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±0.43、3.69±0.30，不同孵育时间（0 、3、6、12、24 h）分别为3.81±0.33、3.75±0.39、3.66±0.33、3.76±0.32、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±0.29，不同pH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、120～140 mg/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（F值分别为1.840、1.321、2.262、0.941、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=6.231，P=0.002）；当以miRNA-126的相对水平lg2^（50-Ct）＞4.19为临界值时，miRNA-126的ROC曲线下面积为0.779，95%可信区间（CI）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 of microRNA-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, 3.76±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±0.30, 3.63±0.38, 3.70±0.50) showed no significantly change. There were no significant differences in the levels of miRNA-126 (F value of 1.840, 1.321,2.262, 0.941, 0.411, all of P＞0.05). The level of urinary miRNA-126 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%CI: 0.681-0.877, P＜0.001) and sensitivity of 61.7%, specificity of 88.9% when the cut-off value was lg2^(50-Ct)＞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.