ABSTRACT: Purpose:The aim of this study was to investigate the potential effect of KeLuoXin on expression of miRNAs in DR with db/db mouse models Meathod: Six db/db mice of Group II were treated with 1560mg/kg KeLuoXin for 20 weeks, intragastrically, once a day, six model control mice were treated with equal volume normal saline.After 12 mice were sacrificed,retinal tissues were removed and stored in cryopreservation tubes. Total RNA was isolated from retina samples and used to prepare the miRNA sequencing library, The libraries were denatured as single-stranded DNA molecules, captured on Illumina flow cells, amplified in situ as clusters and finally sequenced for 51 cycles on Illumina NextSeq 500 according to the manufacturer's instruction. Raw sequencing data generated from Illumina NextSeq 500 that pass the Illumina chastity filter are used for following analysis. Differentially expressed miRNAs analyses were performed with R package edgeR. Fold change (cutoff 1.5), p-value (≤ 0.05) and CPM (≥ 1 mean in one group) were used for filtering differentially expressed miRNAs. Cluster analysis arranges samples into groups based on their expression level (CPM values), which allows us to hypothesize the relationships among samples. The heatmap were performed using significant differentially expressed miRNAs. The Scatter-Plot is a visualization method used for assessing the miRNA expression variation between the two compared groups.The Volcano plot is a visualization method for quick visual identification of miRNAs displaying large magnitude changes which are also statistically significant. MiRNA target prediction analysis for top 10 differentially expressed miRNAs were filtered based on miRNA target databases. Results: We obtained a total of 61,925,224 and 68,473,799 clean reads, 58,159,780 and 61,600,656 adapter-trimmed reads from the T2DM model group and KLX-treatment group. In detail, a total of 52 miRNAs up-regulated and 12 miRNAs were down-regulated in KLX-treatment group. The absolute fold changes ranged from 1.53 to 7.35 for up-regulated miRNAs, and from 0.020 to 0.008 for the down-regulated ones. The differential expression analysis identified 64 miRNAs that were differentially expressed from T2DM model group to KLX-treatment group and potently regulated by Keluoxin capsule. Up-regulated miRNAs targets were assigned to 45 categories (FDR<0.05, up-regulated miRNAs) as follows: 29 were associated with biological processes, 15 were associated with cellular components, 1 were involved in molecular functions. The KEGG pathways analysis indicated that the predicted targets of the miRNAs are involved in several important pathwayssuch as insulin secretion signaling pathway and TGF-beta signaling pathway. Conclusions: Keluoxin capsule may exert the retinal protection in T2DM by targeting miRNAs and regulating miRNAs-mediated signaling pathways downstream.
