汉黄芩素调控miRNA-135b-3p靶向SIX4抑制A549细胞上皮间质转化的分子机制

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

关键词:  汉黄芩素  非小细胞肺癌  侵袭  上皮间质转化  miRNA-135b-3p  SIX4

Key words:  wogonin  non-small cell lung cancer  invasion  epithelial-mesenchymal transition  miRNA-135b-3p  SIX4

摘要:

目的 探究汉黄芩素对人非小细胞肺癌A549细胞侵袭转移的影响及其对上皮间质转化(epithelial mesenchymal transition,EMT)的作用和分子机制。方法 MTT试验检测汉黄芩素对A549细胞的毒性作用,HE染色观察汉黄芩素处理前后A549细胞形态学变化,划痕试验、Transwell小室试验检测侵袭转移能力,Western blotting和qRT-PCR测定EMT相关标志物表达情况。利用高通量测序技术,检测汉黄芩素处理TGF-β1诱导的A549前后差异表达的miRNA,进行qRT-PCR验证;划痕试验、Transwell小室试验分析细胞转染miRNA mimic、inhibitor和NC后A549细胞侵袭转移能力,Western blotting和qRT-PCR测定EMT相关标志物的表达情况。运用GO和KEGG数据库分析,找出目标miRNA作用的靶基因;Western blotting和qRT-PCR验证miRNA mimic、inhibitor和NC转染后靶基因及蛋白的表达情况;双荧光素报告酶基因试验证明该miRNA对靶基因翻译的调控作用。结果 汉黄芩素可抑制A549细胞的生长增殖,上调TGF-β1诱导的A549细胞E-cadherin及下调Vimentin的表达,降低细胞迁移的能力,逆转细胞的形态变化。高通量测序得到差异表达的miRNA,其中汉黄芩素可逆转miRNA-135b-3p在A549细胞EMT过程中的过表达,抑制A549细胞的侵袭和转移。信号通路分析及miRNA数据库靶基因预测miRNA-135b-3p可调控SIX4的表达。双荧光素报告基因试验显示miRNA-135b-3p可结合于SIX4基因的3'-UTR产生调控,Western blotting、qRT-PCR证明过表达miRNA-135b-3p的A549细胞SIX4表达降低,敲低miRNA-135b-3p后可恢复。结论 汉黄芩素调控miRNA-135b-3p靶向SIX4抑制A549细胞的侵袭转移和EMT过程。

Abstract:

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.