Radix Sophorae Flavescentis (Kushen) inhibits NSCLC by targeting miR-183-5p/EGR1 and PTEN/AKt pathway

Abstract

Abstract Background Radix Sophorae Flavescentis (Kushen) is the principal herb consisting of Compound Kushen Injection (CKI), which has been approved for the clinical treatment of tumors. Although CKI has been widely used, especially on non-small cell lung cancer (NSCLC), its underlying mechanisms of Kushen regarding microRNA-target gene-pathway remain unclear. Methods A549 cell line was selected as the NSCLC model and treated with different concentrations of Kushen to obtain the mRNA and miRNA expression profiles. Then differentially expressed (DE) genes and miRNAs were analyzed with miRTarBase, miRDB, and TargetScan 7.0. A DE miRNAs-DEGs network was finally constructed to find the critical miRNA-mRNA interaction influenced by Kushen. Furthermore, clinical significance and prognosis analysis of hub genes based on TCGA and Oncomine datasets were performed further to narrow down the hub genes and corresponding miRNAs. Finally, the effects of Kushen on critical miRNA-mRNA interaction and related pathways were verified by in vitro experiments, including qPCR, Western blot, miRNA mimic transfection assay, and dual-reporter luciferase assay. Results A DE miRNAs-DEGs network was constructed based on miRNA-target databases screening, with 16 DE miRs and 68 DEGs identified. With the help of clinical evaluation and qPCR experiment of hub genes, we focused on miR-183-5p/EGR1 and miR221-3p/FOS interactions. The qPCR results indicated that Kushen induced down-regulation of miR-183-5p and miR-221-3p, and up-regulation of EGR1 and FOS. Dual-luciferase reporter assay results demonstrated that EGR1 and FOS were direct targets of miR-183-5p and miR-221-3p in A549 cells, respectively. Furthermore, the up-regulation of EGR1 by Kushen was also accompanied by an increase in PTEN expression and a decrease in AKt expression. And the opposite effects of EGR1 on A549 were eliminated by miR-183-5p overexpression. However, the overexpression of miR-221-3p cannot induce FOS down-regulation. Conclusions Altogether, the above results supported that miR-183-5p /EGR1 and the PTEN/AKT pathway played a crucial role in the beneficial effects of Kushen on A549, which highlights the therapeutic potential of Kushen in the treatment of NSCLC.