miRNA expression profiles in Smad4-positive and Smad4-negative SW620 human colon cancer cells detected by next-generation small RNA sequencing

摘要

Background and aims: SMAD4, as a tumor suppressive gene in human colon cancer, inhibits the metastasis of colon adenocarcinoma cells. However, the molecular mechanisms are unclear. miRNAs play an important role in the pathogenesis and progression of cancer. Methods: In this study, a deep sequencing technique was used to screen Smad4-regulated miRNAs in human colon cancer SW620 cell line. Using a next-generation small RNA sequencing approach, we compared the miRNA expression profiles of SW620 colon cancer cells transfected with smad4 lentiviral vector with those transfected with control vector. Six samples were selected and sequenced randomly each from control group (smad4-negative cell) and Smad4 group (Smad4-positive cells). Quantitative reverse transcription-PCR (qRT-PCR) and Western blot (WB) was used to validate the results of sequencing. Results: Smad4 reexpression significantly upregulated 43 known miRNAs and downregulated 10 known miRNAs expression. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analysis of predicted miRNAs targets showed that these genes were mainly involved in protein-binding transcription factor activity, vascular smooth muscle contraction, pathways in cancer metastasis, and phosphatidylinositol 3-kinase–Akt signal pathway. qRT-PCR and WB validated the partial results of sequencing. Reexpression of Smad4 inhibited colon cancer cell migration and invasion. Smad4 reexpression increased the expression of E-cadherin (E-cad) and decreased the Vimentin (Vim) and Matrix Metalloproteinase-9 expression. Restoration of SMAD4 results in a marked decrease of Vim by inhibiting p-AKT and p-EPHA2, but significantly increased the E-cad by AKT–EPHA2 pathways. Conclusion: Smad4 inhibits the migration and invasion ability of colon cancer cells in vitro and this is the first report of Smad4-mediated miRNA expression profiling in Smad4-positive and Smad4-negative SW620 human colon cancer cells, which may help us better understand the role of Smad4 in inhibiting the metastasis of colon cancer cells and its possible molecular mechanisms.