微小RNA-320检测在体外循环所致急性呼吸窘迫综合征中的临床价值及其机制研究

目的研究微小RNA（microRNA）在体外循环（CPB）诱导的急性呼吸窘迫综合征（ARDS）患者中表达，并初步探讨其机制。 方法设定体外循环转流开始前（T1）、转流结束后4 h（T2）、术后8 h（T3）、术后16 h（T4）等4个时间点，采用microRNA微阵列芯片分析32例因CPB诱发ARDS患者microRNA变化，并通过实时荧光定量PCR（qRT-PCR）技术加以验证。酶联免疫分析法检测患者肿瘤坏死因子α（TNF-α）、白细胞介素6（IL-6）在T1~T4等4个时间点的水平变化，同时计算呼吸指数和氧合指数。Pearson相关性分析研究microRNA-320（miR-320）与TNF-α、IL-6、呼吸指数、氧合指数、Murray急性肺损伤评分以及急性病生理学和长期健康评价（APACHE）Ⅱ评分的相关性。体外培养人肺腺癌A549细胞，并转染pcDNA3.1-miR-320，采用细胞增殖检测试剂盒8（CCK-8）法和流式细胞术分析pcDNA3.1-miR-320转染对A549细胞48 h存活和凋亡率的影响。 结果在T1与T4时间点，ARDS患者血液标本中共有8个microRNA表达差异有统计学意义（t=28.313、30.014、25.313、20.312、29.442、21.443、18.427、22.369，P均< 0.001），其中5个出现降低，3个出现增高，其中miR-499的降低程度（0.28 ± 0.09）最为显著，而miR-320的增高程度（1.62 ± 0.12）最为显著（P均< 0.05）。qRT-PCR证实从T1至T4时间点，miR-320相对表达水平（1.00、1.14 ± 0.07、1.34 ± 0.06、1.71 ± 0.08）逐渐升高，差异有统计学意义（F=20.648，P < 0.05）。CPB诱发ARDS的患者T1与T4时间点相比，TNF-α[（110 ± 10）ng/L vs.（254 ± 16）ng/L]、IL-6[（86 ± 8）ng/L vs.（165 ± 11）ng/L]、呼吸指数[（0.182 ± 0.021）vs.（0.381 ± 0.032）]均增高，氧合指数[（350 ± 22）vs.（245 ± 18）]下降，差异均有统计学意义（P均< 0.05）。Pearson相关性分析发现，miR-320的表达水平与Murray急性肺损伤评分，APACHEⅡ评分，T4时间点TNF-α、IL-6、呼吸指数均呈正相关（r=0.685、0.744、0.737、0.711、0.846，P均< 0.05），而与氧合指数呈负相关（r=-0.745，P < 0.05）。pcDNA3.1-miR-320转染的A549细胞组与对照组48 h细胞存活率[（82% ± 8%）vs. 100%]、凋亡率[（20.0% ± 1.1%）vs.（9.4% ± 0.8%）]相比，差异均具有统计学意义（P均< 0.05）。 结论miR-320水平与TNF-α、IL-6等肺部损伤指标具有相关性，miR-320的高表达可能介导CPB导致的ARDS，作用机制为诱导肺泡上皮细胞的凋亡，因此miR-320具有临床诊断、评估ARDS生物学指标的潜能。

Clinical value and mechanism research of microRNA-320 in acute respiratory distress syndrome after cardiopulmonary bypass

ObjectiveTo investigate the expression of microRNA in the patients with acute respiratory distress syndrome (ARDS) caused by cardiopulmonary bybass (CPB), and primarily explore the possible mechanism. MethodsFour time points were set up: the beginning of CPB operation (T1), 4 h after the start of operation (T2), 8 h after operation (T3), and 16 h after operation (T4). MicroRNA microarray was used to analyze the expression of microRNAs in 32 patients with ARDS induced by CPB. Quantitative real time polymerase chain reaction (qRT-PCR) was performed to verify the expression of microRNAs. Enzyme linked immunosorbent assay detected tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6) levels of patients in T1-T4 time points. Respiratory index and oxygenation index were also calculated. Pearson correlation analysis investigated the correlation between microRNA-320 (miR-320) expression and TNF-α, IL-6, respiratory index, oxygenation index, Murray acute lung injury score and acute physiology and chronic health evaluation (APACHE) Ⅱ score. Furthermore, human lung adenocarcinoma A549 cells were in vitro cultured, and then pcDNA3.1-miR-320 was transfected into cells; CCK-8 kits and flow cytometry were used to determine the survival and apoptosis rates of A549 cell at 48 h after transfection. ResultsIn patients with ARDS induced by CPB, expressions of eight microRNAs were changed significantly at T1 and T4 time points(t=28.313, 30.014, 25.313, 20.312, 29.442, 21.443, 18.427, 22.369; all P < 0.001); five increased and three decreased. The decrease level of miR-499 (0.28 + 0.09) and increase level of miR-320 (1.62 + 0.12) both changed significantly (all P < 0.05). qRT-PCR found that only the relative expression levels of miR-320 (1.00, 1.14 + 0.07, 1.34 + 0.06, 1.71 + 0.08) gradually increased from T1 to T4 time points, and the difference was statistically significant (F=20.648, P < 0.05). In these CPB induced ARDS patients, TNF-α level [(110 ± 10) ng/L vs. (254 ± 16) ng/L], IL-6 level [(86 ± 8) ng/L vs. (165 ± 11) ng/L], respiratory index [(0.182 ± 0.021) vs. (0.381 ± 0.032)] all dramatically increased, while oxygenation index [(350 ± 22) vs. (245 ± 18)] declined when comparing T1 and T4 time points (all P < 0.05). Pearson correlation analysis found the expression of miR-320 was positively correlated with Murray acute lung injury score, APACHEⅡscore, and TNF-α level, IL-6 level, respiratory index in T4 time point (r=0.685, 0.744, 0.737, 0.711, 0.846; all P < 0.05), and negatively correlated with oxygenation index (r=-0.745, P < 0.05). Moreover, the survival [(82% ± 8%) vs. 100%] and apoptosis [(20.0% ± 1.1%) vs. (9.4% ± 0.8%)] rates after 48 h transfection between the pcDNA3.1-miR-320 transfected A549 cell group and control group were remarkably different (both P < 0.05). ConclusionsMiR-320 expression is correlated with the lung injury indicators such as TNF-α and IL-6, and its high expression may mediate ARDS caused by CPB. The mechanism is to induce apoptosis of alveolar epithelial cells. Thus, miR-320 detection has clinical value for diagnosis and assessment of ARDS.