摘要
目的探讨miR-199a-5p在常氧及低氧环境下的大鼠气道平滑肌细胞(ASMC)中的表达以及miR-199a-5p对ASMC增殖的调控和低氧诱导因子1α(HIF-1α)表达的影响。方法用贴壁法体外培养大鼠ASMC,在常氧及低氧条件下培养24 h,实时荧光定量PCR(qRT-PCR)检测ASMC中miR-199a-5p含量;人工合成大鼠miR-199a-5p的拟似物和抑制物,用脂质体转入低氧培养的ASMC,qRT-PCR检测miR-199a-5p及HIF-1αmRNA的表达,Western blot法检测HIF-1α蛋白的表达,CCK-8法检测ASMC的增殖情况。结果与常氧组相比,低氧处理促进细胞增殖,低氧组HIF-1αmRNA及HIF-1α蛋白的相对水平显著升高;低氧组miR-199a-5p的量较常氧组减少;转染miR-199a-5p拟似物可以显著减缓低氧条件下ASMC的增殖,而其抑制物可促进低氧条件下的细胞增殖。拟似物组HIF-1α蛋白较对照组降低,抑制物组则升高,而低氧条件下各组HIF-1αmRNA水平无明显差异。结论 miR-199a-5p能抑制低氧条件下ASMC的增殖和HIF-1α蛋白的表达。
Objective To investigate the expression of miR-199a-5p in airway smooth muscle cells（ASMCs） of rats in normoxia and hypoxia and its role in the regulation of proliferation and the expression of hypoxia inducible factor 1 alpha（HIF-1α） of ASMCs. Methods ASMCs were prepared by means of adherent culture in vitro. After ASMCs were cultured under normoxia and hypoxia conditions for 24 hours,the content of miR-199a-5p was detected by real-time quantitative PCR（qRT-PCR）. The mimic or inhibitor of miR-199a-5p were artificially synthesized and transferred into ASMCs in hypoxia via liposomes. The expressions of miR-199a-5p and HIF-1α mRNA were detected by qRT-PCR. Western blotting and CCK-8assay were applied to detect the expression levels of HIF-1α protein and the proliferation of ASMCs,respectively. Results Compared with the normoxia group,hypoxia significantly promoted cell proliferation and increased the levels of HIF-1α mRNA and protein. The level of miR-199a-5p decreased in the hypoxia group compared with the normoxia group. The proliferation rate of ASMCs under hypoxia conditions was significantly attenuated by transfection of miR-199a-5p mimic,while it was significantly lifted by transfection of miR-199a-5p inhibitor. Compared with control group,the expression of HIF-1α protein was reduced in the mimic group and raised in the inhibitor group. There was no significant difference in the content of HIF-1αmRNA among groups under hypoxia conditions. Conclusion miR-199a-5p can inhibit the proliferation of ASMCs and the expression of HIF-1α protein in vitro under hypoxia conditions.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2015年第9期1183-1188,共6页
Chinese Journal of Cellular and Molecular Immunology
