=7.092, P=0.002; 2.5% glucose: 2.910 ± 0.199 vs. 1, F=4.000, P＜0.001; 4.25% glucose: 3.601±0.301 vs. 1, F=5.575, P＜0.001; high glucose for 6h:1.761±0.172 vs. 1, F=7.144, P=0.002; high glucose for 12h: 2.390±0.170 vs. 1, F=4.499, P＜0.001; high glucose for 24h: 2.910±0.199 vs. 1, F=4.000, P＜0.001; high glucose for 48h: 3.601±0.300 vs. 1, F=4.570, P＜0.001), but inhibited the expression of E-cadherin (1.5% glucose: 0.693±0.065 vs. 1, F=4.665, P=0.001; 2.5% glucose: 0.452±0.045 vs. 1, F=4.994, P＜0.001; 4.25% glucose: 0.302±0.030 vs. 1, F=4.000, P＜0.001; high glucose for 6h: 0.802±0.084 vs. 1, F=4.000, P=0.012; high glucose for 12h: 0.630±0.070 vs. 1, F=7.030, P=0.001; high glucose for 24h: 0.452±0.045 vs. 1, F=4.994, P＜0.001; high glucose for 48h: 0.290±0.030 vs. 1, F=4.000, P＜0.001), suggesting that EMT occurred in HPMCs stimulated with high glucose. Moreover, miR-29a expression increased in HMPCs along with the increase of glucose concentration and stimulation period (1.5% glucose: 1.452 ± 0.147 vs. 1, F=13.411, P=0.001; 2.5% glucose: 3.120 ± 0.320 vs. 1, F=10.372, P＜0.001; 4.25% glucose: 4.130±0.368 vs. 1, F=12.497, P＜0.001; high glucose for 6h: 1.330±0.114 vs. 1, F=5.788, P=0.001; high glucose for 12h: 2.310±0.173 vs. 1, F=5.546, P＜0.001; high glucose for 24h: 3.100±0.236 vs. 1, F=10.372, P＜0.001; high glucose for 48h: 3.310 ± 0.211 vs. 1, F=4.929, P＜0.001). When HPMCs were transfected with the miR-29a inhibitor to inhibit the expression of miR-29a, the cells showed decreased expression of α-SMA and FN and increased expression of E- cadherin, indicating that the EMT induced by high glucose was inhibited in these cells (α-SMA: 1.871±0.206 vs. 3.291±0.265, F=0.104, P=0.002; FN: 1.782±0.156 vs. 2.910±0.199, F=0.091, P=0.002; E-cadherin: 0.873±0.085 vs. 0.452±0.045, F=0.760, P=0.002). Conclusion High glucose induced EMT in dose dependent and time dependent manner. In addition, there was a positive correlation between miR-29a and EMT; EMT induced by high glucose was attenuated in HPMCs in which miR-29a was downregulated.

Key words: HPMCs,  Epithelial to mesenchymal transition, miR-29a