人肺动脉平滑肌细胞中微小RNA-210通过MKP-1负性调节低氧下细胞的增殖

 目的:研究低氧条件下人肺动脉平滑肌细胞中微小RNA-210(miR-210)与丝裂原活化蛋白激酶磷酸酶1 (MKP-1)的关系，是否通过MKP-1调节肺动脉平滑肌细胞增殖及其机制。方法:选用4~8代的人肺动脉平滑肌细胞，共分为12组，其中21% O2正常氧及1% O2低氧培养箱各6组，分别给予转染miR-210抑制剂、增强剂、MKP-1 siRNA等处理，提取RNA和miRNA，并采用实时定量PCR法检测各组平滑肌细胞中miR-210和MKP-1 mRNA的表达，提取蛋白，并用Western blotting法比较各组MKP-1蛋白水平的表达，MTT法检测肺动脉平滑肌细胞的增殖情况。结果:低氧下，人肺动脉平滑肌细胞中miR-210及MKP-1的表达均明显增加，抑制miR-210表达使MKP-1的表达增加并可抑制低氧诱导的细胞增殖，miR-210的过表达可抑制低氧诱导的MKP-1表达上调，但不影响细胞增殖，MKP-1基因沉默后，低氧下miR-210抑制剂对细胞增殖的抑制作用消失。结论: 低氧下的人肺动脉平滑肌细胞中，MKP-1是miR-210的一个新的靶基因，MKP-1可以介导miR-210抑制剂对人肺动脉平滑肌细胞增殖的负性调节作用。

MicroRNA-210 negatively regulates hypoxic hPASMC proliferation by targeting MKP-1

AIM:To investigate the possible interactions between microRNA-210 (miR-210) and mitogen-activated protein kinase phosphatase  1 (MKP-1) and the effect on the proliferation of hypoxic human pulmonary artery smooth muscle cells (hPASMCs). METHODS:hPASMCs were cultured in 21% O2 and 5% CO2 (normoxia) or 1% O2 and 5% CO2 (hypoxia) for 48 h, and then transfected with mimic or inhibitor of miR-210 or MKP-1 small interfering RNA (si-RNA). The levels of RNA, miRNA and protein were isolated separately. RESULTS:The level of miR-210 was significantly increased in cultured hPASMCs exposed to 1% O2 for 48 h, and the expression of MKP-1 at mRNA and protein levels was also increased. Furthermore, inhibition of miR-210 expression increased the mRNA and protein levels of MKP-1 in the hPASMCs and decreased the cell proliferation under hypoxia. Conversely, over-expression of miR-210 prevented hypoxia-induced MKP-1 expression with no effect on the cell proliferation. Knockdown of MKP-1by siRNA abolished the preventive effect of miR-210 inhibitor on the cell proliferation under hypoxia. CONCLUSION: MKP-1 is a target of miR-210 and mediates the negative regulation of miR-210 inhibitor in hypoxic hPASMCs.