miR-20b对小鼠RAW264.7细胞血管内皮生长因子表达的调控作用

Effects of miR-20b on regulation of expression of vascular endothelial growth factor in RAW264.7 cells of mice

目的探讨miR-20b对小鼠RAW264．7细胞血管内皮生长因子（VEGF）表达的影响。方法以不同浓度（1、10、50、100、200ng／mL）TNF-α刺激小鼠RAW264．7细胞24h，ELISA法检测上清中VEGF的分泌情况。TNF-α（50ng／mL）刺激RAW264．7细胞6h，Real-TimePCR检测miR．20b和VEGFmRNA的表达。使用PicTar算法预测VEGF是否为miR-20b的调控靶点。利用脂质体Lipofectamine2000介导miR-20b模拟物及其对照转染RAW264．7细胞，用TNF-α（100ng／mL）刺激24h，ELISA法检测VEGF的产生情况。结果TNF-α能够诱导小鼠RAW264．7细胞分泌VEGF，50-100ng／mLTNF-α．是最适合的刺激浓度；50ng／mLTNF-α刺激RAW264．7细胞6h，VEGFmRNA的相对表达量升高至对照组的（1．60±0．85）倍，而miR-20b的相对表达量降至对照组（0．55±0．33）倍。PicTar算法表明，小鼠VEGF的3’-UTR区含有miR-20b种子区域AAAGUGC的互补序列GCACUUU。转染miR-20b模拟物后，TNF-α诱导的VEGF蛋白表达升高被完全抑制（P〈0．01），但转染miR-20b模拟物对照对VEGF的产生无明显影响（P〉0．05）。结论小鼠RAW264．7细胞中miR-20b负性调节VEGF的表达。

Objective To explore the effects of miR-20b on the expression of vascular endothelial growth factor （VEGF） in RAW264. 7 cells of rats. Methods RAW264. 7 cells of mice were stimulated by different concentrations （1, 10, 50, 100, and 200 ng/mL） of TNF-α for 24 h and the amount of VEGF in the supernatant was detected by the ELISA. The miR-20b and VEGF mRNA expressions were detected by the Real- Time PCR for RAW264.7 ceils stimulated by TNF-α （50 ng/mL） for 6 h. The Pictar algorithm was used to predict whether VEGF was a regulating target of miR-20b. MiR-20b mimics and its control were transfected into RAW264.7 cells by Lipofectamine 2000. ELISA was used to detect the amount of VEGF in the supernatant after the transfected RAW264.7 cells were stimulated by TNF-α （100 ng/mL） for 24 h. Results TNF-α induced the RAW264. 7 cells to secret VEGF and 50 -100 ng/mL TNF-α was the most suitable concentration for stimulation. After RAW264.7 cells were stimulated by TNF-α （50 ng/mL） for 6 h, the expression of VEGF was elevated to （ 1.60±0. 85） times of that of the control group, while the expression of miR-20b was decreased to （0.55 ±0.33） times of that of the control group. The Pictar algorithm showed that the VEGF 3＇-UTR contained the complementary sequences ＇ GCACUUU＇ of miR-20b seed region ＇ AAAGUGC＇. The increased expression of VEGF protein was completely inhibited after TNF-α-stimulated RAW264.7 cells were transfectedwith miR-20b mimics （P 〈 0.01）, while for the control group, transfected with miR-20b mimics had no significant effect on the production of VEGF （P〉0.05）. Conclusion MiR-20b can negatively regulate the expression of VEGF in RAW264.7 cells of mice.