miR-20b对小鼠RAW264.7细胞血管内皮生长因子表达的调控作用

发布时间：2018-07-17 03:02

【摘要】：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、200 ng/mL)TNF-α刺激小鼠RAW264.7细胞24 h,ELISA法检测上清中VEGF的分泌情况。TNF-α(50 ng/mL)刺激RAW264.7细胞6 h,Real-Time PCR检测miR-20b和VEGF mRNA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Lipofectamine 2000介导miR-20b模拟物及其对照转染RAW264.7细胞,用TNF-α(100 ng/mL)刺激24 h,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,50~100 ng/mL TNF-α是最适合的刺激浓度;50 ng/mL TNF-α刺激RAW264.7细胞6 h,VEGF mRNA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(P0.01),但转染miR-20b模拟物对照对VEGF的产生无明显影响(P0.05)。结论小鼠RAW264.7细胞中miR-20b负性调节VEGF的表达。
[Abstract]:Objective to investigate the effect of miR-20b on the expression of vascular endothelial growth factor (VEGF) in mouse RAW264.7 cells. Methods the 1,10,50100200 ng/mL TNF- alpha was used to stimulate the 24 h of mouse RAW264.7 cells, and ELISA method was used to detect the secretion of VEGF in the supernatant. The expression of.TNF- alpha (50 ng/mL) was 6. Using the PicTar algorithm to predict whether VEGF is a regulatory target for miR-20b. Using liposome Lipofectamine 2000 to mediate miR-20b analogue and its control transfected RAW264.7 cells, TNF- alpha (100 ng/mL) stimulates 24 h, ELISA method is used to detect the production of VEGF. 50 ng/mL TNF- alpha stimulated RAW264.7 cells to 6 h, and the relative expression of VEGF mRNA increased to (1.60 + 0.85) times of the control group, while the relative expression of miR-20b decreased to the control group (0.55 + 0.33) times.PicTar algorithm, indicating that the 3'-UTR region of VEGF contained the complementary sequence of AAAGUGC in the miR-20b seed region. After that, the expression of VEGF protein induced by TNF- alpha was completely inhibited (P0.01), but the transfection of miR-20b analogue control had no significant effect on the production of VEGF (P0.05). Conclusion miR-20b negatively regulated the expression of VEGF in mouse RAW264.7 cells.
【作者单位】：蚌埠医学院免疫学教研室安徽省感染与免疫重点实验室;蚌埠医学院科研中心;
【基金】：国家自然科学基金(81273273) 安徽省自然科学基金(1308085MH114)~~
【分类号】：R346

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