Zhang Hui, Zhu Shuyang, Zhang Cantang, Liu Wenjing, Zhu Jiechen
Department of Respiratory Medicine, Hospital Affiliated to Xuzhou Medical College, Xuzhou 221006, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2015 Sep;31(9):1183-8.
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.
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-8 assay were applied to detect the expression levels of HIF-1α protein and the proliferation of ASMCs, respectively.
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-1α mRNA among groups under hypoxia conditions.
miR-199a-5p can inhibit the proliferation of ASMCs and the expression of HIF-1α protein in vitro under hypoxia conditions.
探讨miR-199a-5p在常氧和低氧条件下大鼠气道平滑肌细胞（ASMCs）中的表达及其在调节ASMCs增殖和低氧诱导因子1α（HIF-1α）表达中的作用。
采用体外贴壁培养法制备ASMCs。将ASMCs在常氧和低氧条件下培养24小时后，通过实时定量PCR（qRT-PCR）检测miR-199a-5p的含量。人工合成miR-199a-5p的模拟物或抑制剂，并通过脂质体将其转入低氧条件下的ASMCs中。通过qRT-PCR检测miR-199a-5p和HIF-1α mRNA的表达。分别采用蛋白质免疫印迹法和CCK-8法检测HIF-1α蛋白的表达水平和ASMCs的增殖情况。
与常氧组相比，低氧显著促进细胞增殖，增加HIF-1α mRNA和蛋白水平。低氧组miR-199a-5p水平较常氧组降低。转染miR-199a-5p模拟物可显著减弱低氧条件下ASMCs的增殖率，而转染miR-199a-5p抑制剂则可显著提高其增殖率。与对照组相比，模拟物组HIF-1α蛋白表达降低，抑制剂组升高。低氧条件下各组间HIF-1α mRNA含量无显著差异。
miR-199a-5p在体外低氧条件下可抑制ASMCs的增殖及HIF-1α蛋白的表达。
