对糖尿病肾脏疾病湿热证患者与正常人群尿液外泌体微RNA差异表达的研究

Differential expression of urinary exosome miRNAs in diabetes kidney disease patients with dampness-heat syndrome

目的通过外泌体小RNA测序技术,比较慢性肾脏病3~4期糖尿病肾脏疾病(DKD)湿热证患者与正常人群尿液外泌体微RNA(miRNA)的差异表达。方法2021年4月—2022年4月于安徽中医药大学第一附属医院招募受试者,DKD湿热证患者40例,健康体检正常者40名。2组分别任选10名/例受试者,对尿液外泌体进行miRNA高通量测序,以log2(Fold Change)绝对值≥1及q值<0.05为条件筛选出所有差异表达miRNA,然后进一步筛选出10个miRNA进行验证,筛选条件为:(1)2组样本中,至少1组组内平均表达量(TPM)>2;(2)log2(Fold Change)绝对值≥2;(3)DKD湿热证组中至少有1个样本TPM>0。最后通过定量RT-PCR在2组受试者中进行验证。结果将DKD湿热证组及正常对照组的测序数据与数据库miRNA成熟序列进行对比,正常对照组表达的miRNA为302个,DKD湿热证组表达的miRNA为386个,2组共同表达的miRNA为263个。得出97个有差异表达的miRNA,其中上调的84个,下调的13个。DKD湿热证组患者尿液miR-664a-5p、miR-320b及miR-199a-5p表达水平较正常对照组上调(P<0.001)。miR-664a-5p、miR-320b及miR-199a-5p在诊断DKD湿热证中的受试者工作特征曲线下面积分别为0.886、0.923及0.761。结论慢性肾脏病3~4期DKD湿热证患者尿液外泌体miRNA与正常人群比较存在差异表达;尿液外泌体miR-664a-5p、miR-320b和miR-199a-5p有助于DKD湿热证患者的诊断。

Objective We aimed to analyze the differential expression of urinary exosome miRNAs in diabetes kidney disease(DKD)patients with dampness-heat syndrome in chronic kidney diseaseⅢtoⅣstage by small RNA sequencing technology.Methods This study was conducted at the First Affiliated Hospital of Anhui University of Chinese Medicine from April 2021 to April 2022.In total,80 subjects were enrolled,including 40 DKD patients with dampness-heat syndrome and 40 healthy controls.The urinary exosomes of 10 subjects in both groups were selected for miRNA high-throughput sequencing,and selected the differentially expressed miRNAs were selected according to■.Differentially expressed miRNAs were selected by the following criteria:the mean expression level(TPM)in at least one group■,and at least one sample in the DKD group has TPM>0 and verified by quantitative RT-PCR.Results By comparing the 80 subjects’data to the database,302 miRNAs expressed in healthy controls and 386 miRNAs expressed in DKD patients.In total,97 differentially expressed miRNAs were screened,in including 84 upregulated and 13 downregulated miRNAs.Of these,10 miRNAs were selected for verification.RT-PCR result showed that the expression levels of miR-664 a-5 p,miR-320 b,and miR-199 a-5 p were significantly different between the two groups(P<0.01).The areas under the ROC curve of miR-664 a-5 p,miR-320 b,and miR-199 a-5 p in the diagnosis of dampness-heat syndrome in DKD patients were 0.886,0.923,and 0.761,respectively.Conclusion We found differences in miRNA expression levels in urinary exosomes between DKD patients with dampness-heat syndrome and controls.The urinary exosome levels of miR-664 a-5 p,miR-320 b,and miR-199 a-5 p can be used for the diagnosis of DKD patients with dampness-heat syndrome.