人尿中微小RNA的稳定性及其诊断肾损伤的价值

The stability of human urine microRNA-126 and its diagnosis value for the renal injury

目的分析多种实验因素对微小RNA（microRNA，miRNA）．126的影响，并探讨其在临床诊断肾损伤的价值。方法采用配对实验设计和单变量方差分析方法评估不同孵育时间、储存温度、反复冻融次数、pH值、微量蛋白浓度等因素对尿液中miRNA．126稳定性的影响：用实时定量PCR检测139例肾损伤患者和69名健康体检者尿miRNA．126的表达水平。然后，分别用t检验和ROC曲线评价尿中miRNA．126在肾损伤组与健康对照组的差异程度及其对临床肾损伤诊断的价值。结果尿标本中miRNA．126在不同存储温度（室温、4、-20、-80℃）分别为3．81±0．33、3．78±0．32、3．83±O．43、3．69±0．30，不同孵育时间（O、3、6、12、24h）分别为3．81±0．33、3．75±0．39、3．66±0．33、3．76±O．32、3．62±0．39，不同反复冻融次数（0、2、4、8次）分别为3．75±0．29、3．70±0．32、3．70±0-31、3．56±O．29，不同DH值（4．5、7．0、9．0）分别为3．76±0．28、3．72-~0．28、3．65±0．33，不同微量蛋白的浓度（0-20、40-80、120-140mg／L）分别为3．72±0．30、3．63±0．38、3．70±0．50；经单因素方差分析，同因素不同水平下miRNA-126含量差异均无统计学意义（，值分别为1．840、1．321、2．262、O．941、0．411，P均〉0．05）。肾损伤组尿中miRNA-126表达水平（4．55±0．63）显著高于健康对照组（3．75±0．39），且差异有统计学意义（t=6．23l，P=0．002）：当以miRNA-126的相对水平182（50-Cr）〉4．19为临界值时，miRNA-126的ROC曲线下面积为0．779，95％可信区间（a）为0．681-0．877，P〈0．001，预测。肾损伤的敏感度为61．7％，特异度为88．9％。结论miRNA．126在尿中相对稳定，有望成为一项新的临床诊断肾损伤指标。

Objective To analyze the stability ofmicroRNA-126 （miRNA-126） in human urine and evaluate its possibility in diagnosis of renal injury. Methods The influence of incubation time, test temperature, numbers of freeze-thaw cycles, pH value and different albumin concentration on stability of microRNA-126 in the urine was evaluated by one variable Chi-Square analysis. Real-time PCR was used to compare the levels of urinary miRNA-126 between 69 healthy volunteers and 139 patients with renal injury. T test was used to compare the variance of urinary miRNA- 126 levels between the healthy and patient groups. ROC curve was used to evaluate the value of urine miRNA-126 in diagnosis of renal injury. Results The levels of urinary miRNA- 126 were 3.81 ± 0.33, 3.78 ± 0.32, 3.83 ± 0.43, 3.69± 0.30 when the samples were incubated at room temperature, 4 ℃, -20 ℃, -80 ℃ for 24 hours respectively. The concentration of urinary miRNA-126 were 3.81 ±0.33, 3.75±0.39, 3.66±0.33, 3.764-0.32, 3.62±0.39 when the samples were placed at room temperature for 0, 3, 6, 12, 24 h. The levels of urinary miRNA-126 were 3.75±0.29, 3.70±0.32, 3.70±0.31, 3.56±0.29 when urinary specimens were subjected to different freeze-thaw cycles（0, 2, 4, 8）. The levels of urinary miRNA- 126 were 3.76± 0.28, 3.72± 0.28, 3.65 ± 0.33 when urinary specimens were subjected to different pH values （4.5, 7.0, 9.0）. The concentrations of urinary miRNA-126 in groups （0-20,40-80, 120-140 mg/L） containing different urinary albumins （3.72-t-0.30, 3.63 ＋0.38, 3.70-t-0.50） showed no significantly change. There were no significant differences in the levels ofmiRNA-126 （F value of 1.840, 1.321, 2.262, 0.941, 0.411, all of P〉0.05）. The level of urinary miRNA-t26 in patients with renal injury （4.55 ＋ 0.63） was higher than that in healthy volunteers （3.75 -＋- 0.39, t=6.231, P=-0.002）. ROC analysis revealed that miRNA- 126 had an AUC of 0.779 （95%C1： 0.681-0.877, P〈0.001） and sensitivity of 61.7%, specificity of 88.9% when the cut-off value was lg2（50-Cr 〉 4.19. Conclusion These results indicated that miRNA-126 in urine is relatively stable in urine. It has the potential to become a new molecular biomarker for early diagnosis of renal injury.