Prediction Of Cfa-let-7b MicroRNA Cluster And Cfa-miR-23a Cluster And Validation Of Related Target Proteins During Aristolochic Acid Ⅰ Induced Liver Cancer Stem/progenitor-like Cell Formation In Canines

Objective:To study and predict the differentially expressed miRNAs and miRNA clusters, and validate the related target proteins of predicted miRNAs during the formation of hepatic cancer progenitor-like cells (HCPLCs) induced by aristolochic acid I (AAⅠ) in canine liver.Methods:(1) 8 healthy adult canines were randomized into two groups (4 in each group). The AAⅠ group was orally administrated capsules containing AAⅠ (3 mg/kg/day), and the control group was given the capsules containing control filler for 10 days. Canines were anesthetized by pentobarbital sodium 2 days after the withdrawal of AAⅠ. The eight samples of canine liver (control group C1, C2, C3, C4 and AAⅠ group A1, A2, A3, A4) were analyzed by microarray to detect the differentially expressed miRNAs. Bioinformatics softwares were used to further predict the close miRNA clusters based on the differentially expressed miRNAs, and obtain the post-transcriptional regulatory networks of bioinformatics during the formation of HCPLCs. To find all the miRNA clusters less than 40 kb by searching the miRBase database (http://mirbase.org/). The results were compared with that of miRNA microarray, and the other neighboring miRNAs were aslo selected for qRT-PCR validation. The qRT-PCR results were analyzed statistically using unmatched Mann Whitney test, and P＜0.05 represented significant difference. (2) TargetScan (www.targetscan.org) and miRDB (www.mirdb.org) were used to predict the target genes of different miRNAs, and the overlapping parts of the predicted results by two databases were imported into DAVID (http://david.abcc.ncifcrf.gov) to analyze the KEGG metabolic pathways (www.genome.jp/kegg) which were possibly influenced. The predicted results of metabolic pathways were analyzed accurately by Fisher’s test for statistical analysis. Finally, the number of target genes in the metabolic pathways was tested with the number of genes known in the metabolic pathways. (3) Western blot was used to detect the expressions of FOXO1 in the nucleus and p-FOXO1 in the cytoplasm. (4) Immunohistochemical assay was used to detect the distribution changes of p-FOXO1 in liver tissue to clarify the possible pathologic mechanisms of HCPLCs formation induced by AAⅠ.Results:(1) miRNA microarray and qRT-PCR results showed that compared with CON group, miR-378, let-7b, and miR-33a expressions in the AAⅠ group were downregulated, while miR-200c, miR-24, and miR-660 expressions were upregulated (fold of upregulation> 1.6, fold of downregulation> 1.7, P<0.05). Cfa-let-7a-l-let-7b and cfa-miR-23a-27a-24-2 clusters were found to show consistent changes based on the qRT-PCR validation and the miRBase database, with the former cluster downregulated and the latter one upregulated respectively. (2)The above miRNA clusters were involved in the regulation of apoptosis, immune signal transduction and some other important pathways. The MAPK. signaling pathway is potentially regulated by miR-27a, indicating that the cfa-let-7b and cfa-miR-23a clusters participated in the regulation of apoptosis, and immune signal transduction in the liver injury by AAⅠ. (3) Western blot result found that compared with CON group, the miR-27a target protein FOXO1 in the AAⅠ group was downregulated in the nucleus, while p-FOXO1 expression was significantly upregulated in the cytoplasm (P< 0.05). (4) Immunohistochemical results showed that p-FOXOl in the control group only existed in the nucleus, and p-FOXO1 in the AAⅠ group is significantly translocated into the cytoplasm, indicating that miR-27a targeted FOXO1 and phophorylation of FOXO1 enabled the cells to escape from apoptosis and to maintain the premalignant state.Conclusion:Cfa-let-7a-l-let-7b and cfa-miR-23a-27a-24-2 clusters were involved in the regulation of apoptosis and immune signal transduction in the AAⅠ induced liver injury in canines. Among them, cfa-miR-23a cluster member miR-27a regulated the dynamic states of the downstream target proteins FOXO1/p-FOXO1, and activated p-FOXO1 enabled a few cells to escape from apoptosis and to maintain the state of liver stem-like cells, eventually making these cells become HCPLCs.