Molecular Mechanism of Wogonin Inhibits EMT of A549 Cells by the Regulation of miRNA-135b-3p Targeting SIX4

OBJECTIVE To explore the influence of wogonin on cell migration and invasion in human non-small cell lung cancer cells A549 and its role in epithelial-mesenchymal transition(EMT) and its molecular mechanism. METHODS Using MTT assay to detect the toxic effect of wogonin on A549 cells. HE staining was used to observe morphology change of the treatment of TGF-β1 induced A549 by wogonin. Using scratch assay and Transwell assay to detect the alterration of migration and invasion ability of wogonin on TGF-β1-induced A549 cells. The expression of EMT-related markers was analyzed by Western blotting and qRT-PCR after wogonin treatment TGF-β1 induced A549 cells. High-throughput sequencing technology was used to detect the expression of miRNA in TGF-β1-induced A549 cells before and after treatment with wogonin, data enrichment analysis showed that miRNA gene differentially expressed, it was verified by qRT-PCR. After A549 cells were transfected with miRNA mimic, inhibitor and NC, scratch and Transwell assays were used to analyze the changes of A549 cell invasion and metastasis. The expression of EMT-related markers in A549 cells was determined by Western blotting and qRT-PCR. Using GO and KEGG database analysis to identify the target gene of miRNA. Western blotting and qRT-PCR confirmed that the expression of target gene and target protein after the miRNA mimic, inhibitor and NC transfected A549 cells. The Dual-Luciferase Assay demonstrates the regulation of the target gene translation by the miRNA. RESULTS Wogonin could inhibit the growth and proliferation of A549 cells. Wogonin significantly reversed the TGF-β1 decrease in E-cadherin expression and increase in Vimentin expression. Wogonin could inhibit migration and invasion of TGF-β1 induced A549 cells. Wogonin could reverse morphological changed in TGF-b1-induced A549 cells. High-throughput sequencing results showed that there were many differentially expressed miRNAs, in which miRNA-135b-3p was significantly increased in A549 cells during EMT, and after treatment with wogonin the expression was significantly decreased. Pathway analysis and miRNA database predicted that miRNA-135b-3p can regulate the expression of SIX4. Dual-Luciferase assay showed that miRNA-135b-3p binds to the 3'-UTR of the SIX4. Both Western blotting and qRT-PCR showed that the expression of SIX4 was significantly decreased in overexpressing miRNA-135b-3p A549 cells, while knockdown miRNA-135b-3p, the expression of SIX4 was restored in A549 cells. CONCLUSION Wogonin inhibits invasion and metastasis and EMT of A549 cells by the regulation of miRNA-135b-3p targeting SIX4.