| Objective: This article investigates the molecular mechanism of wogonin on the Epithelial-mesenchymal transition of non-small cell lung cancer cell line A549.According this molecular mechanism,we look forward to finding potential miRNA markers and therapeutic targets for the treatment of non-small cell lung cancer.It can provide more experiment data for clinical use of anti-tumor activity of Chinese herbal extracts.Methods:(1)Inhibitory effect of wogonin on growth,invasion and metastasis of non-small cell lung cancer cell line A549:Using MTT assay to detect the toxic effect of wogonin on A549 cells;The effects of Wogonin on morphological changes of A549 cells induced by TGF-β1 were observed by HE staining;Using scratch assay and transwell assay to detect the effects of wogonin on ability of migration and invasion in TGF-β1-induced A549 cells;the expression of EMT-related markers are analyzed by Western blot assay and qRT-PCR assay in TGF-β1 induced A549 cells after treatment by wogonin.(2)The role of miRNA in the regulation of EMT in A549 cells by wogonin:High-throughput sequencing technology was used to detect the expression of miRNA in TGF-β1-induced non-small cell lung cancer cell A549 before and after treatment with Wogonin,data enrichment analysis showed that miRNA gene differentially expressed.Using GO and KEGG database analysis to identify the target gene of miRNA;Using Western Blot and qRT-PCR observe the expression of target gene of miRNA after TGF –β1 induced A549 cell with treatment of wogonin;The expression of miRNA was detected by qRT-PCR after A549 cells were transfected with miRNA mimic,inhibitor and NC 48h;The expression of EMT-related markers and target gene in A549 cells was also determined by Western Blot and qRT-PCR,and transwell assays were used to analyze the changes of A549 cell invasion and metastasis,both after A549 cells were transfected with miRNA mimic、 inhibitor and NC 48h;The Dual-Luciferase Assay demonstrates the regulation of the target gene translation by the miRNA.Results:(1)Wogonin can inhibit the growth and proliferation of A549 cells;Wogonin can reverse the morphological changes of TGF-β1-induced A549 cells;Wogonin inhibits the migration and invasion of TGF-β1-induced A549 cells,promoted the expression of E-cadherin in EMT cells,and inhibited the expression of Vimentin in EMT cells.(2)High-throughput sequencing results showed that there are many differentially expressed miRNAs verified by qRT-PCR,in which miRNA-135b-3p was significantly increased in A549 cells during EMT,and after treatment with wogonin the expression was significantly decreased;GO and miRNA database predicted that miRNA-135b-3p can regulate the expression of SIX4;The expression of SIX4 was recovered in TGF-β1 induced A549 cell with treatment of wogonin;After overexpressing miRNA-135b-3p in A549 cells,E-cadherin decreased and Vimentin increased at both gene and protein levels,whereas E-cadherin were increased and Vimentin was down-regulated at both gene and protein levels after knocking down miRNA-135b-3p in A549 cells.Both Western Blot 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;After A549 cells were transfected with miR-135b-3p mimic、miR-135b-3p inhibitor and NC,Transwell assay showed that miR-135b-3p can promote invasion and metastasis of A549 cells;Dual-Luciferase assay showed that miRNA-135b-3p binds to the 3’-UTR of the SIX4.Conclusion:Wogonin can inhibit the invasion,metastasis and EMT of A549 cells in non-small cell lung cancer.This effect was related to baicalin inhibiting miRNA-135b-3p expression and then promoting SIX4 expression.miRNA-135b-3p should a potential target for recurrence and metastasis treatment of non-small cell lung cancer. |
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